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Phospho-Akt (Ser473) antibody

RRID:AB_2315049

Antibody ID

AB_2315049

Target Antigen

Ser473

Proper Citation

(Cell Signaling Technology Cat# 4060, RRID:AB_2315049)

Clonality

monoclonal antibody

Comments

Applications: W, IP, IHC-P, IF-IC, F. Consolidation on 7/2016: AB_916027, AB_2341228, AB_10234665. Info: Rated by ISCC, Intestinal Stem Cell Consortium (check ras https://iscc.coh.org/).

Clone ID

Clone D9E

Host Organism

rabbit

Vendor

Cell Signaling Technology

Cat Num

4060 also 4060L, 4060S, 4060P

Publications that use this research resource

A Role for Hypocretin/Orexin in Metabolic and Sleep Abnormalities in a Mouse Model of Non-metastatic Breast Cancer.

  • Borniger JC
  • Cell Metab.
  • 2018 Jul 3

Literature context:


Abstract:

We investigated relationships among immune, metabolic, and sleep abnormalities in mice with non-metastatic mammary cancer. Tumor-bearing mice displayed interleukin-6 (IL-6)-mediated peripheral inflammation, coincident with altered hepatic glucose processing and sleep. Tumor-bearing mice were hyperphagic, had reduced serum leptin concentrations, and enhanced sensitivity to exogenous ghrelin. We tested whether these phenotypes were driven by inflammation using neutralizing monoclonal antibodies against IL-6; despite the reduction in IL-6 signaling, metabolic and sleep abnormalities persisted. We next investigated neural populations coupling metabolism and sleep, and observed altered activity within lateral-hypothalamic hypocretin/orexin (HO) neurons. We used a dual HO-receptor antagonist to test whether increased HO signaling was causing metabolic abnormalities. This approach rescued metabolic abnormalities and enhanced sleep quality in tumor-bearing mice. Peripheral sympathetic denervation prevented tumor-induced increases in serum glucose. Our results link metabolic and sleep abnormalities via the HO system, and provide evidence that central neuromodulators contribute to tumor-induced changes in metabolism.

Funding information:
  • NCI NIH HHS - R01 CA194924()
  • NCI NIH HHS - R21 CA191846()
  • NHLBI NIH HHS - R01 HL095372(United States)

Encoding Growth Factor Identity in the Temporal Dynamics of FOXO3 under the Combinatorial Control of ERK and AKT Kinases.

  • Sampattavanich S
  • Cell Syst
  • 2018 Jun 27

Literature context:


Abstract:

Extracellular growth factors signal to transcription factors via a limited number of cytoplasmic kinase cascades. It remains unclear how such cascades encode ligand identities and concentrations. In this paper, we use live-cell imaging and statistical modeling to study FOXO3, a transcription factor regulating diverse aspects of cellular physiology that is under combinatorial control. We show that FOXO3 nuclear-to-cytosolic translocation has two temporally distinct phases varying in magnitude with growth factor identity and cell type. These phases comprise synchronous translocation soon after ligand addition followed by an extended back-and-forth shuttling; this shuttling is pulsatile and does not have a characteristic frequency, unlike a simple oscillator. Early and late dynamics are differentially regulated by Akt and ERK and have low mutual information, potentially allowing the two phases to encode different information. In cancer cells in which ERK and Akt are dysregulated by oncogenic mutation, the diversity of states is lower.

Funding information:
  • British Heart Foundation - RG/11/17/29256(United Kingdom)
  • NHLBI NIH HHS - U54 HL127365()
  • NIGMS NIH HHS - P50 GM107618()

mTORC1 Promotes Metabolic Reprogramming by the Suppression of GSK3-Dependent Foxk1 Phosphorylation.

  • He L
  • Mol. Cell
  • 2018 Jun 7

Literature context:


Abstract:

The mammalian Target of Rapamycin Complex 1 (mTORC1)-signaling system plays a critical role in the maintenance of cellular homeostasis by sensing and integrating multiple extracellular and intracellular cues. Therefore, uncovering the effectors of mTORC1 signaling is pivotal to understanding its pathophysiological effects. Here we report that the transcription factor forkhead/winged helix family k1 (Foxk1) is a mediator of mTORC1-regulated gene expression. Surprisingly, Foxk1 phosphorylation is increased upon mTORC1 suppression, which elicits a 14-3-3 interaction, a reduction of DNA binding, and nuclear exclusion. Mechanistically, this occurs by mTORC1-dependent suppression of nuclear signaling by the Foxk1 kinase, Gsk3. This pathway then regulates the expression of multiple genes associated with glycolysis and downstream anabolic pathways directly modulated by Foxk1 and/or by Foxk1-regulated expression of Hif-1α. Thus, Foxk1 mediates mTORC1-driven metabolic rewiring, and it is likely to be critical for metabolic diseases where improper mTORC1 signaling plays an important role.

Funding information:
  • NHLBI NIH HHS - R01 HL121266()
  • NIGMS NIH HHS - P41 GM103533(United States)
  • NIGMS NIH HHS - R01 GM051405()

Spred1 Safeguards Hematopoietic Homeostasis against Diet-Induced Systemic Stress.

  • Tadokoro Y
  • Cell Stem Cell
  • 2018 May 3

Literature context:


Abstract:

Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis.

Funding information:
  • Arthritis Research UK - 17522(United Kingdom)

R-Ras1 and R-Ras2 Are Essential for Oligodendrocyte Differentiation and Survival for Correct Myelination in the Central Nervous System.

  • Sanz-Rodriguez M
  • J. Neurosci.
  • 2018 May 30

Literature context:


Abstract:

Rapid and effective neural transmission of information requires correct axonal myelination. Modifications in myelination alter axonal capacity to transmit electric impulses and enable pathological conditions. In the CNS, oligodendrocytes (OLs) myelinate axons, a complex process involving various cellular interactions. However, we know little about the mechanisms that orchestrate correct myelination. Here, we demonstrate that OLs express R-Ras1 and R-Ras2. Using female and male mutant mice to delete these proteins, we found that activation of the PI3K/Akt and Erk1/2-MAPK pathways was weaker in mice lacking one or both of these GTPases, suggesting that both proteins coordinate the activity of these two pathways. Loss of R-Ras1 and/or R-Ras2 diminishes the number of OLs in major myelinated CNS tracts and increases the proportion of immature OLs. In R-Ras1-/- and R-Ras2-/--null mice, OLs show aberrant morphologies and fail to differentiate correctly into myelin-forming phenotypes. The smaller OL population and abnormal OL maturation induce severe hypomyelination, with shorter nodes of Ranvier in R-Ras1-/- and/or R-Ras2-/- mice. These defects explain the slower conduction velocity of myelinated axons that we observed in the absence of R-Ras1 and R-Ras2. Together, these results suggest that R-Ras1 and R-Ras2 are upstream elements that regulate the survival and differentiation of progenitors into OLs through the PI3K/Akt and Erk1/2-MAPK pathways for proper myelination.SIGNIFICANCE STATEMENT In this study, we show that R-Ras1 and R-Ras2 play essential roles in regulating myelination in vivo and control fundamental aspects of oligodendrocyte (OL) survival and differentiation through synergistic activation of PI3K/Akt and Erk1/2-MAPK signaling. Mice lacking R-Ras1 and/or R-Ras2 show a diminished OL population with a higher proportion of immature OLs, explaining the observed hypomyelination in main CNS tracts. In vivo electrophysiology recordings demonstrate a slower conduction velocity of nerve impulses in the absence of R-Ras1 and R-Ras2. Therefore, R-Ras1 and R-Ras2 are essential for proper axonal myelination and accurate neural transmission.

Funding information:
  • Intramural NIH HHS - ZIA BC011010-06(United States)

Exogenous H2 S exerts biphasic effects on porcine mammary epithelial cells proliferation through PI3K/Akt-mTOR signaling pathway.

  • Zhang J
  • J. Cell. Physiol.
  • 2018 May 11

Literature context:


Abstract:

This study aimed to investigate the effects of exogenous H2 S on the proliferation of porcine mammary gland epithelial cells (PMECs) and explore the underlying mechanisms. We found that exposure of PMECs to NaHS, at concentrations ranging from 10 to 200 µM, stimulated cell proliferation. However, high concentration of NaHS (600 µM) inhibited PMECs proliferation. Accordingly, 10 µM NaHS significantly increased the percentage of cells undergoing DNA replication, elevated the mRNA and/or protein expression of Cyclin A2, Cyclin D1/3, Cyclin E2 and PCNA, and decreased p21 mRNA expression. In contrast, 600 µM NaHS elicited the opposite effects to that of 10 µM NaHS. In addition, PI3 K/Akt and mTOR signaling pathways were activated or inhibited in response to 10 or 600 µM NaHS, respectively. Furthermore, the promotion of PMECs proliferation, the change of proliferative genes expression, and the activation of mTOR signaling pathway induced by 10 µM NaHS were effectively blocked by PI3 K inhibitor Wortmannin. Similarly, inhibition of mTOR with Rapamycin totally abolished the 10 µM NaHS-induced stimulation of PMECs proliferation and alteration of proliferative genes expression, with no influence on PI3 K/Akt signaling pathway. Moreover, constitutive activation of Akt pathway via transfection of Akt-CA completely eliminated the inhibition of PMECs proliferation and mTOR signaling pathway, and the change of proliferative genes expression induced by 600 µM NaHS. In conclusion, our findings provided evidence that exogenous H2 S supplied by NaHS exerted biphasic effects on PMECs proliferation, with stimulation at lower doses and suppression at high dose, through the intracellular PI3 K/Akt-mTOR signaling pathway.

Funding information:
  • European Research Council - 232933(International)

Helicobacter pylori Infection Modulates Host Cell Metabolism through VacA-Dependent Inhibition of mTORC1.

  • Kim IJ
  • Cell Host Microbe
  • 2018 May 9

Literature context:


Abstract:

Helicobacter pylori (Hp) vacuolating cytotoxin (VacA) is a bacterial exotoxin that enters host cells and induces mitochondrial dysfunction. However, the extent to which VacA-dependent mitochondrial perturbations affect overall cellular metabolism is poorly understood. We report that VacA perturbations in mitochondria are linked to alterations in cellular amino acid homeostasis, which results in the inhibition of mammalian target of rapamycin complex 1 (mTORC1) and subsequent autophagy. mTORC1, which regulates cellular metabolism during nutrient stress, is inhibited during Hp infection by a VacA-dependent mechanism. This VacA-dependent inhibition of mTORC1 signaling is linked to the dissociation of mTORC1 from the lysosomal surface and results in activation of cellular autophagy through the Unc 51-like kinase 1 (Ulk1) complex. VacA intoxication results in reduced cellular amino acids, and bolstering amino acid pools prevents VacA-mediated mTORC1 inhibition. Overall, these studies support a model that Hp modulate host cell metabolism through the action of VacA at mitochondria.

Funding information:
  • NIAID NIH HHS - R01 AI045928()
  • NIAID NIH HHS - R21 AI117497()
  • NIAID NIH HHS - U19 AI106772(United States)
  • NIGMS NIH HHS - R01 GM089771()

Endothelial insulin receptor restoration rescues vascular function in male insulin receptor haploinsufficient mice.

  • Sengupta A
  • Endocrinology
  • 2018 May 15

Literature context:


Abstract:

Reduced systemic insulin signaling promotes endothelial dysfunction and diminished endogenous vascular repair. We asked whether restoration of endothelial insulin receptor expression could rescue this phenotype. Insulin receptor haploinsufficient mice (IRKO) were crossed with mice expressing a human insulin receptor transgene in the endothelium (hIRECO), to produce IRKO-hIRECO progeny. No metabolic differences were noted between IRKO and IRKO-hIRECO in glucose- and insulin-tolerance tests. In contrast with control IRKO littermates, IRKO-hIRECO exhibited normal blood pressure and aortic vasodilatation in response to acetylcholine, comparable to parameters noted in wild-type littermates. These phenotypic changes were associated with enhanced basal- and insulin-stimulated nitric oxide production. IRKO-hIRECO also demonstrated normalized endothelial repair after denuding arterial injury, which was associated with rescued endothelial cell migration in vitro, but not with changes in circulating progenitor populations or culture-derived myeloid angiogenic cells. These data show that restoration of endothelial insulin receptor expression alone is sufficient to prevent the vascular dysfunction caused by systemically reduced insulin signaling.

Funding information:
  • NIAID NIH HHS - SC1-AI-078559(United States)

Unbiased Combinatorial Screening Identifies a Bispecific IgG1 that Potently Inhibits HER3 Signaling via HER2-Guided Ligand Blockade.

  • Geuijen CAW
  • Cancer Cell
  • 2018 May 14

Literature context:


Abstract:

HER2-driven cancers require phosphatidylinositide-3 kinase (PI3K)/Akt signaling through HER3 to promote tumor growth and survival. The therapeutic benefit of HER2-targeting agents, which depend on PI3K/Akt inhibition, can be overcome by hyperactivation of the heregulin (HRG)/HER3 pathway. Here we describe an unbiased phenotypic combinatorial screening approach to identify a bispecific immunoglobulin G1 (IgG1) antibody against HER2 and HER3. In tumor models resistant to HER2-targeting agents, the bispecific IgG1 potently inhibits the HRG/HER3 pathway and downstream PI3K/Akt signaling via a "dock & block" mechanism. This bispecific IgG1 is a potentially effective therapy for breast cancer and other tumors with hyperactivated HRG/HER3 signaling.

Funding information:
  • Wellcome Trust - (United Kingdom)

Juvenile treatment with mGluR2/3 agonist prevents schizophrenia-like phenotypes in adult by acting through GSK3β.

  • Xing B
  • Neuropharmacology
  • 2018 May 14

Literature context:


Abstract:

Prodromal memory deficits represent an important marker for the development of schizophrenia (SZ), in which glutamatergic hypofunction occurs in the prefrontal cortex (PFC). The mGluR2/3 agonist LY379268 (LY37) attenuates excitatory N-methyl-D-aspartate receptor (NMDAR)-induced neurotoxicity, a central pathological characteristic of glutamatergic hypofunction. We therefore hypothesized that early treatment with LY37 would rescue cognitive deficits and confer benefits for SZ-like behaviors in adults. To test this, we assessed whether early intervention with LY37 would improve learning outcomes in the Morris Water Maze for rats prenatally exposed to methylazoxymethanol acetate (MAM), a neurodevelopmental SZ model. We found that a medium dose of LY37 prevents learning deficits in MAM rats. These effects were mediated through postsynaptic mGluR2/3 via improving GluN2B-NMDAR function by inhibiting glycogen synthase kinase-3β (GSK3β). Furthermore, dendritic spine loss and learning and memory deficits observed in adult MAM rats were restored by juvenile LY37 treatment, which did not change prefrontal neuronal excitability and glutamatergic synaptic transmission in adult normal rats. Our results provide a mechanism for mGluR2/3 agonists against NMDAR hypofunction, which may prove to be beneficial in the prophylactic treatment of SZ.

Funding information:
  • NIAID NIH HHS - R56AI1691785(United States)
  • NIMH NIH HHS - R01 MH085666()

Catch-Up Growth in Zebrafish Embryo Requires Neural Crest Cells Sustained by Irs1 Signaling.

  • Kamei H
  • Endocrinology
  • 2018 Apr 1

Literature context:


Abstract:

Most animals display retarded growth in adverse conditions; however, upon the removal of unfavorable factors, they often show quick growth restoration, which is known as "catch-up" growth. In zebrafish embryos, hypoxia causes growth arrest, but subsequent reoxygenation induces catch-up growth. Here, we report the role of insulin receptor substrate (Irs)1-mediated insulin/insulinlike growth factor signaling (IIS) and the involvement of stem cells in catch-up growth in reoxygenated zebrafish embryos. Disturbed irs1 expression attenuated IIS, resulting in greater inhibition in catch-up growth than in normal growth and forced IIS activation‒restored catch-up growth. The irs1 knockdown induced noticeable cell death in neural crest cells (NCCs; multipotent stem cells) under hypoxia, and the pharmacological/genetic ablation of NCCs hindered catch-up growth. Furthermore, inhibition of the apoptotic pathway by pan-caspase inhibition or forced activation of Akt signaling in irs1 knocked-down embryos blocked NCC cell death and rescued catch-up growth. Our data indicate that this multipotent stem cell is indispensable for embryonic catch-up growth and that Irs1-mediated IIS is a prerequisite for its survival under severe adverse environments such as prolonged hypoxia.

EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation.

  • Lee JH
  • Mol. Cell
  • 2018 Apr 19

Literature context:


Abstract:

EGFR activates phosphatidylinositide 3-kinase (PI3K), but the mechanism underlying this activation is not completely understood. We demonstrated here that EGFR activation resulted in lysine acetyltransferase 5 (KAT5)-mediated K395 acetylation of the platelet isoform of phosphofructokinase 1 (PFKP) and subsequent translocation of PFKP to the plasma membrane, where the PFKP was phosphorylated at Y64 by EGFR. Phosphorylated PFKP binds to the N-terminal SH2 domain of p85α, which is distinct from binding of Gab1 to the C-terminal SH2 domain of p85α, and recruited p85α to the plasma membrane resulting in PI3K activation. PI3K-dependent AKT activation results in enhanced phosphofructokinase 2 (PFK2) phosphorylation and production of fructose-2,6-bisphosphate, which in turn promotes PFK1 activation. PFKP Y64 phosphorylation-enhanced PI3K/AKT-dependent PFK1 activation and GLUT1 expression promoted the Warburg effect, tumor cell proliferation, and brain tumorigenesis. These findings underscore the instrumental role of PFKP in PI3K activation and enhanced glycolysis through PI3K/AKT-dependent positive-feedback regulation.

Funding information:
  • NCI NIH HHS - T32 CA121938(United States)

Coordinated Control of mRNA and rRNA Processing Controls Embryonic Stem Cell Pluripotency and Differentiation.

  • Corsini NS
  • Cell Stem Cell
  • 2018 Apr 5

Literature context:


Abstract:

Stem cell-specific transcriptional networks are well known to control pluripotency, but constitutive cellular processes such as mRNA splicing and protein synthesis can add complex layers of regulation with poorly understood effects on cell-fate decisions. Here, we show that the RNA binding protein HTATSF1 controls embryonic stem cell differentiation by regulating multiple aspects of RNA processing during ribosome biogenesis. HTATSF1, in a complex with splicing factor SF3B1, controls intron removal from ribosomal protein transcripts and regulates ribosomal RNA transcription and processing, thereby controlling 60S ribosomal abundance and protein synthesis. HTATSF1-dependent protein synthesis is essential for naive pre-implantation epiblast to transition into post-implantation epiblast, a stage with transiently low protein synthesis, and further differentiation toward neuroectoderm. Together, these results identify coordinated regulation of ribosomal RNA and protein synthesis by HTATSF1 and show that this essential mechanism controls protein synthesis during early mammalian embryogenesis.

Funding information:
  • NIGMS NIH HHS - GM077403(United States)

Synergy between the KEAP1/NRF2 and PI3K Pathways Drives Non-Small-Cell Lung Cancer with an Altered Immune Microenvironment.

  • Best SA
  • Cell Metab.
  • 2018 Apr 3

Literature context:


Abstract:

The lung presents a highly oxidative environment, which is tolerated through engagement of tightly controlled stress response pathways. A critical stress response mediator is the transcription factor nuclear factor erythroid-2-related factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-associated protein 1 (KEAP1). Alterations in the KEAP1/NRF2 pathway have been identified in 23% of lung adenocarcinomas, suggesting that deregulation of the pathway is a major cancer driver. We demonstrate that inactivation of Keap1 and Pten in the mouse lung promotes adenocarcinoma formation. Notably, metabolites identified in the plasma of Keap1f/f/Ptenf/f tumor-bearing mice indicate that tumorigenesis is associated with reprogramming of the pentose phosphate pathway. Furthermore, the immune milieu was dramatically changed by Keap1 and Pten deletion, and tumor regression was achieved utilizing immune checkpoint inhibition. Thus, our study highlights the ability to exploit both metabolic and immune characteristics in the detection and treatment of lung tumors harboring KEAP1/NRF2 pathway alterations.

Funding information:
  • NHGRI NIH HHS - U54 HG004592(United States)

The Dietary Supplement Chondroitin-4-Sulfate Exhibits Oncogene-Specific Pro-tumor Effects on BRAF V600E Melanoma Cells.

  • Lin R
  • Mol. Cell
  • 2018 Mar 15

Literature context:


Abstract:

Dietary supplements such as vitamins and minerals are widely used in the hope of improving health but may have unidentified risks and side effects. In particular, a pathogenic link between dietary supplements and specific oncogenes remains unknown. Here we report that chondroitin-4-sulfate (CHSA), a natural glycosaminoglycan approved as a dietary supplement used for osteoarthritis, selectively promotes the tumor growth potential of BRAF V600E-expressing human melanoma cells in patient- and cell line-derived xenograft mice and confers resistance to BRAF inhibitors. Mechanistically, chondroitin sulfate glucuronyltransferase (CSGlcA-T) signals through its product CHSA to enhance casein kinase 2 (CK2)-PTEN binding and consequent phosphorylation and inhibition of PTEN, which requires CHSA chains and is essential to sustain AKT activation in BRAF V600E-expressing melanoma cells. However, this CHSA-dependent PTEN inhibition is dispensable in cancer cells expressing mutant NRAS or PI3KCA, which directly activate the PI3K-AKT pathway. These results suggest that dietary supplements may exhibit oncogene-dependent pro-tumor effects.

Funding information:
  • NCI NIH HHS - R01 CA140515()
  • NCI NIH HHS - R01 CA174786()
  • NCI NIH HHS - R01 CA183594()
  • Wellcome Trust - 090532(United Kingdom)

B Cell Receptor and CD40 Signaling Are Rewired for Synergistic Induction of the c-Myc Transcription Factor in Germinal Center B Cells.

  • Luo W
  • Immunity
  • 2018 Feb 20

Literature context:


Abstract:

Positive selection of germinal center (GC) B cells is driven by B cell receptor (BCR) affinity and requires help from follicular T helper cells. The transcription factors c-Myc and Foxo1 are critical for GC B cell selection and survival. However, how different affinity-related signaling events control these transcription factors in a manner that links to selection is unknown. Here we showed that GC B cells reprogram CD40 and BCR signaling to transduce via NF-κB and Foxo1, respectively, whereas naive B cells propagate both signals downstream of either receptor. Although either BCR or CD40 ligation induced c-Myc in naive B cells, both signals were required to highly induce c-Myc, a critical mediator of GC B cell survival and cell cycle reentry. Thus, GC B cells rewire their signaling to enhance selection stringency via a requirement for both antigen receptor- and T cell-mediated signals to induce mediators of positive selection.

Funding information:
  • NIMH NIH HHS - R01MH091115(United States)

Direct Activation of Angiotensin II Type 2 Receptors Enhances Muscle Microvascular Perfusion, Oxygenation, and Insulin Delivery in Male Rats.

  • Yan F
  • Endocrinology
  • 2018 Feb 1

Literature context:


Abstract:

Angiotensin II receptors regulate muscle microvascular recruitment and the delivery of nutrients, oxygen, and insulin to muscle. Although angiotensin type 1 receptor antagonism increases muscle microvascular perfusion and insulin action, angiotensin type 2 receptor blockade markedly restricts muscle microvascular blood volume and decreases muscle delivery of insulin. To examine the effects of direct type 2 receptor stimulation using Compound 21 (C21) on microvascular perfusion, insulin delivery and action, and tissue oxygenation in muscle, overnight-fasted adult male rats were infused with C21 systemically. C21 potently increased microvascular blood volume without altering microvascular flow velocity or blood pressure, resulting in a net increase in microvascular blood flow in muscle. This was associated with a substantial increase in muscle interstitial oxygen saturation and insulin delivery into the skeletal and cardiac muscle. These effects were neutralized by coinfusion of the type 2 receptor antagonist or nitric oxide synthase inhibitor. Superimposing C21 infusion on insulin infusion increased insulin-mediated whole body glucose disposal by 50%. C21 significantly relaxed the preconstricted distal saphenous artery ex vivo. We have concluded that direct type 2 receptor stimulation markedly increases muscle microvascular perfusion through nitric oxide biosynthesis and enhances insulin delivery and action in muscle. These findings provide a physiologic mechanistic insight into type 2 receptor modulation of insulin action and suggest that type 2 receptor agonists might have therapeutic potential in the management of diabetes and its associated complications.

Funding information:
  • NHLBI NIH HHS - R01 HL094722()
  • NICHD NIH HHS - U54 HD052668(United States)
  • NIDDK NIH HHS - R01 DK102359()

mTOR-dependent alterations of Kv1.1 subunit expression in the neuronal subset-specific Pten knockout mouse model of cortical dysplasia with epilepsy.

  • Nguyen LH
  • Sci Rep
  • 2018 Feb 23

Literature context:


Abstract:

Cortical dysplasia (CD) is a common cause for intractable epilepsy. Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway has been implicated in CD; however, the mechanisms by which mTOR hyperactivation contribute to the epilepsy phenotype remain elusive. Here, we investigated whether constitutive mTOR hyperactivation in the hippocampus is associated with altered voltage-gated ion channel expression in the neuronal subset-specific Pten knockout (NS-Pten KO) mouse model of CD with epilepsy. We found that the protein levels of Kv1.1, but not Kv1.2, Kv1.4, or Kvβ2, potassium channel subunits were increased, along with altered Kv1.1 distribution, within the hippocampus of NS-Pten KO mice. The aberrant Kv1.1 protein levels were present in young adult (≥postnatal week 6) but not juvenile (≤postnatal week 4) NS-Pten KO mice. No changes in hippocampal Kv1.1 mRNA levels were found between NS-Pten KO and WT mice. Interestingly, mTOR inhibition with rapamycin treatment at early and late stages of the pathology normalized Kv1.1 protein levels in NS-Pten KO mice to WT levels. Together, these studies demonstrate altered Kv1.1 protein expression in association with mTOR hyperactivation in NS-Pten KO mice and suggest a role for mTOR signaling in the modulation of voltage-gated ion channel expression in this model.

Funding information:
  • NIAID NIH HHS - R01AI067979(United States)
  • NICHD NIH HHS - U54 HD083092()
  • NINDS NIH HHS - R01 NS081053()

Insulinlike Growth Factor-Binding Protein-1 Improves Vascular Endothelial Repair in Male Mice in the Setting of Insulin Resistance.

  • Aziz A
  • Endocrinology
  • 2018 Feb 1

Literature context:


Abstract:

Insulin resistance is associated with impaired endothelial regeneration in response to mechanical injury. We recently demonstrated that insulinlike growth factor-binding protein-1 (IGFBP1) ameliorated insulin resistance and increased nitric oxide generation in the endothelium. In this study, we hypothesized that IGFBP1 would improve endothelial regeneration and restore endothelial reparative functions in the setting of insulin resistance. In male mice heterozygous for deletion of insulin receptors, endothelial regeneration after femoral artery wire injury was enhanced by transgenic expression of human IGFBP1 (hIGFBP1). This was not explained by altered abundance of circulating myeloid angiogenic cells. Incubation of human endothelial cells with hIGFBP1 increased integrin expression and enhanced their ability to adhere to and repopulate denuded human saphenous vein ex vivo. In vitro, induction of insulin resistance by tumor necrosis factor α (TNFα) significantly inhibited endothelial cell migration and proliferation. Coincubation with hIGFBP1 restored endothelial migratory and proliferative capacity. At the molecular level, hIGFBP1 induced phosphorylation of focal adhesion kinase, activated RhoA and modulated TNFα-induced actin fiber anisotropy. Collectively, the effects of hIGFBP1 on endothelial cell responses and acceleration of endothelial regeneration in mice indicate that manipulating IGFBP1 could be exploited as a putative strategy to improve endothelial repair in the setting of insulin resistance.

Funding information:
  • NIGMS NIH HHS - GM076990(United States)

The Response of Prostate Smooth Muscle Cells to Testosterone Is Determined by the Subcellular Distribution of the Androgen Receptor.

  • Peinetti N
  • Endocrinology
  • 2018 Feb 1

Literature context:


Abstract:

Androgen signaling in prostate smooth muscle cells (pSMCs) is critical for the maintenance of prostate homeostasis, the alterations of which are a central aspect in the development of pathological conditions. Testosterone can act through the classic androgen receptor (AR) in the cytoplasm, eliciting genomic signaling, or through different types of receptors located at the plasma membrane for nongenomic signaling. We aimed to find evidence of nongenomic testosterone-signaling mechanisms in pSMCs and their participation in cell proliferation, differentiation, and the modulation of the response to lipopolysaccharide. We demonstrated that pSMCs can respond to testosterone by a rapid activation of ERK1/2 and Akt. Furthermore, a pool of ARs localized at the cell surface of pSMCs is responsible for a nongenomic testosterone-induced increase in cell proliferation. Through membrane receptor stimulation, testosterone favors a muscle phenotype, indicated by an increase in smooth muscle markers. We also showed that the anti-inflammatory effects of testosterone, capable of attenuating lipopolysaccharide-induced proinflammatory actions, are promoted only by receptors located inside the cell. We postulate that testosterone might perform prohomeostatic effects through intracellular-initiated mechanisms by modulating cell proliferation and inflammation, whereas some pathological, hyperproliferative actions would be induced by membrane-initiated nongenomic signaling in pSMCs.

Funding information:
  • Intramural NIH HHS - ZIA AG000741-09(United States)

KRAS Dimerization Impacts MEK Inhibitor Sensitivity and Oncogenic Activity of Mutant KRAS.

  • Ambrogio C
  • Cell
  • 2018 Feb 8

Literature context:


Abstract:

The mechanism by which the wild-type KRAS allele imparts a growth inhibitory effect to oncogenic KRAS in various cancers, including lung adenocarcinoma (LUAD), is poorly understood. Here, using a genetically inducible model of KRAS loss of heterozygosity (LOH), we show that KRAS dimerization mediates wild-type KRAS-dependent fitness of human and murine KRAS mutant LUAD tumor cells and underlies resistance to MEK inhibition. These effects are abrogated when wild-type KRAS is replaced by KRASD154Q, a mutant that disrupts dimerization at the α4-α5 KRAS dimer interface without changing other fundamental biochemical properties of KRAS, both in vitro and in vivo. Moreover, dimerization has a critical role in the oncogenic activity of mutant KRAS. Our studies provide mechanistic and biological insights into the role of KRAS dimerization and highlight a role for disruption of dimerization as a therapeutic strategy for KRAS mutant cancers.

RHOA G17V Induces T Follicular Helper Cell Specification and Promotes Lymphomagenesis.

  • Cortes JR
  • Cancer Cell
  • 2018 Feb 12

Literature context:


Abstract:

Angioimmunoblastic T cell lymphoma (AITL) is an aggressive tumor derived from malignant transformation of T follicular helper (Tfh) cells. AITL is characterized by loss-of-function mutations in Ten-Eleven Translocation 2 (TET2) epigenetic tumor suppressor and a highly recurrent mutation (p.Gly17Val) in the RHOA small GTPase. Yet, the specific role of RHOA G17V in AITL remains unknown. Expression of Rhoa G17V in CD4+ T cells induces Tfh cell specification; increased proliferation associated with inducible co-stimulator (ICOS) upregulation and increased phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase signaling. Moreover, RHOA G17V expression together with Tet2 loss resulted in development of AITL in mice. Importantly, Tet2-/-RHOA G17V tumor proliferation in vivo can be inhibited by ICOS/PI3K-specific blockade, supporting a driving role for ICOS signaling in Tfh cell transformation.

Funding information:
  • NCI NIH HHS - R01 CA197945()
  • NIDDK NIH HHS - R37 DK44746(United States)

c-RAF Ablation Induces Regression of Advanced Kras/Trp53 Mutant Lung Adenocarcinomas by a Mechanism Independent of MAPK Signaling.

  • Sanclemente M
  • Cancer Cell
  • 2018 Feb 12

Literature context:


Abstract:

A quarter of all solid tumors harbor KRAS oncogenes. Yet, no selective drugs have been approved to treat these malignancies. Genetic interrogation of the MAPK pathway revealed that systemic ablation of MEK or ERK kinases in adult mice prevent tumor development but are unacceptably toxic. Here, we demonstrate that ablation of c-RAF expression in advanced tumors driven by KrasG12V/Trp53 mutations leads to significant tumor regression with no detectable appearance of resistance mechanisms. Tumor regression results from massive apoptosis. Importantly, systemic abrogation of c-RAF expression does not inhibit canonical MAPK signaling, hence, resulting in limited toxicities. These results are of significant relevance for the design of therapeutic strategies to treat K-RAS mutant cancers.

Funding information:
  • NHLBI NIH HHS - HL076604(United States)

TrkB-mediated activation of the phosphatidylinositol-3-kinase/Akt cascade reduces the damage inflicted by oxygen-glucose deprivation in area CA3 of the rat hippocampus.

  • Tecuatl C
  • Eur. J. Neurosci.
  • 2018 Feb 27

Literature context:


Abstract:

The selective vulnerability of hippocampal area CA1 to ischemia-induced injury is a well-known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen-glucose deprivation-reperfusion (OGD-RP), an in vitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain-derived neurotrophic factor (BDNF) or 7,8-dihydroxyflavone (7,8-DHF) exhibited reduced depression of the electrical activity triggered by OGD-RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD-RP. The protective effect of TrkB activation was limited to area CA3, as OGD-RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3-kinase/Akt (PI-3-K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD-RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase-3 activity in slices pretreated with BDNF and exposed to OGD-RP. We propose that PI-3-K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.

Funding information:
  • NIGMS NIH HHS - GM 052880(United States)
  • NIGMS NIH HHS - R01 GM044842()
  • NIGMS NIH HHS - R01 GM066018()
  • NINDS NIH HHS - R01 NS037459()
  • NINDS NIH HHS - R56 NS037459()

PTEN negatively regulates the cell lineage progression from NG2+ glial progenitor to oligodendrocyte via mTOR-independent signaling.

  • González-Fernández E
  • Elife
  • 2018 Feb 20

Literature context:


Abstract:

Oligodendrocytes (OLs), the myelin-forming CNS glia, are highly vulnerable to cellular stresses, and a severe myelin loss underlies numerous CNS disorders. Expedited OL regeneration may prevent further axonal damage and facilitate functional CNS repair. Although adult OL progenitors (OPCs) are the primary players for OL regeneration, targetable OPC-specific intracellular signaling mechanisms for facilitated OL regeneration remain elusive. Here, we report that OPC-targeted PTEN inactivation in the mouse, in contrast to OL-specific manipulations, markedly promotes OL differentiation and regeneration in the mature CNS. Unexpectedly, an additional deletion of mTOR did not reverse the enhanced OL development from PTEN-deficient OPCs. Instead, ablation of GSK3β, another downstream signaling molecule that is negatively regulated by PTEN-Akt, enhanced OL development. Our results suggest that PTEN persistently suppresses OL development in an mTOR-independent manner, and at least in part, via controlling GSK3β activity. OPC-targeted PTEN-GSK3β inactivation may benefit facilitated OL regeneration and myelin repair.

Funding information:
  • Ellison Medical Foundation - AG-NS-1101-13()
  • National Institute of Neurological Disorders and Stroke - R01NS07693()
  • National Institute of Neurological Disorders and Stroke - R01NS089586()
  • NIH HHS - DP2 OD006740(United States)
  • Shriners Hospitals for Children - 84298-PHI()
  • Shriners Hospitals for Children - 85500-PHI-14()
  • Shriners Hospitals for Children - 86600()

IL-10 Signaling Remodels Adipose Chromatin Architecture to Limit Thermogenesis and Energy Expenditure.

  • Rajbhandari P
  • Cell
  • 2018 Jan 11

Literature context:


Abstract:

Signaling pathways that promote adipose tissue thermogenesis are well characterized, but the limiters of energy expenditure are largely unknown. Here, we show that ablation of the anti-inflammatory cytokine IL-10 improves insulin sensitivity, protects against diet-induced obesity, and elicits the browning of white adipose tissue. Mechanistic studies define bone marrow cells as the source of the IL-10 signal and adipocytes as the target cell type mediating these effects. IL-10 receptor alpha is highly enriched in mature adipocytes and is induced in response to differentiation, obesity, and aging. Assay for transposase-accessible chromatin sequencing (ATAC-seq), ChIP-seq, and RNA-seq reveal that IL-10 represses the transcription of thermogenic genes in adipocytes by altering chromatin accessibility and inhibiting ATF and C/EBPβ recruitment to key enhancer regions. These findings expand our understanding of the relationship between inflammatory signaling pathways and adipose tissue function and provide insight into the physiological control of thermogenesis that could inform future therapy.

Funding information:
  • Howard Hughes Medical Institute - (United States)
  • NHLBI NIH HHS - K08 HL128822()
  • NHLBI NIH HHS - P01 HL090553()
  • NIAID NIH HHS - T32 AI007323()
  • NIDDK NIH HHS - F32 DK104484()
  • NIDDK NIH HHS - F32 DK109601()
  • NIDDK NIH HHS - P30 DK063491()
  • NIGMS NIH HHS - R01 GM086372()
  • NIGMS NIH HHS - T32 GM008042()

Notch Signaling Regulates Differentiation and Steroidogenesis in Female Mouse Ovarian Granulosa Cells.

  • Prasasya RD
  • Endocrinology
  • 2018 Jan 1

Literature context:


Abstract:

The Notch pathway is a highly conserved juxtacrine signaling mechanism that is important for many cellular processes during development, including differentiation and proliferation. Although Notch is important during ovarian follicle formation and early development, its functions during the gonadotropin-dependent stages of follicle development are largely unexplored. We observed positive regulation of Notch activity and expression of Notch ligands and receptors following activation of the luteinizing hormone-receptor in prepubertal mouse ovary. JAG1, the most abundantly expressed Notch ligand in mouse ovary, revealed a striking shift in localization from oocytes to somatic cells following hormone stimulation. Using primary cultures of granulosa cells, we investigated the functions of Jag1 using small interfering RNA knockdown. The loss of JAG1 led to suppression of granulosa cell differentiation as marked by reduced expression of enzymes and factors involved in steroid biosynthesis, and in steroid secretion. Jag1 knockdown also resulted in enhanced cell proliferation. These phenotypes were replicated, although less robustly, following knockdown of the obligate canonical Notch transcription factor RBPJ. Intracellular signaling analysis revealed increased activation of the mitogenic phosphatidylinositol 3-kinase/protein kinase B and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways following Notch knockdown, with a mitogen-activated protein kinase kinase inhibitor blocking the enhanced proliferation observed in Jag1 knockdown granulosa cells. Activation of YB-1, a known regulator of granulosa cell differentiation genes, was suppressed by Jag1 knockdown. Overall, this study reveals a role of Notch signaling in promoting the differentiation of preovulatory granulosa cells, adding to the diverse functions of Notch in the mammalian ovary.

Funding information:
  • NIA NIH HHS - R56 AG016379(United States)
  • NICHD NIH HHS - P01 HD021921(United States)
  • NICHD NIH HHS - P50 HD028934(United States)
  • NIGMS NIH HHS - T32 GM008061(United States)

Oncogenic KRAS Regulates Amino Acid Homeostasis and Asparagine Biosynthesis via ATF4 and Alters Sensitivity to L-Asparaginase.

  • Gwinn DM
  • Cancer Cell
  • 2018 Jan 8

Literature context:


Abstract:

KRAS is a regulator of the nutrient stress response in non-small-cell lung cancer (NSCLC). Induction of the ATF4 pathway during nutrient depletion requires AKT and NRF2 downstream of KRAS. The tumor suppressor KEAP1 strongly influences the outcome of activation of this pathway during nutrient stress; loss of KEAP1 in KRAS mutant cells leads to apoptosis. Through ATF4 regulation, KRAS alters amino acid uptake and asparagine biosynthesis. The ATF4 target asparagine synthetase (ASNS) contributes to apoptotic suppression, protein biosynthesis, and mTORC1 activation. Inhibition of AKT suppressed ASNS expression and, combined with depletion of extracellular asparagine, decreased tumor growth. Therefore, KRAS is important for the cellular response to nutrient stress, and ASNS represents a promising therapeutic target in KRAS mutant NSCLC.

Funding information:
  • Howard Hughes Medical Institute - (United States)
  • NCI NIH HHS - R01 CA129562()
  • NCI NIH HHS - T32 CA009302()
  • NINDS NIH HHS - R01 NS089868()

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

  • Janes MR
  • Cell
  • 2018 Jan 25

Literature context:


Abstract:

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Funding information:
  • NCI NIH HHS - R01-CA093678(United States)

Iron promotes α-synuclein aggregation and transmission by inhibiting TFEB-mediated autophagosome-lysosome fusion.

  • Xiao Y
  • J. Neurochem.
  • 2018 Jan 25

Literature context:


Abstract:

Recent studies have strongly shown that cell-to-cell transmission of neuropathogenic proteins is a common mechanism for the development of neurodegenerative diseases. However, the underlying cause is complex and little is known. Although distinct processes are involved in the pathogenesis of various diseases, they all share the common feature of iron accumulation, an attribute that is particularly prominent in synucleinopathies. However, whether iron is a cofactor in facilitating the spread of α-synuclein remains unclear. Here, we constructed a cell-to-cell transmission model of α-synuclein using SN4741 cell line based on adenovirus vectors. Cells were treated with FeCl2, and α-synuclein aggregation and transmission were then evaluated. In addition, the possible mechanisms were investigated through gene knockdown or over-expression. Our results demonstrated that iron promoted α-synuclein aggregation and transmission by inhibiting autophagosome-lysosome fusion. Furthermore, iron decreased the expression of nuclear transcription factor EB (TFEB), a master transcriptional regulator of autophagosome-lysosome fusion, and inhibited its nuclear translocation through activating AKT/mTORC1 signaling. After silencing TFEB, ratios of α-synuclein aggregation and transmission were not significantly altered by the presence of iron; on the other hand, when TFEB was over-expressed, the transmission of α-synuclein induced by iron was obviously reversed; suggesting the mechanism by which iron promotes α-synuclein transmission may be mediated by TFEB. Taken together, our data reveal a previously unknown relationship between iron and α-synuclein, and identify TFEB as not only a potential target for preventing α-synuclein transmission, but also a critical factor for iron-induced α-synuclein aggregation and transmission. Indeed, this newly discovered role of iron and TFEB in synucleinopathies may provide novel targets for developing therapeutic strategies to prevent α-synuclein transmission in Parkinson's disease.

Funding information:
  • NINDS NIH HHS - R01 NS078283(United States)

Repression of Adipose Tissue Fibrosis through a PRDM16-GTF2IRD1 Complex Improves Systemic Glucose Homeostasis.

  • Hasegawa Y
  • Cell Metab.
  • 2018 Jan 9

Literature context:


Abstract:

Adipose tissue fibrosis is a hallmark of malfunction that is linked to insulin resistance and type 2 diabetes; however, what regulates this process remains unclear. Here we show that the PRDM16 transcriptional complex, a dominant activator of brown/beige adipocyte development, potently represses adipose tissue fibrosis in an uncoupling protein 1 (UCP1)-independent manner. By purifying the PRDM16 complex, we identified GTF2IRD1, a member of the TFII-I family of DNA-binding proteins, as a cold-inducible transcription factor that mediates the repressive action of the PRDM16 complex on fibrosis. Adipocyte-selective expression of GTF2IRD1 represses adipose tissue fibrosis and improves systemic glucose homeostasis independent of body-weight loss, while deleting GTF2IRD1 promotes fibrosis in a cell-autonomous manner. GTF2IRD1 represses the transcription of transforming growth factor β-dependent pro-fibrosis genes by recruiting PRDM16 and EHMT1 onto their promoter/enhancer regions. These results suggest a mechanism by which repression of obesity-associated adipose tissue fibrosis through the PRDM16 complex leads to an improvement in systemic glucose homeostasis.

Funding information:
  • Intramural NIH HHS - (United States)
  • NIDDK NIH HHS - P30 DK098722()
  • NIDDK NIH HHS - R01 DK097441()
  • NIDDK NIH HHS - R01 DK103175()
  • NIDDK NIH HHS - R01 DK108822()
  • NIDDK NIH HHS - R01 DK112304()
  • NIDDK NIH HHS - T32 DK007418()

Lessons in PROTAC Design from Selective Degradation with a Promiscuous Warhead.

  • Bondeson DP
  • Cell Chem Biol
  • 2018 Jan 18

Literature context:


Abstract:

Inhibiting protein function selectively is a major goal of modern drug discovery. Here, we report a previously understudied benefit of small molecule proteolysis-targeting chimeras (PROTACs) that recruit E3 ubiquitin ligases to target proteins for their ubiquitination and subsequent proteasome-mediated degradation. Using promiscuous CRBN- and VHL-recruiting PROTACs that bind >50 kinases, we show that only a subset of bound targets is degraded. The basis of this selectivity relies on protein-protein interactions between the E3 ubiquitin ligase and the target protein, as illustrated by engaged proteins that are not degraded as a result of unstable ternary complexes with PROTAC-recruited E3 ligases. In contrast, weak PROTAC:target protein affinity can be stabilized by high-affinity target:PROTAC:ligase trimer interactions, leading to efficient degradation. This study highlights design guidelines for generating potent PROTACs as well as possibilities for degrading undruggable proteins immune to traditional small-molecule inhibitors.

Cooperative Epigenetic Remodeling by TET2 Loss and NRAS Mutation Drives Myeloid Transformation and MEK Inhibitor Sensitivity.

  • Kunimoto H
  • Cancer Cell
  • 2018 Jan 8

Literature context:


Abstract:

Mutations in epigenetic modifiers and signaling factors often co-occur in myeloid malignancies, including TET2 and NRAS mutations. Concurrent Tet2 loss and NrasG12D expression in hematopoietic cells induced myeloid transformation, with a fully penetrant, lethal chronic myelomonocytic leukemia (CMML), which was serially transplantable. Tet2 loss and Nras mutation cooperatively led to decrease in negative regulators of mitogen-activated protein kinase (MAPK) activation, including Spry2, thereby causing synergistic activation of MAPK signaling by epigenetic silencing. Tet2/Nras double-mutant leukemia showed preferential sensitivity to MAPK kinase (MEK) inhibition in both mouse model and patient samples. These data provide insights into how epigenetic and signaling mutations cooperate in myeloid transformation and provide a rationale for mechanism-based therapy in CMML patients with these high-risk genetic lesions.

Funding information:
  • NCI NIH HHS - K08 CA169055()
  • NCI NIH HHS - K08 CA181507()
  • NCI NIH HHS - P30 CA008748()
  • NCI NIH HHS - R01 CA173636()
  • NCI NIH HHS - R01 CA198089()
  • NCI NIH HHS - R35 CA197594()
  • NCI NIH HHS - T32 CA009156(United States)
  • NIH HHS - U54 OD020355()

Region-Specific Suppression of Hypothalamic Responses to Insulin To Adapt to Elevated Maternal Insulin Secretion During Pregnancy.

  • Ladyman SR
  • Endocrinology
  • 2017 Dec 1

Literature context:


Abstract:

As part of the adaptation of maternal glucose regulation during pregnancy to ensure glucose provision to the fetus, maternal insulin concentrations become elevated. However, increased central actions of insulin, such as suppression of appetite, would be maladaptive during pregnancy. We hypothesized that central nervous system targets of insulin become less responsive during pregnancy to prevent overstimulation by the increased circulating insulin concentrations. To test this hypothesis, we have measured insulin-induced phosphorylation of Akt (pAkt) in specific hypothalamic nuclei as an index of hypothalamic insulin responsiveness. Despite higher endogenous insulin concentrations following feeding, arcuate nucleus pAkt levels were significantly lower in the pregnant group compared with the nonpregnant group. In response to an intracerebroventricular injection of insulin, insulin-induced pAkt was significantly reduced in the arcuate nucleus and ventromedial nucleus of pregnant rats compared with nonpregnant rats. Similar levels of insulin receptor β and PTEN, a negative regulator of the phosphoinositide 3-kinase/Akt pathway, were detected in hypothalamic areas of nonpregnant and pregnant rats. In the ventromedial nucleus, however, levels of phosphorylated PTEN were significantly lower in pregnancy, suggesting that reduced inactivation of PTEN may contribute to the attenuated insulin signaling in this area during pregnancy. In conclusion, these results demonstrate region-specific changes in responsiveness to insulin in the hypothalamus during pregnancy that may represent an adaptive response to minimize the impact of elevated circulating insulin on the maternal brain.

Funding information:
  • NINDS NIH HHS - NS052671(United States)

A Method for the Acute and Rapid Degradation of Endogenous Proteins.

  • Clift D
  • Cell
  • 2017 Dec 14

Literature context:


Abstract:

Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.

Funding information:
  • NIDCD NIH HHS - P30 DC04657(United States)

mTORC1-Mediated Inhibition of 4EBP1 Is Essential for Hedgehog Signaling-Driven Translation and Medulloblastoma.

  • Wu CC
  • Dev. Cell
  • 2017 Dec 18

Literature context:


Abstract:

Mechanistic target of rapamycin (MTOR) cooperates with Hedgehog (HH) signaling, but the underlying mechanisms are incompletely understood. Here we provide genetic, biochemical, and pharmacologic evidence that MTOR complex 1 (mTORC1)-dependent translation is a prerequisite for HH signaling. The genetic loss of mTORC1 function inhibited HH signaling-driven growth of the cerebellum and medulloblastoma. Inhibiting translation or mTORC1 blocked HH signaling. Depleting 4EBP1, an mTORC1 target that inhibits translation, alleviated the dependence of HH signaling on mTORC1. Consistent with this, phosphorylated 4EBP1 levels were elevated in HH signaling-driven medulloblastomas in mice and humans. In mice, an mTORC1 inhibitor suppressed medulloblastoma driven by a mutant SMO that is inherently resistant to existing SMO inhibitors, prolonging the survival of the mice. Our study reveals that mTORC1-mediated translation is a key component of HH signaling and an important target for treating medulloblastoma and other cancers driven by HH signaling.

Funding information:
  • NCI NIH HHS - P30 CA021765()

MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma.

  • Huang T
  • Cancer Cell
  • 2017 Dec 11

Literature context:


Abstract:

ATG4B stimulates autophagy by promoting autophagosome formation through reversible modification of ATG8. We identify ATG4B as a substrate of mammalian sterile20-like kinase (STK) 26/MST4. MST4 phosphorylates ATG4B at serine residue 383, which stimulates ATG4B activity and increases autophagic flux. Inhibition of MST4 or ATG4B activities using genetic approaches or an inhibitor of ATG4B suppresses autophagy and the tumorigenicity of glioblastoma (GBM) cells. Furthermore, radiation induces MST4 expression, ATG4B phosphorylation, and autophagy. Inhibiting ATG4B in combination with radiotherapy in treating mice with intracranial GBM xenograft markedly slows tumor growth and provides a significant survival benefit. Our work describes an MST4-ATG4B signaling axis that influences GBM autophagy and malignancy, and whose therapeutic targeting enhances the anti-tumor effects of radiotherapy.

Funding information:
  • NCI NIH HHS - P01 CA163205()
  • NCI NIH HHS - R01 CA159467()
  • NCI NIH HHS - R21 CA175875()
  • NCI NIH HHS - T32 CA070085()
  • NIAAA NIH HHS - R01 AA021751()
  • NIGMS NIH HHS - R01 GM038660(United States)
  • NIMHD NIH HHS - L32 MD010147()
  • NINDS NIH HHS - P30 NS081774()
  • NINDS NIH HHS - R01 NS080619()
  • NINDS NIH HHS - R01 NS083767()
  • NINDS NIH HHS - R01 NS093843()
  • NINDS NIH HHS - R01 NS095634()
  • NINDS NIH HHS - R01 NS102669()
  • NLM NIH HHS - K99 LM011673()
  • NLM NIH HHS - R00 LM011673()
  • NLM NIH HHS - R01 LM012011()

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

  • Essig K
  • Immunity
  • 2017 Dec 19

Literature context:


Abstract:

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

Funding information:
  • NHLBI NIH HHS - R37 HL036982(United States)

Divergent JNK Phosphorylation of HDAC3 in Triple-Negative Breast Cancer Cells Determines HDAC Inhibitor Binding and Selectivity.

  • Hanigan TW
  • Cell Chem Biol
  • 2017 Nov 16

Literature context:


Abstract:

Histone deacetylase (HDAC) catalytic activity is regulated by formation of co-regulator complexes and post-translational modification. Whether these mechanisms are transformed in cancer and how this affects the binding and selectivity of HDAC inhibitors (HDACis) is unclear. In this study, we developed a method that identified a 3- to 16-fold increase in HDACi selectivity for HDAC3 in triple-negative breast cancer (TNBC) cells in comparison with luminal subtypes that was not predicted by current practice measurements with recombinant proteins. We found this increase was caused by c-Jun N-terminal kinase (JNK) phosphorylation of HDAC3, was independent of HDAC3 complex composition or subcellular localization, and was associated with a 5-fold increase in HDAC3 enzymatic activity. This study points to HDAC3 and the JNK axes as targets in TNBC, highlights how HDAC phosphorylation affects HDACi binding and selectivity, and outlines a method to identify changes in individual HDAC isoforms catalytic activity, applicable to any disease state.

Funding information:
  • NCI NIH HHS - R01 CA131970()
  • NCI NIH HHS - R21 CA183627()

Tumor-Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer.

  • Bajikar SS
  • Dev. Cell
  • 2017 Nov 20

Literature context:


Abstract:

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous carcinoma in which various tumor-suppressor genes are lost by mutation, deletion, or silencing. Here we report a tumor-suppressive mode of action for growth-differentiation factor 11 (GDF11) and an unusual mechanism of its inactivation in TNBC. GDF11 promotes an epithelial, anti-invasive phenotype in 3D triple-negative cultures and intraductal xenografts by sustaining expression of E-cadherin and inhibitor of differentiation 2 (ID2). Surprisingly, clinical TNBCs retain the GDF11 locus and expression of the protein itself. GDF11 bioactivity is instead lost because of deficiencies in its convertase, proprotein convertase subtilisin/kexin type 5 (PCSK5), causing inactive GDF11 precursor to accumulate intracellularly. PCSK5 reconstitution mobilizes the latent TNBC reservoir of GDF11 in vitro and suppresses triple-negative mammary cancer metastasis to the lung of syngeneic hosts. Intracellular GDF11 retention adds to the concept of tumor-suppressor inactivation and reveals a cell-biological vulnerability for TNBCs lacking therapeutically actionable mutations.

Funding information:
  • NIDCD NIH HHS - R01 DC011184(United States)

Notch Signaling Controls Transdifferentiation of Pulmonary Neuroendocrine Cells in Response to Lung Injury.

  • Yao E
  • Stem Cells
  • 2017 Nov 18

Literature context:


Abstract:

Production of an appropriate number of distinct cell types in precise locations during embryonic development is critical for proper tissue function. Homeostatic renewal or repair of damaged tissues in adults also requires cell expansion and transdifferentiation to replenish lost cells. However, the responses of diverse cell types to tissue injury are not fully elucidated. Moreover, the molecular mechanisms underlying transdifferentiation remain poorly understood. This knowledge is essential for harnessing the regenerative potential of individual cell types. This study investigated the fate of pulmonary neuroendocrine cells (PNECs) following lung damage to understand their plasticity and potential. PNECs are proposed to carry out diverse physiological functions in the lung and can also be the cells of origin of human small cell lung cancer. We found that Notch signaling is activated in proliferating PNECs in response to epithelial injury. Forced induction of high levels of Notch signaling in PNECs in conjunction with lung injury results in extensive proliferation and transdifferentiation of PNECs toward the fate of club cells, ciliated cells and goblet cells. Conversely, inactivating Notch signaling in PNECs abolishes their ability to switch cell fate following lung insult. We also established a connection between PNEC transdifferentiation and epigenetic modification mediated by the polycomb repressive complex 2 and inflammatory responses that involve the IL6-STAT3 pathway. These studies not only reveal a major pathway that controls PNEC fate change following lung injury but also provide tools to uncover the molecular basis of cell proliferation and fate determination in response to lung injury. Stem Cells 2018;36:377-391.

Funding information:
  • NIAID NIH HHS - R01 AI083450-04(United States)

Mouse Cutaneous Melanoma Induced by Mutant BRaf Arises from Expansion and Dedifferentiation of Mature Pigmented Melanocytes.

  • Köhler C
  • Cell Stem Cell
  • 2017 Nov 2

Literature context:


Abstract:

To identify the cells at the origin of melanoma, we combined single-cell lineage-tracing and transcriptomics approaches with time-lapse imaging. A mouse model that recapitulates key histopathological features of human melanomagenesis was created by inducing a BRafV600E-driven melanomagenic program in tail interfollicular melanocytes. Most targeted mature, melanin-producing melanocytes expanded clonally within the epidermis before losing their differentiated features through transcriptional reprogramming and eventually invading the dermis. Tumors did not form within interscales, which contain both mature and dormant amelanotic melanocytes. The hair follicle bulge, which contains melanocyte stem cells, was also refractory to melanomagenesis. These studies identify varying tumor susceptibilities within the melanocytic lineage, highlighting pigment-producing cells as the melanoma cell of origin, and indicate that regional variation in tumor predisposition is dictated by microenvironmental cues rather than intrinsic differences in cellular origin. Critically, this work provides in vivo evidence that differentiated somatic cells can be reprogrammed into cancer initiating cells.

PTEN Regulates PI(3,4)P2 Signaling Downstream of Class I PI3K.

  • Malek M
  • Mol. Cell
  • 2017 Nov 2

Literature context:


Abstract:

The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.

Funding information:
  • NINDS NIH HHS - R01 NS037462(United States)

A Split-Abl Kinase for Direct Activation in Cells.

  • Diaz JE
  • Cell Chem Biol
  • 2017 Oct 19

Literature context:


Abstract:

To dissect the cellular roles of individual kinases, it is useful to design tools for their selective activation. We describe the engineering of a split-cAbl kinase (sKin-Abl) that is rapidly activated in cells with rapamycin and allows temporal, dose, and compartmentalization control. Our design strategy involves an empirical screen in mammalian cells and identification of split site in the N lobe. This split site leads to complete loss of activity, which can be restored upon small-molecule-induced dimerization in cells. Remarkably, the split site is transportable to the related Src Tyr kinase and the distantly related Ser/Thr kinase, AKT, suggesting broader applications to kinases. To quantify the fold induction of phosphotyrosine (pTyr) modification, we employed quantitative proteomics, NeuCode SILAC. We identified a number of known Abl substrates, including autophosphorylation sites and novel pTyr targets, 432 pTyr sites in total. We believe that this split-kinase technology will be useful for direct activation of protein kinases in cells.

Funding information:
  • NCI NIH HHS - R01 CA191018()
  • NIGMS NIH HHS - F32 GM089082()
  • NIGMS NIH HHS - P41 GM108538()
  • NIGMS NIH HHS - R01 GM097316()
  • NLM NIH HHS - T15 LM007359()

Hydrogen Sulfide and Sulfate Prebiotic Stimulates the Secretion of GLP-1 and Improves Glycemia in Male Mice.

  • Pichette J
  • Endocrinology
  • 2017 Oct 1

Literature context:


Abstract:

Recently, the gastrointestinal microbiome, and its metabolites, has emerged as a potential regulator of host metabolism. However, to date little is known on the precise mechanisms of how this regulation occurs. Hydrogen sulfide (H2S) is abundantly produced in the colon by sulfate-reducing bacteria (SRB). H2S is a bioactive gas that plays regulatory roles in many systems, including metabolic hormone regulation. This gas metabolite is produced in close proximity to the glucagonlike peptide-1 (GLP-1)-secreting cells in the gut epithelium. GLP-1 is a peptide hormone that plays pivotal roles in both glucose homeostasis and appetite regulation. We hypothesized that H2S can directly regulate GLP-1 secretion. We demonstrated that H2S donors (NaHS and GYY4137) directly stimulate GLP-1 secretion in murine L-cells (GLUTag) and that this occurs through p38 mitogen-activated protein kinase without affecting cell viability. We then increased SRB in mice by supplementing the diet with a prebiotic chondroitin sulfate for 4 weeks. Mice treated with chondroitin sulfate had elevated Desulfovibrio piger levels in the feces and increased colonic and fecal H2S concentration. These animals also had enhanced GLP-1 and insulin secretion, improved oral glucose tolerance, and reduced food consumption. These results indicate that H2S plays a stimulatory role in GLP-1 secretion and that sulfate prebiotics can enhance GLP-1 release and its downstream metabolic actions.

Binding of PLD2-Generated Phosphatidic Acid to KIF5B Promotes MT1-MMP Surface Trafficking and Lung Metastasis of Mouse Breast Cancer Cells.

  • Wang Z
  • Dev. Cell
  • 2017 Oct 23

Literature context:


Abstract:

Little is known about the cellular events promoting metastasis. We show that knockout of phospholipase D2 (PLD2), which generates the signaling lipid phosphatidic acid (PA), inhibits lung metastases in the mammary tumor virus (MMTV)-Neu transgenic mouse breast cancer model. PLD2 promotes local invasion through the regulation of the plasma membrane targeting of MT1-MMP and its associated invadopodia. A liposome pull-down screen identifies KIF5B, the heavy chain of the motor protein kinesin-1, as a new PA-binding protein. In vitro assays reveal that PA specifically and directly binds to the C terminus of KIF5B. The binding between PLD2-generated PA and KIF5B is required for the vesicular association of KIF5B, surface localization of MT1-MMP, invadopodia, and invasion in cancer cells. Taken together, these results identify a role of PLD2-generated PA in the regulation of kinesin-1 motor functions and breast cancer metastasis and suggest PLD2 as a potential therapeutic target for metastatic breast cancer.

Funding information:
  • NCI NIH HHS - R01 CA112403()
  • NCI NIH HHS - R01 CA193455()
  • NHLBI NIH HHS - R01 HL119478()
  • NIGMS NIH HHS - R01 GM114260()

Reactive Neutrophil Responses Dependent on the Receptor Tyrosine Kinase c-MET Limit Cancer Immunotherapy.

  • Glodde N
  • Immunity
  • 2017 Oct 17

Literature context:


Abstract:

Inhibitors of the receptor tyrosine kinase c-MET are currently used in the clinic to target oncogenic signaling in tumor cells. We found that concomitant c-MET inhibition promoted adoptive T cell transfer and checkpoint immunotherapies in murine cancer models by increasing effector T cell infiltration in tumors. This therapeutic effect was independent of tumor cell-intrinsic c-MET dependence. Mechanistically, c-MET inhibition impaired the reactive mobilization and recruitment of neutrophils into tumors and draining lymph nodes in response to cytotoxic immunotherapies. In the absence of c-MET inhibition, neutrophils recruited to T cell-inflamed microenvironments rapidly acquired immunosuppressive properties, restraining T cell expansion and effector functions. In cancer patients, high serum levels of the c-MET ligand HGF correlated with increasing neutrophil counts and poor responses to checkpoint blockade therapies. Our findings reveal a role for the HGF/c-MET pathway in neutrophil recruitment and function and suggest that c-MET inhibitor co-treatment may improve responses to cancer immunotherapy in settings beyond c-MET-dependent tumors.

Funding information:
  • NIAID NIH HHS - 2-U54-AI-057153(United States)

Ubiquilin1 promotes antigen-receptor mediated proliferation by eliminating mislocalized mitochondrial proteins.

  • Whiteley AM
  • Elife
  • 2017 Sep 21

Literature context:


Abstract:

Ubiquilins (Ubqlns) are a family of ubiquitin receptors that promote the delivery of hydrophobic and aggregated ubiquitinated proteins to the proteasome for degradation. We carried out a proteomic analysis of a B cell lymphoma-derived cell line, BJAB, that requires UBQLN1 for survival to identify UBQLN1 client proteins. When UBQLN1 expression was acutely inhibited, 120 mitochondrial proteins were enriched in the cytoplasm, suggesting that the accumulation of mitochondrial client proteins in the absence of UBQLN1 is cytostatic. Using a Ubqln1-/- mouse strain, we found that B cell receptor (BCR) ligation of Ubqln1-/- B cells led to a defect in cell cycle entry. As in BJAB cells, mitochondrial proteins accumulated in BCR-stimulated cells, leading to protein synthesis inhibition and cell cycle block. Thus, UBQLN1 plays an important role in clearing mislocalized mitochondrial proteins upon cell stimulation, and its absence leads to suppression of protein synthesis and cell cycle arrest.

Funding information:
  • NIDDK NIH HHS - K01 DK098285()
  • NINDS NIH HHS - R01 NS078279(United States)

YAP/TAZ Orchestrate VEGF Signaling during Developmental Angiogenesis.

  • Wang X
  • Dev. Cell
  • 2017 Sep 11

Literature context:


Abstract:

Vascular endothelial growth factor (VEGF) is a major driver of blood vessel formation. However, the signal transduction pathways culminating in the biological consequences of VEGF signaling are only partially understood. Here, we show that the Hippo pathway effectors YAP and TAZ work as crucial signal transducers to mediate VEGF-VEGFR2 signaling during angiogenesis. We demonstrate that YAP/TAZ are essential for vascular development as endothelium-specific deletion of YAP/TAZ leads to impaired vascularization and embryonic lethality. Mechanistically, we show that VEGF activates YAP/TAZ via its effects on actin cytoskeleton and that activated YAP/TAZ induce a transcriptional program to further control cytoskeleton dynamics and thus establish a feedforward loop that ensures a proper angiogenic response. Lack of YAP/TAZ also results in altered cellular distribution of VEGFR2 due to trafficking defects from the Golgi apparatus to the plasma membrane. Altogether, our study identifies YAP/TAZ as central mediators of VEGF signaling and therefore as important regulators of angiogenesis.

The Kinase mTORC1 Promotes the Generation and Suppressive Function of Follicular Regulatory T Cells.

  • Xu L
  • Immunity
  • 2017 Sep 19

Literature context:


Abstract:

Follicular regulatory T (Tfr) cells differentiate from conventional regulatory T (Treg) cells and suppress excessive germinal center (GC) responses by acting on both GC B cells and T follicular helper (Tfh) cells. Here, we examined the impact of mTOR, a serine/threonine protein kinase that senses and integrates diverse environmental cues, on the differentiation and functional competency of Tfr cells in response to protein immunization or viral infection. By genetically deleting Rptor or Rictor, essential components for mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), respectively, we found that mTORC1 but not mTORC2 is essential for Tfr differentiation. Mechanistically, mTORC1-mediated phosphorylation of the transcription factor STAT3 induced the expression of the transcription factor TCF-1 by promoting STAT3 binding to the Tcf7 5'-regulatory region. Subsequently, TCF-1 bound to the Bcl6 promoter to induce Bcl6 expression, which launched the Tfr cell differentiation program. Thus, mTORC1 initiates Tfr cell differentiation by activating the TCF-1-Bcl-6 axis during immunization or infection.

PTEN Loss Increases the Connectivity of Fast Synaptic Motifs and Functional Connectivity in a Developing Hippocampal Network.

  • Barrows CM
  • J. Neurosci.
  • 2017 Sep 6

Literature context:


Abstract:

Changes in synaptic strength and connectivity are thought to be a major mechanism through which many gene variants cause neurological disease. Hyperactivation of the PI3K-mTOR signaling network, via loss of function of repressors such as PTEN, causes epilepsy in humans and animal models, and altered mTOR signaling may contribute to a broad range of neurological diseases. Changes in synaptic transmission have been reported in animal models of PTEN loss; however, the full extent of these changes, and their effect on network function, is still unknown. To better understand the scope of these changes, we recorded from pairs of mouse hippocampal neurons cultured in a two-neuron microcircuit configuration that allowed us to characterize all four major connection types within the hippocampus. Loss of PTEN caused changes in excitatory and inhibitory connectivity, and these changes were postsynaptic, presynaptic, and transynaptic, suggesting that disruption of PTEN has the potential to affect most connection types in the hippocampal circuit. Given the complexity of the changes at the synaptic level, we measured changes in network behavior after deleting Pten from neurons in an organotypic hippocampal slice network. Slices containing Pten-deleted neurons showed increased recruitment of neurons into network bursts. Importantly, these changes were not confined to Pten-deleted neurons, but involved the entire network, suggesting that the extensive changes in synaptic connectivity rewire the entire network in such a way that promotes a widespread increase in functional connectivity.SIGNIFICANCE STATEMENT Homozygous deletion of the Pten gene in neuronal subpopulations in the mouse serves as a valuable model of epilepsy caused by mTOR hyperactivation. To better understand how gene deletions lead to altered neuronal activity, we investigated the synaptic and network effects that occur 1 week after Pten deletion. PTEN loss increased the connectivity of all four types of hippocampal synaptic connections, including two forms of increased inhibition of inhibition, and increased network functional connectivity. These data suggest that single gene mutations that cause neurological diseases such as epilepsy may affect a surprising range of connection types. Moreover, given the robustness of homeostatic plasticity, these diverse effects on connection types may be necessary to cause network phenotypes such as increased synchrony.

Spatial Activation of TORC1 Is Regulated by Hedgehog and E2F1 Signaling in the Drosophila Eye.

  • Kim W
  • Dev. Cell
  • 2017 Aug 21

Literature context:


Abstract:

Target of rapamycin complex 1 (TORC1) regulates cell growth in response to nutrients and growth factors. Although TORC1 signaling has been thoroughly studied at the cellular level, the regulation of TORC1 in multicellular tissues and organs has remained elusive. Here we found that TORC1 is selectively activated in the second mitotic wave (SMW), the terminal synchronous cell division, of the developing Drosophila eye. We demonstrated that Hedgehog (Hh) signaling regulates TORC1 through E2F1 and the cyclin D/Cdk4 complex in the SMW, and this regulation is independent from insulin and amino acid signaling pathways. TORC1 is necessary for the proper G1/S transition of the cells, and the activation of TORC1 rescues the cell-cycle defect of Hh signaling-deficient cells in the SMW. Based on this evolutionarily conserved regulation of TORC1 by Hh signaling, we propose that Hh-dependent developmental signaling pathways spatially regulate TORC1 activity in multicellular organisms.

Funding information:
  • NIMHD NIH HHS - G12 MD007592(United States)

Focal Adhesion- and IGF1R-Dependent Survival and Migratory Pathways Mediate Tumor Resistance to mTORC1/2 Inhibition.

  • Yoon SO
  • Mol. Cell
  • 2017 Aug 3

Literature context:


Abstract:

Aberrant signaling by the mammalian target of rapamycin (mTOR) contributes to the devastating features of cancer cells. Thus, mTOR is a critical therapeutic target and catalytic inhibitors are being investigated as anti-cancer drugs. Although mTOR inhibitors initially block cell proliferation, cell viability and migration in some cancer cells are quickly restored. Despite sustained inhibition of mTORC1/2 signaling, Akt, a kinase regulating cell survival and migration, regains phosphorylation at its regulatory sites. Mechanistically, mTORC1/2 inhibition promotes reorganization of integrin/focal adhesion kinase-mediated adhesomes, induction of IGFR/IR-dependent PI3K activation, and Akt phosphorylation via an integrin/FAK/IGFR-dependent process. This resistance mechanism contributes to xenograft tumor cell growth, which is prevented with mTOR plus IGFR inhibitors, supporting this combination as a therapeutic approach for cancers.

Neurosecretory protein GL stimulates food intake, de novo lipogenesis, and onset of obesity.

  • Iwakoshi-Ukena E
  • Elife
  • 2017 Aug 11

Literature context:


Abstract:

Mechanisms underlying the central regulation of food intake and fat accumulation are not fully understood. We found that neurosecretory protein GL (NPGL), a newly-identified neuropeptide, increased food intake and white adipose tissue (WAT) in rats. NPGL-precursor gene overexpression in the hypothalamus caused increases in food intake, WAT, body mass, and circulating insulin when fed a high calorie diet. Intracerebroventricular administration of NPGL induced de novo lipogenesis in WAT, increased insulin, and it selectively induced carbohydrate intake. Neutralizing antibody administration decreased the size of lipid droplets in WAT. Npgl mRNA expression was upregulated by fasting and low insulin levels. Additionally, NPGL-producing cells were responsive to insulin. These results point to NPGL as a novel neuronal regulator that drives food intake and fat deposition through de novo lipogenesis and acts to maintain steady-state fat level in concert with insulin. Dysregulation of NPGL may be a root cause of obesity.

Molecular mechanisms underlying protective effects of quercetin against mitochondrial dysfunction and progressive dopaminergic neurodegeneration in cell culture and MitoPark transgenic mouse models of Parkinson's Disease.

  • Ay M
  • J. Neurochem.
  • 2017 Aug 17

Literature context:


Abstract:

Quercetin, one of the major flavonoids in plants, has been recently reported to have neuroprotective effects against neurodegenerative processes. However, since the molecular signaling mechanisms governing these effects are not well clarified, we evaluated quercetin's effect on the neuroprotective signaling events in dopaminergic neuronal models and further tested its efficacy in the MitoPark transgenic mouse model of Parkinson's disease (PD). Western blot analysis revealed that quercetin significantly induced the activation of two major cell survival kinases, protein kinase D1 (PKD1) and Akt in MN9D dopaminergic neuronal cells. Furthermore, pharmacological inhibition or siRNA knockdown of PKD1 blocked the activation of Akt, suggesting that PKD1 acts as an upstream regulator of Akt in quercetin-mediated neuroprotective signaling. Quercetin also enhanced cAMP response-element binding protein phosphorylation and expression of the cAMP response-element binding protein target gene brain-derived neurotrophic factor. Results from qRT-PCR, Western blot analysis, mtDNA content analysis, and MitoTracker assay experiments revealed that quercetin augmented mitochondrial biogenesis. Quercetin also increased mitochondrial bioenergetics capacity and protected MN9D cells against 6-hydroxydopamine-induced neurotoxicity. To further evaluate the neuroprotective efficacy of quercetin against the mitochondrial dysfunction underlying PD, we used the progressive dopaminergic neurodegenerative MitoPark transgenic mouse model of PD. Oral administration of quercetin significantly reversed behavioral deficits, striatal dopamine depletion, and TH neuronal cell loss in MitoPark mice. Together, our findings demonstrate that quercetin activates the PKD1-Akt cell survival signaling axis and suggest that further exploration of quercetin as a promising neuroprotective agent for treating PD may offer clinical benefits.

Heparan sulfate alterations in extracellular matrix structures and fibroblast growth factor-2 signaling impairment in the aged neurogenic niche.

  • Yamada T
  • J. Neurochem.
  • 2017 Aug 26

Literature context:


Abstract:

Adult neurogenesis in the subventricular zone of the lateral ventricle decreases with age. In the subventricular zone, the specialized extracellular matrix structures, known as fractones, contact neural stem cells and regulate neurogenesis. Fractones are composed of extracellular matrix components, such as heparan sulfate proteoglycans. We previously found that fractones capture and store fibroblast growth factor 2 (FGF-2) via heparan sulfate binding, and may deliver FGF-2 to neural stem cells in a timely manner. The heparan sulfate (HS) chains in the fractones of the aged subventricular zone are modified based on immunohistochemistry. However, how aging affects fractone composition and subsequent FGF-2 signaling and neurogenesis remains unknown. The formation of the FGF-fibroblast growth factor receptor-HS complex is necessary to activate FGF-2 signaling and induce the phosphorylation of extracellular signal-regulated kinase (Erk1/2). In this study, we observed a reduction in HS 6-O-sulfation, which is critical for FGF-2 signal transduction, and failure of the FGF-2-induced phosphorylation of Erk1/2 in the aged subventricular zone. In addition, we observed increased HS 6-O-endo-sulfatase, an enzyme that may be responsible for the HS modifications in aged fractones. In conclusion, the data revealed that heparan sulfate 6-O-sulfation is reduced and FGF-2-dependent Erk1/2 signaling is impaired in the aged subventricular zone. HS modifications in fractones might play a role in the reduced neurogenic activity in aging brains.

Protective Role of Complement C3 Against Cytokine-Mediated β-Cell Apoptosis.

  • Dos Santos RS
  • Endocrinology
  • 2017 Aug 1

Literature context:


Abstract:

Type 1 diabetes is a chronic autoimmune disease characterized by pancreatic islet inflammation and β-cell destruction by proinflammatory cytokines and other mediators. Based on RNA sequencing and protein-protein interaction analyses of human islets exposed to proinflammatory cytokines, we identified complement C3 as a hub for some of the effects of cytokines. The proinflammatory cytokines interleukin-1β plus interferon-γ increase C3 expression in rodent and human pancreatic β-cells, and C3 is detected by histology in and around the islets of diabetic patients. Surprisingly, C3 silencing exacerbates apoptosis under both basal condition and following exposure to cytokines, and it increases chemokine expression upon cytokine treatment. C3 exerts its prosurvival effects via AKT activation and c-Jun N-terminal kinase inhibition. Exogenously added C3 also protects against cytokine-induced β-cell death and partially rescues the deleterious effects of inhibition of endogenous C3. These data suggest that locally produced C3 is an important prosurvival mechanism in pancreatic β-cells under a proinflammatory assault.

Effects of the Activation of Three Major Hepatic Akt Substrates on Glucose Metabolism in Male Mice.

  • Sakai G
  • Endocrinology
  • 2017 Aug 1

Literature context:


Abstract:

Insulin suppresses glucose output from the liver via Akt activation; however, which substrate of Akt plays the major role in transducing this effect is unclear. We tested the postnatal expression of Akt-unresponsive, constitutively active mutants of three major Akt substrates widely considered to regulate glucose metabolism [i.e., FoxO1, PGC1α, and glycogen synthase kinase-3β (GSK3β)] using adenoviral gene delivery to the mouse liver. We performed physiological hyperinsulinemic-euglycemic clamp studies using these mice under awake and nonrestrained conditions with blood sampling via an arterial catheter. Hepatic expression of constitutively active FoxO1 induced significant hepatic and systemic insulin resistance. However, neither the expression of constitutively active PGC1α nor that of GSK3β significantly changed insulin sensitivity. Simultaneous expression of all three mutants together induced no further insulin resistance compared with that of the FoxO1 mutant. The glycogen content in the liver was significantly reduced by constitutively active GSK3β expression. In cultured hepatocytes, constitutively active PGC1α induced markedly stronger transcriptional enhancement of gluconeogenic key enzymes than did constitutively active FoxO1. From these results, we conclude that FoxO1 has the most prominent role in transducing insulin's effect downstream from Akt to suppress hepatic glucose output, involving mechanisms independent of the transcriptional regulation of key gluconeogenic enzymes.

Autophagy impairment by caspase-1-dependent inflammation mediates memory loss in response to β-Amyloid peptide accumulation.

  • Álvarez-Arellano L
  • J. Neurosci. Res.
  • 2017 Aug 13

Literature context:


Abstract:

β-Amyloid peptide accumulation in the cortex and in the hippocampus results in neurodegeneration and memory loss. Recently, it became evident that the inflammatory response triggered by β-Amyloid peptides promotes neuronal cell death and degeneration. In addition to inflammation, β-Amyloid peptides also induce alterations in neuronal autophagy, eventually leading to neuronal cell death. Thus, here we evaluated whether the inflammatory response induced by the β-Amyloid peptides impairs memory via disrupting the autophagic flux. We show that male mice overexpressing β-Amyloid peptides (5XFAD) but lacking caspase-1, presented reduced β-Amyloid plaques in the cortex and in the hippocampus; restored brain autophagic flux and improved learning and memory capacity. At the molecular level, inhibition of the inflammatory response in the 5XFAD mice restored LC3-II levels and prevented the accumulation of oligomeric p62 and ubiquitylated proteins. Furthermore, caspase-1 deficiency reinstates activation of the AMPK/Raptor pathway while down-regulating AKT/mTOR pathway. Consistent with this, we found an inverse correlation between the increase of autophagolysosomes in the cortex of 5XFAD mice lacking caspase-1 and the presence of mitochondria with altered morphology. Together our results indicate that β-Amyloid peptide-induced caspase-1 activation, disrupts autophagy in the cortex and in the hippocampus resulting in neurodegeneration and memory loss.

Funding information:
  • NCATS NIH HHS - UL1 TR001439(United States)

A Hypothalamic Phosphatase Switch Coordinates Energy Expenditure with Feeding.

  • Dodd GT
  • Cell Metab.
  • 2017 Aug 1

Literature context:


Abstract:

Beige adipocytes can interconvert between white and brown-like states and switch between energy storage versus expenditure. Here we report that beige adipocyte plasticity is important for feeding-associated changes in energy expenditure and is coordinated by the hypothalamus and the phosphatase TCPTP. A fasting-induced and glucocorticoid-mediated induction of TCPTP, inhibited insulin signaling in AgRP/NPY neurons, repressed the browning of white fat and decreased energy expenditure. Conversely feeding reduced hypothalamic TCPTP, to increase AgRP/NPY neuronal insulin signaling, white adipose tissue browning and energy expenditure. The feeding-induced repression of hypothalamic TCPTP was defective in obesity. Mice lacking TCPTP in AgRP/NPY neurons were resistant to diet-induced obesity and had increased beige fat activity and energy expenditure. The deletion of hypothalamic TCPTP in obesity restored feeding-induced browning and increased energy expenditure to promote weight loss. Our studies define a hypothalamic switch that coordinates energy expenditure with feeding for the maintenance of energy balance.

Funding information:
  • NEI NIH HHS - R01 EY017097(United States)

mTORC2 Regulates Amino Acid Metabolism in Cancer by Phosphorylation of the Cystine-Glutamate Antiporter xCT.

  • Gu Y
  • Mol. Cell
  • 2017 Jul 6

Literature context:


Abstract:

Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic inhibition of the mammalian target of rapamycin (mTOR) kinase, promotes glutamate secretion, cystine uptake, and incorporation into glutathione, linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism regulating amino acid metabolism in cancer, enabling tumor cells to adapt to changing environmental conditions.

Funding information:
  • NCI NIH HHS - F31 CA186668()
  • NIGMS NIH HHS - R01 GM116897()
  • NINDS NIH HHS - R01 NS073831()

IL-6/Stat3-Dependent Induction of a Distinct, Obesity-Associated NK Cell Subpopulation Deteriorates Energy and Glucose Homeostasis.

  • Theurich S
  • Cell Metab.
  • 2017 Jul 5

Literature context:


Abstract:

Natural killer (NK) cells contribute to the development of obesity-associated insulin resistance. We demonstrate that in mice obesity promotes expansion of a distinct, interleukin-6 receptor (IL6R)a-expressing NK subpopulation, which also expresses a number of other myeloid lineage genes such as the colony-stimulating factor 1 receptor (Csf1r). Selective ablation of this Csf1r-expressing NK cell population prevents obesity and insulin resistance. Moreover, conditional inactivation of IL6Ra or Stat3 in NK cells limits obesity-associated formation of these myeloid signature NK cells, protecting from obesity, insulin resistance, and obesity-associated inflammation. Also in humans IL6Ra+ NK cells increase in obesity and correlate with markers of systemic low-grade inflammation, and their gene expression profile overlaps with characteristic gene sets of NK cells in obese mice. Collectively, we demonstrate that obesity-associated inflammation and metabolic disturbances depend on interleukin-6/Stat3-dependent formation of a distinct NK population, which may provide a target for the treatment of obesity, metaflammation-associated pathologies, and diabetes.

Funding information:
  • NHLBI NIH HHS - P01 HL096571(United States)
  • PHS HHS - HHSN268201100037C(United States)

IGF1R Expression in Ovarian Granulosa Cells Is Essential for Steroidogenesis, Follicle Survival, and Fertility in Female Mice.

  • Baumgarten SC
  • Endocrinology
  • 2017 Jul 1

Literature context:


Abstract:

Folliculogenesis is a lengthy process that requires the proliferation and differentiation of granulosa cells (GCs) for preovulatory follicle formation. The most crucial endocrine factor involved in this process is follicle-stimulating hormone (FSH). Interestingly, previous in vitro studies indicated that FSH does not stimulate GC proliferation in the absence of the insulinlike growth factor 1 receptor (IGF1R). To determine the role of the IGF1R in vivo, female mice with a conditional knockdown of the IGF1R in the GCs were produced and had undetectable levels of IGF1R mRNA and protein in the GCs. These animals were sterile, and their ovaries were smaller than those of control animals and contained no antral follicles even after gonadotropin stimulation. The lack of antral follicles correlated with a 90% decrease in serum estradiol levels. In addition, under a superovulation protocol no oocytes were found in the oviducts of these animals. Accordingly, the GCs of the mutant females expressed significantly lower levels of preovulatory markers including aromatase, luteinizing hormone receptor, and inhibin α. In contrast, no alterations in FSH receptor expression were observed in GCs lacking IGF1R. Immunohistochemistry studies demonstrated that ovaries lacking IGF1R had higher levels of apoptosis in follicles from the primary to the large secondary stages. Finally, molecular studies determined that protein kinase B activation was significantly impaired in mutant females when compared with controls. These in vivo findings demonstrate that IGF1R has a crucial role in GC function and, consequently, in female fertility.

Long-Term Exposure of Pancreatic β-Cells to Palmitate Results in SREBP-1C-Dependent Decreases in GLP-1 Receptor Signaling via CREB and AKT and Insulin Secretory Response.

  • Natalicchio A
  • Endocrinology
  • 2017 Jun 5

Literature context:


Abstract:

The effects of prolonged exposure of pancreatic β-cells to high saturated fatty acids on glucagon-like peptide-1 (GLP-1) action were investigated. Murine islets, human pancreatic 1.1B4 cells, and rat INS-1E cells were exposed to palmitate for 24 hours. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting, respectively. Specific short interfering RNAs were used to knockdown expression of the GLP-1 receptor (Glp1r) and Srebf1. Insulin release was assessed with a specific ELISA. Exposure of murine islets, as well as of human and INS-1E β-cells, to palmitate reduced the ability of exendin-4 to augment insulin mRNA levels, protein content, and release. In addition, palmitate blocked exendin-4-stimulated cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, whereas phosphorylation of MAPK-ERK kinase-1/2 and ERK-1/2 was not altered. Similarly, RNA interference-mediated suppression of Glp1r expression prevented exendin-4-induced cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, but did not impair exendin-4 stimulation of MAPK-ERK kinase-1/2 and ERK-1/2. Both islets from mice fed a high fat diet and human and INS-1E β-cells exposed to palmitate showed reduced GLP-1 receptor and pancreatic duodenal homeobox-1 (PDX-1) and increased sterol regulatory element-binding protein (SREBP-1C) mRNA and protein levels. Furthermore, suppression of SREBP-1C protein expression prevented the reduction of PDX-1 and GLP-1 receptor levels and restored exendin-4 signaling and action. Finally, treatment of INS-1E cells with metformin for 24 h resulted in inhibition of SREBP-1C expression, increased PDX-1 and GLP-1 receptor levels, consequently, enhancement of exendin-4-induced insulin release. Palmitate impairs exendin-4 effects on β-cells by reducing PDX-1 and GLP-1 receptor expression and signaling in a SREBP-1C-dependent manner. Metformin counteracts the impairment of GLP-1 receptor signaling induced by palmitate.

Funding information:
  • NIMH NIH HHS - R01 MH066332(United States)

Formin 1 Regulates Microtubule and F-Actin Organization to Support Spermatid Transport During Spermatogenesis in the Rat Testis.

  • Li N
  • Endocrinology
  • 2017 Jun 5

Literature context:


Abstract:

Formin 1 confers actin nucleation by generating long stretches of actin microfilaments to support cell movement, cell shape, and intracellular protein trafficking. Formin 1 is likely involved in microtubule (MT) dynamics due to the presence of a MT binding domain near its N terminus. Here, formin 1 was shown to structurally interact with α-tubulin, the building block of MT, and also end-binding protein 1 (a MT plus [+]-end-binding protein that stabilizes MT) in the testis. Knockdown of formin 1 in Sertoli cells with an established tight junction barrier was found to induce down-regulation of detyrosinated MT (a stabilized form of MT), and disorganization of MTs, in which MTs were retracted from the cell cortical zone, mediated through a loss of MT polymerization and down-regulation of Akt1/2 signaling kinase. An efficient knockdown of formin 1 in the testis reduced the number of track-like structures conferred by MTs and F-actin considerably, causing defects in spermatid and phagosome transport across the seminiferous epithelium. In summary, formin1 maintains MT and F-actin track-like structures to support spermatid and phagosome transport across the seminiferous epithelium during spermatogenesis.

Funding information:
  • NEI NIH HHS - T32 EY015387(United States)

Progesterone-Dependent Induction of Phospholipase C-Related Catalytically Inactive Protein 1 (PRIP-1) in Decidualizing Human Endometrial Stromal Cells.

  • Muter J
  • Endocrinology
  • 2017 Jun 5

Literature context:


Abstract:

Decidualization denotes the transformation of endometrial stromal cells into specialized decidual cells. In pregnancy, decidual cells form a protective matrix around the implanting embryo, enabling coordinated trophoblast invasion and formation of a functional placenta. Continuous progesterone (P4) signaling renders decidual cells resistant to various environmental stressors, whereas withdrawal inevitably triggers tissue breakdown and menstruation or miscarriage. Here, we show that PLCL1, coding phospholipase C (PLC)-related catalytically inactive protein 1 (PRIP-1), is highly induced in response to P4 signaling in decidualizing human endometrial stromal cells (HESCs). Knockdown experiments in undifferentiated HESCs revealed that PRIP-1 maintains basal phosphoinositide 3-kinase/Protein kinase B activity, which in turn prevents illicit nuclear translocation of the transcription factor forkhead box protein O1 and induction of the apoptotic activator BIM. By contrast, loss of this scaffold protein did not compromise survival of decidual cells. PRIP-1 knockdown did also not interfere with the responsiveness of HESCs to deciduogenic cues, although the overall expression of differentiation markers, such as PRL, IGFBP1, and WNT4, was blunted. Finally, we show that PRIP-1 in decidual cells uncouples PLC activation from intracellular Ca(2+) release by attenuating inositol 1,4,5-trisphosphate signaling. In summary, PRIP-1 is a multifaceted P4-inducible scaffold protein that gates the activity of major signal transduction pathways in the endometrium. It prevents apoptosis of proliferating stromal cells and contributes to the relative autonomy of decidual cells by silencing PLC signaling downstream of Gq protein-coupled receptors.

Funding information:
  • NEI NIH HHS - R01 EY012135(United States)
  • NHLBI NIH HHS - HL58144(United States)

Kupffer Cell-Derived Tnf Triggers Cholangiocellular Tumorigenesis through JNK due to Chronic Mitochondrial Dysfunction and ROS.

  • Yuan D
  • Cancer Cell
  • 2017 Jun 12

Literature context:


Abstract:

Intrahepatic cholangiocarcinoma (ICC) is a highly malignant, heterogeneous cancer with poor treatment options. We found that mitochondrial dysfunction and oxidative stress trigger a niche favoring cholangiocellular overgrowth and tumorigenesis. Liver damage, reactive oxygen species (ROS) and paracrine tumor necrosis factor (Tnf) from Kupffer cells caused JNK-mediated cholangiocellular proliferation and oncogenic transformation. Anti-oxidant treatment, Kupffer cell depletion, Tnfr1 deletion, or JNK inhibition reduced cholangiocellular pre-neoplastic lesions. Liver-specific JNK1/2 deletion led to tumor reduction and enhanced survival in Akt/Notch- or p53/Kras-induced ICC models. In human ICC, high Tnf expression near ICC lesions, cholangiocellular JNK-phosphorylation, and ROS accumulation in surrounding hepatocytes are present. Thus, Kupffer cell-derived Tnf favors cholangiocellular proliferation/differentiation and carcinogenesis. Targeting the ROS/Tnf/JNK axis may provide opportunities for ICC therapy.

Funding information:
  • NIDDK NIH HHS - R01 DK107220()

Ywhaz/14-3-3ζ Deletion Improves Glucose Tolerance Through a GLP-1-Dependent Mechanism.

  • Lim GE
  • Endocrinology
  • 2017 Jun 5

Literature context:


Abstract:

Multiple signaling pathways mediate the actions of metabolic hormones to control glucose homeostasis, but the proteins that coordinate such networks are poorly understood. We previously identified the molecular scaffold protein, 14-3-3ζ, as a critical regulator of in vitro β-cell survival and adipogenesis, but its metabolic roles in glucose homeostasis have not been studied in depth. Herein, we report that Ywhaz gene knockout mice (14-3-3ζKO) exhibited elevated fasting insulin levels while maintaining normal β-cell responsiveness to glucose when compared with wild-type littermate controls. In contrast with our observations after an ip glucose bolus, glucose tolerance was significantly improved in 14-3-3ζKO mice after an oral glucose gavage. This improvement in glucose tolerance was associated with significantly elevated fasting glucagon-like peptide-1 (GLP-1) levels. 14-3-3ζ knockdown in GLUTag L cells elevated GLP-1 synthesis and increased GLP-1 release. Systemic inhibition of the GLP-1 receptor attenuated the improvement in oral glucose tolerance that was seen in 14-3-3ζKO mice. When taken together these findings demonstrate novel roles of 14-3-3ζ in the regulation of glucose homeostasis and suggest that modulating 14-3-3ζ levels in intestinal L cells may have beneficial metabolic effects through GLP-1-dependent mechanisms.

Funding information:
  • NIAID NIH HHS - R01 AI118985(United States)
  • NIDDK NIH HHS - DK43140(United States)

Germinal Center Selection and Affinity Maturation Require Dynamic Regulation of mTORC1 Kinase.

  • Ersching J
  • Immunity
  • 2017 Jun 20

Literature context:


Abstract:

During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection.

Funding information:
  • NCI NIH HHS - R00 CA151827()
  • NCI NIH HHS - R01 CA103866()
  • NIAID NIH HHS - R01 AI047389()
  • NIAID NIH HHS - R01 AI119006()
  • NIAID NIH HHS - R37 AI047389()
  • NIH HHS - DP5 OD012146()

JMJD3 Is Crucial for the Female AVPV RIP-Cre Neuron-Controlled Kisspeptin-Estrogen Feedback Loop and Reproductive Function.

  • Song A
  • Endocrinology
  • 2017 Jun 1

Literature context:


Abstract:

The hypothalamic-pituitary-gonadal axis controls development, reproduction, and metabolism. Although most studies have focused on the hierarchy from the brain to the gonad, many questions remain unresolved concerning the feedback from the gonad to the central nervous system, especially regarding the potential epigenetic modifications in hypothalamic neurons. In the present report, we generated genetically modified mice lacking histone H3 lysine 27 (H3K27) demethylase Jumonji domain-containing 3 (JMJD3) in hypothalamic rat-insulin-promoter-expressing neurons (RIP-Cre neurons). The female mutant mice displayed late-onset obesity owing to reduced locomotor activity and decreased energy expenditure. JMJD3 deficiency in RIP-Cre neurons also results in delayed pubertal onset, an irregular estrous cycle, impaired fertility, and accelerated ovarian failure in female mice owing to the dysregulation of the hypothalamic-ovarian axis. We found that JMJD3 directly regulates Kiss1 gene expression by binding to the Kiss1 promoter and triggering H3K27me3 demethylation in the anteroventral periventricular (AVPV) nucleus. Further study confirmed that the aberrations arose from impaired kisspeptin signaling in the hypothalamic AVPV nucleus and subsequent estrogen deficiency. Estrogen replacement therapy can reverse obesity in mutant mice. Moreover, we demonstrated that Jmjd3 is an estrogen target gene in the hypothalamus. These results provide direct genetic and molecular evidence that JMJD3 is a key mediator for the kisspeptin-estrogen feedback loop.

Manganese-Stimulated Kisspeptin Is Mediated by the IGF-1/Akt/Mammalian Target of Rapamycin Pathway in the Prepubertal Female Rat.

  • Srivastava VK
  • Endocrinology
  • 2017 May 31

Literature context:


Abstract:

Low-dose administration of manganese chloride (MnCl2) causes release of hypothalamic LH-releasing hormone (LHRH) and advances puberty in rat. Recently, this element was shown to up-regulate mammalian target of rapamycin (mTOR), kisspeptin gene (KiSS-1), and LHRH gene expressions in the brain preoptic area (POA)/anteroventral periventricular (AVPV) nucleus. Because these genes are critical for puberty, this study was conducted to identify the upstream mechanism by which Mn activates the mTOR/KiSS-1 pathway. On day 12, immature female rats began receiving a daily supplemental dose of 10 mg/kg of MnCl2 or saline by gavage, and POA/AVPV tissues were collected on day 29 for specific protein assessments. Another experiment assessed in vitro IGF-1 release in response to Mn and assessed signal transduction pathways in the POA/AVPV region after Mn delivery into the third ventricle. Chronic Mn exposure increased (P < .05) basal expressions of mTOR and kisspeptin proteins. Mn increased protein kinase B (Akt) and Ras homolog enriched in brain, both capable of activating mTOR. Central Mn delivery increased expressions of phosphorylated IGF-1 receptor (IGF-1R) (P < .05) and Akt (P < .01) in the POA/AVPV region. The previous central delivery of JB1, an IGF-1R antagonist, blocked Mn-induced expressions of both phosphorylated IGF-1R and Akt. Downstream to Akt, centrally administered Mn increased tuberous sclerosis complex 2 (P < .05), Ras homolog enriched in brain (P < .01), mTOR (P < .05), and kisspeptin (P < .05). Finally, we observed that the early puberty induced by Mn was blocked by the administration of an mTOR inhibitor. These results suggest that Mn acts, at least in part, through the IGF-1/Akt/mTOR pathway to influence prepubertal kisspeptin and LHRH.

Funding information:
  • NIDDK NIH HHS - R37 DK33165(United States)

Glucose Sensing by Skeletal Myocytes Couples Nutrient Signaling to Systemic Homeostasis.

  • Meng ZX
  • Mol. Cell
  • 2017 May 4

Literature context:


Abstract:

Skeletal muscle is a major site of postprandial glucose disposal. Inadequate insulin action in skeletal myocytes contributes to hyperglycemia in diabetes. Although glucose is known to stimulate insulin secretion by β cells, whether it directly engages nutrient signaling pathways in skeletal muscle to maintain systemic glucose homeostasis remains largely unexplored. Here we identified the Baf60c-Deptor-AKT pathway as a target of muscle glucose sensing that augments insulin action in skeletal myocytes. Genetic activation of this pathway improved postprandial glucose disposal in mice, whereas its muscle-specific ablation impaired insulin action and led to postprandial glucose intolerance. Mechanistically, glucose triggers KATP channel-dependent calcium signaling, which promotes HDAC5 phosphorylation and nuclear exclusion, leading to Baf60c induction and insulin-independent AKT activation. This pathway is engaged by the anti-diabetic sulfonylurea drugs to exert their full glucose-lowering effects. These findings uncover an unexpected mechanism of glucose sensing in skeletal myocytes that contributes to homeostasis and therapeutic action.

Funding information:
  • NIDDK NIH HHS - P30 DK020572()
  • NIDDK NIH HHS - P30 DK089503()
  • NIDDK NIH HHS - R01 DK102456()
  • NIDDK NIH HHS - R01 DK112800()

The Sec14-like phosphatidylinositol transfer proteins Sec14l3/SEC14L2 act as GTPase proteins to mediate Wnt/Ca2+ signaling.

  • Gong B
  • Elife
  • 2017 May 2

Literature context:


Abstract:

The non-canonical Wnt/Ca2+ signaling pathway plays important roles in embryonic development, tissue formation and diseases. However, it is unclear how the Wnt ligand-stimulated, G protein-coupled receptor Frizzled activates phospholipases for calcium release. Here, we report that the zebrafish/human phosphatidylinositol transfer protein Sec14l3/SEC14L2 act as GTPase proteins to transduce Wnt signals from Frizzled to phospholipase C (PLC). Depletion of sec14l3 attenuates Wnt/Ca2+ responsive activity and causes convergent and extension (CE) defects in zebrafish embryos. Biochemical analyses in mammalian cells indicate that Sec14l3-GDP forms complex with Frizzled and Dishevelled; Wnt ligand binding of Frizzled induces translocation of Sec14l3 to the plasma membrane; and then Sec14l3-GTP binds to and activates phospholipase Cδ4a (Plcδ4a); subsequently, Plcδ4a initiates phosphatidylinositol-4,5-bisphosphate (PIP2) signaling, ultimately stimulating calcium release. Furthermore, Plcδ4a can act as a GTPase-activating protein to accelerate the hydrolysis of Sec14l3-bound GTP to GDP. Our data provide a new insight into GTPase protein-coupled Wnt/Ca2+ signaling transduction.

Hyperhomocysteinemia Promotes Insulin Resistance and Adipose Tissue Inflammation in PCOS Mice Through Modulating M2 Macrophage Polarization via Estrogen Suppression.

  • Qi X
  • Endocrinology
  • 2017 May 1

Literature context:


Abstract:

It has been shown that serum homocysteine (Hcy) levels are higher in women with polycystic ovary syndrome (PCOS). However, the specific role of hyperhomocysteinemia (HHcy) in the development of PCOS has never been reported. Adipose tissue inflammation is featured by the infiltration of macrophages, which plays a critical role in the pathogenesis of glucose and insulin intolerance. In this study, C57BL/6 mice were treated with dehydroepiandrosterone (DHEA) and/or a high methionine diet to induce PCOS and HHcy mice models. We showed that DHEA induced a PCOS-like phenotypes, irregular estrous cycles, weight gain, abnormal sex hormone production, glucose and insulin resistance, and polycystic ovaries. HHcy further intensified the effects DHEA on the metabolic, endocrinal, hormonal, and morphological changes in PCOS-like mice. In addition, HHcy attenuated the DHEA-induced increase in serum estrogen levels in mice. Furthermore, HHcy may exacerbate the insulin resistance in PCOS-like mice, most likely through modulating the macrophage M1/M2 polarization pathways via the suppression of estrogen. Most important, our clinical data showed that there were increases in serum Hcy levels in patients with PCOS. These findings deepen our understanding of the pathological roles of HHcy in the development of PCOS and provide a promising target for PCOS therapy in clinical application.

Aging Triggers Cytoplasmic Depletion and Nuclear Translocation of the E3 Ligase Mahogunin: A Function for Ubiquitin in Neuronal Survival.

  • Benvegnù S
  • Mol. Cell
  • 2017 May 4

Literature context:


Abstract:

A decline in proteasome function is causally connected to neuronal aging and aging-associated neuropathologies. By using hippocampal neurons in culture and in vivo, we show that aging triggers a reduction and a cytoplasm-to-nucleus redistribution of the E3 ubiquitin ligase mahogunin (MGRN1). Proteasome impairment induces MGRN1 monoubiquitination, the key post-translational modification for its nuclear entry. One potential mechanism for MGRN1 monoubiquitination is via progressive deubiquitination at the proteasome of polyubiquitinated MGRN1. Once in the nucleus, MGRN1 potentiates the transcriptional cellular response to proteotoxic stress. Inhibition of MGRN1 impairs ATF3-mediated neuronal responsiveness to proteosomal stress and increases neuronal stress, while increasing MGRN1 ameliorates signs of neuronal aging, including cognitive performance in old animals. Our results imply that, among others, the strength of neuronal survival in a proteasomal deterioration background, like during aging, depends on the fine-tuning of ubiquitination-deubiquitination.

SIRT6 regulates Ras-related protein R-Ras2 by lysine defatty-acylation.

  • Zhang X
  • Elife
  • 2017 Apr 13

Literature context:


Abstract:

The Ras family of GTPases are important in cell signaling and frequently mutated in human tumors. Understanding their regulation is thus important for studying biology and human diseases. Here, we report that a novel posttranslational mechanism, reversible lysine fatty acylation, regulates R-Ras2, a member of the Ras family. SIRT6, a sirtuin with established tumor suppressor function, regulates the lysine fatty acylation of R-Ras2. In mouse embryonic fibroblasts (MEFs), Sirt6 knockout (KO) increased R-Ras2 lysine fatty acylation. Lysine fatty acylation promotes the plasma membrane localization of R-Ras2 and its interaction with phosphatidylinositol 3-kinase PI3K, leading to activated Akt and increased cell proliferation. Our study establishes lysine fatty acylation as a previously unknown mechanism that regulates the Ras family of GTPases and provides an important mechanism by which SIRT6 functions as a tumor suppressor.

Funding information:
  • NIDDK NIH HHS - R01 DK107868()
  • NIGMS NIH HHS - R01 GM098596()
  • NIGMS NIH HHS - T32 GM008500()
  • NIH HHS - S10 OD018516()

Impact of High-Fat Diet and Early Stress on Depressive-Like Behavior and Hippocampal Plasticity in Adult Male Rats.

  • Arcego DM
  • Mol. Neurobiol.
  • 2017 Apr 30

Literature context:


Abstract:

During development, the brain goes through fundamental processes, including organization of neural networks and plasticity. Environmental interventions may change initial brain programming, leading to long-lasting effects and altering the susceptibility to psychopathologies, including depression disorder. It is known that depression is a psychiatric disorder with a high prevalence worldwide, including high rates among adolescents. In this study, we evaluated whether social isolation in the prepubertal period and chronic use of high-fat diet (HFD) may induce depressive-like behavior in male adult rats. We also investigated hippocampal plasticity markers and neurotransmitter systems. We found both social isolation and HFD induced a depressive-like behavior in the forced swimming task. Moreover, chronic HFD reduced synaptic markers in hippocampus, demonstrated by reductions in βIII-tubulin (neuronal marker), PSD-95, SNAP-25, and neurotrophin-3. The HFD group also presented decreased glutamatergic and GABAergic receptors subunits. On the other hand, stress affected hippocampal brain-derived neurotrophic factor (BDNF) signaling pathways, and increased expression of subunit of the NMDA receptor (NR2A). Both factors (stress and diet) decreased GR in the hippocampus without affecting plasma corticosterone at basal levels. Interactions between early stress and HFD access were observed only in the BNDF receptor (tropomyosin receptor kinase B; TrkB) and synaptophysin. In summary, these findings showed that a brief social isolation and chronic HFD, during a sensitive developmental period, cause depressive-like behavior in adulthood. The mechanisms underlying these behavioral effects may involve changes in the levels of synaptic proteins in hippocampus: HFD consumption appears to affect synaptic markers, while social isolation affected BDNF signaling more significantly.

The Ubiquitin Ligase CHIP Integrates Proteostasis and Aging by Regulation of Insulin Receptor Turnover.

  • Tawo R
  • Cell
  • 2017 Apr 20

Literature context:


Abstract:

Aging is attended by a progressive decline in protein homeostasis (proteostasis), aggravating the risk for protein aggregation diseases. To understand the coordination between proteome imbalance and longevity, we addressed the mechanistic role of the quality-control ubiquitin ligase CHIP, which is a key regulator of proteostasis. We observed that CHIP deficiency leads to increased levels of the insulin receptor (INSR) and reduced lifespan of worms and flies. The membrane-bound INSR regulates the insulin and IGF1 signaling (IIS) pathway and thereby defines metabolism and aging. INSR is a direct target of CHIP, which triggers receptor monoubiquitylation and endocytic-lysosomal turnover to promote longevity. However, upon proteotoxic stress conditions and during aging, CHIP is recruited toward disposal of misfolded proteins, reducing its capacity to degrade the INSR. Our study indicates a competitive relationship between proteostasis and longevity regulation through CHIP-assisted proteolysis, providing a mechanistic concept for understanding the impact of proteome imbalance on aging.

Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G0/G1 Arrest by Inhibiting Deubiquitinase USP2a.

  • Magiera K
  • Cell Chem Biol
  • 2017 Apr 20

Literature context:


Abstract:

USP2a is a deubiquitinase responsible for stabilization of cyclin D1, a crucial regulator of cell-cycle progression and a proto-oncoprotein overexpressed in numerous cancer types. Here we report that lithocholic acid (LCA) derivatives are inhibitors of USP proteins, including USP2a. The most potent LCA derivative, LCA hydroxyamide (LCAHA), inhibits USP2a, leading to a significant Akt/GSK3β-independent destabilization of cyclin D1, but does not change the expression of p27. This leads to the defects in cell-cycle progression. As a result, LCAHA inhibits the growth of cyclin D1-expressing, but not cyclin D1-negative cells, independently of the p53 status. We show that LCA derivatives may be considered as future therapeutics for the treatment of cyclin D1-addicted p53-expressing and p53-defective cancer types.

Selective Chemical Inhibition of PGC-1α Gluconeogenic Activity Ameliorates Type 2 Diabetes.

  • Sharabi K
  • Cell
  • 2017 Mar 23

Literature context:


Abstract:

Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.

Funding information:
  • NHGRI NIH HHS - U54 HG005032()
  • NIDA NIH HHS - R03 DA032468()
  • NIDDK NIH HHS - F32 DK102293()
  • NIDDK NIH HHS - R01 DK040936()
  • NIDDK NIH HHS - R01 DK069966()
  • NIDDK NIH HHS - R24 DK080261()
  • NIDDK NIH HHS - U2C DK059635()

The PERK arm of the unfolded protein response regulates satellite cell-mediated skeletal muscle regeneration.

  • Xiong G
  • Elife
  • 2017 Mar 23

Literature context:


Abstract:

Regeneration of skeletal muscle in adults is mediated by satellite stem cells. Accumulation of misfolded proteins triggers endoplasmic reticulum stress that leads to unfolded protein response (UPR). The UPR is relayed to the cell through the activation of PERK, IRE1/XBP1, and ATF6. Here, we demonstrate that levels of PERK and IRE1 are increased in satellite cells upon muscle injury. Inhibition of PERK, but not the IRE1 arm of the UPR in satellite cells inhibits myofiber regeneration in adult mice. PERK is essential for the survival and differentiation of activated satellite cells into the myogenic lineage. Deletion of PERK causes hyper-activation of p38 MAPK during myogenesis. Blocking p38 MAPK activity improves the survival and differentiation of PERK-deficient satellite cells in vitro and muscle formation in vivo. Collectively, our results suggest that the PERK arm of the UPR plays a pivotal role in the regulation of satellite cell homeostasis during regenerative myogenesis.

Funding information:
  • NIA NIH HHS - R01 AG029623()
  • NIAMS NIH HHS - R01 AR059810()
  • NIAMS NIH HHS - R01 AR068313()

Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy.

  • Qian X
  • Mol. Cell
  • 2017 Mar 2

Literature context:


Abstract:

Autophagy is crucial for maintaining cell homeostasis. However, the precise mechanism underlying autophagy initiation remains to be defined. Here, we demonstrate that glutamine deprivation and hypoxia result in inhibition of mTOR-mediated acetyl-transferase ARD1 S228 phosphorylation, leading to ARD1-dependent phosphoglycerate kinase 1 (PGK1) K388 acetylation and subsequent PGK1-mediated Beclin1 S30 phosphorylation. This phosphorylation enhances ATG14L-associated class III phosphatidylinositol 3-kinase VPS34 activity by increasing the binding of phosphatidylinositol to VPS34. ARD1-dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumorigenesis. Furthermore, PGK1 K388 acetylation levels correlate with Beclin1 S30 phosphorylation levels and poor prognosis in glioblastoma patients. Our study unearths an important mechanism underlying cellular-stress-induced autophagy initiation in which the protein kinase activity of the metabolic enzyme PGK1 plays an instrumental role and reveals the significance of the mutual regulation of autophagy and cell metabolism in maintaining cell homeostasis.

Funding information:
  • NCI NIH HHS - P30 CA016672()
  • NCI NIH HHS - P50 CA127001()
  • NCI NIH HHS - R01 CA109035()
  • NCI NIH HHS - R01 CA169603()
  • NINDS NIH HHS - R01 NS089754()

Angiotensin-II-induced Muscle Wasting is Mediated by 25-Hydroxycholesterol via GSK3β Signaling Pathway.

  • Shen C
  • EBioMedicine
  • 2017 Feb 5

Literature context:


Abstract:

While angiotensin II (ang II) has been implicated in the pathogenesis of cardiac cachexia (CC), the molecules that mediate ang II's wasting effect have not been identified. It is known TNF-α level is increased in patients with CC, and TNF-α release is triggered by ang II. We therefore hypothesized that ang II induced muscle wasting is mediated by TNF-α. Ang II infusion led to skeletal muscle wasting in wild type (WT) but not in TNF alpha type 1 receptor knockout (TNFR1KO) mice, suggesting that ang II induced muscle loss is mediated by TNF-α through its type 1 receptor. Microarray analysis identified cholesterol 25-hydroxylase (Ch25h) as the down stream target of TNF-α. Intraperitoneal injection of 25-hydroxycholesterol (25-OHC), the product of Ch25h, resulted in muscle loss in C57BL/6 mice, accompanied by increased expression of atrogin-1, MuRF1 and suppression of IGF-1/Akt signaling pathway. The identification of 25-OHC as an inducer of muscle wasting has implications for the development of specific treatment strategies in preventing muscle loss.

Crosstalk between CLCb/Dyn1-Mediated Adaptive Clathrin-Mediated Endocytosis and Epidermal Growth Factor Receptor Signaling Increases Metastasis.

  • Chen PH
  • Dev. Cell
  • 2017 Feb 6

Literature context:


Abstract:

Signaling receptors are internalized and regulated by clathrin-mediated endocytosis (CME). Two clathrin light chain isoforms, CLCa and CLCb, are integral components of the endocytic machinery whose differential functions remain unknown. We report that CLCb is specifically upregulated in non-small-cell lung cancer (NSCLC) cells and is associated with poor patient prognosis. Engineered single CLCb-expressing NSCLC cells, as well as "switched" cells that predominantly express CLCb, exhibit increased rates of CME and altered clathrin-coated pit dynamics. This "adaptive CME" resulted from upregulation of dynamin-1 (Dyn1) and its activation through a positive feedback loop involving enhanced epidermal growth factor (EGF)-dependent Akt/GSK3β phosphorylation. CLCb/Dyn1-dependent adaptive CME selectively altered EGF receptor trafficking, enhanced cell migration in vitro, and increased the metastatic efficiency of NSCLC cells in vivo. We define molecular mechanisms for adaptive CME in cancer cells and a role for the reciprocal crosstalk between signaling and CME in cancer progression.

Funding information:
  • NIGMS NIH HHS - R01 GM042455()
  • NIGMS NIH HHS - R01 GM073165()

Adaptive Chromatin Remodeling Drives Glioblastoma Stem Cell Plasticity and Drug Tolerance.

  • Liau BB
  • Cell Stem Cell
  • 2017 Feb 2

Literature context:


Abstract:

Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors. In this state, GSCs upregulate primitive developmental programs and are dependent upon Notch signaling. This transition is accompanied by widespread redistribution of repressive histone methylation. Accordingly, persister GSCs upregulate, and are dependent on, the histone demethylases KDM6A/B. Slow-cycling cells with high Notch activity and histone demethylase expression are present in primary glioblastomas before treatment, potentially contributing to relapse. Our findings illustrate how cancer cells may hijack aspects of native developmental programs for deranged proliferation, adaptation, and tolerance. They also suggest strategies for eliminating refractory tumor cells by targeting epigenetic and developmental pathways.

Funding information:
  • NCI NIH HHS - DP1 CA216873()
  • NCI NIH HHS - P50 CA165962()
  • NHGRI NIH HHS - U54 HG006991()
  • NINDS NIH HHS - R25 NS065743()

Soluble cpg15 from Astrocytes Ameliorates Neurite Outgrowth Recovery of Hippocampal Neurons after Mouse Cerebral Ischemia.

  • Zhao JJ
  • J. Neurosci.
  • 2017 Feb 8

Literature context:


Abstract:

The present study focuses on the function of cpg15, a neurotrophic factor, in ischemic neuronal recovery using transient global cerebral ischemic (TGI) mouse model and oxygen-glucose deprivation (OGD)-treated primary cultured cells. The results showed that expression of cpg15 proteins in astrocytes, predominantly the soluble form, was significantly increased in mouse hippocampus after TGI and in the cultured astrocytes after OGD. Addition of the medium from the cpg15-overexpressed astrocytic culture into the OGD-treated hippocampal neuronal cultures reduces the neuronal injury, whereas the recovery of neurite outgrowths of OGD-injured neurons was prevented when cpg15 in the OGD-treated astrocytes was knocked down, or the OGD-treated-astrocytic medium was immunoadsorbed by cpg15 antibody. Furthermore, lentivirus-delivered knockdown of cpg15 expression in mouse hippocampal astrocytes diminishes the dendritic branches and exacerbates injury of neurons in CA1 region after TGI. In addition, treatment with inhibitors of MEK1/2, PI3K, and TrkA decreases, whereas overexpression of p-CREB, but not dp-CREB, increases the expression of cpg15 in U118 or primary cultured astrocytes. Also, it is observed that the Flag-tagged soluble cpg15 from the astrocytes transfected with Flag-tagged cpg15-expressing plasmids adheres to the surface of neuronal bodies and the neurites. In conclusion, our results suggest that the soluble cpg15 from astrocytes induced by ischemia could ameliorate the recovery of the ischemic-injured hippocampal neurons via adhering to the surface of neurons. The upregulated expression of cpg15 in astrocytes may be activated via MAPK and PI3K signal pathways, and regulation of CREB phosphorylation.SIGNIFICANCE STATEMENT Neuronal plasticity plays a crucial role in the amelioration of neurological recovery of ischemic injured brain, which remains a challenge for clinic treatment of cerebral ischemia. cpg15 as a synaptic plasticity-related factor may participate in promoting the recovery process; however, the underlying mechanisms are still largely unknown. The objective of this study is to reveal the function and mechanism of neuronal-specific cpg15 expressed in astrocytes after ischemia induction, in promoting the recovery of injured neurons. Our findings provided new mechanistic insight into the neurological recovery, which might help develop novel therapeutic options for cerebral ischemia via astrocytic-targeting interference of gene expression.

EGFR Induces E2F1-Mediated Corticotroph Tumorigenesis.

  • Araki T
  • J Endocr Soc
  • 2017 Feb 1

Literature context:


Abstract:

The epidermal growth factor receptor (EGFR), expressed in adrenocorticotrophic hormone (ACTH)-secreting pituitary adenomas causing Cushing disease, regulates ACTH production and corticotroph proliferation. To elucidate the utility of EGFR as a therapeutic target for Cushing disease, we generated transgenic (Tg) mice with corticotroph-specific human EGFR expression (corti-EGFR-Tg) using a newly constructed corticotroph-specific promoter. Pituitary-specific EGFR expression was observed by 2.5 months, and aggressive ACTH-secreting pituitary adenomas with features of Crooke's cells developed by 8 months with 65% penetrance observed. Features consistent with the Cushing phenotype included elevated plasma ACTH and corticosterone levels, increased body weight, glucose intolerance, and enlarged adrenal cortex. Gefitinib, an EGFR tyrosine kinase inhibitor, suppressed tumor POMC expression and downstream EGFR tumor signaling, and ACTH and corticosterone levels were attenuated by 80% and 78%, respectively. Both E2F1 and phosphorylated Ser-337 E2F1 were increased in corti-EGFR-Tg mice and also colocalized with human POMC (hPOMC) in human pituitary corticotroph tumor samples. EGFR inhibition reversed E2F1 activity in vivo, whereas E2F1 inhibition suppressed POMC and ACTH in cultured human pituitary tumor cells. The corti-EGFR-Tg phenotype recapitulates ACTH-secreting pituitary adenomas and Cushing disease, validating the relevance of EGFR to corticotroph tumorigenesis. E2F1 is identified as a promising corticotroph-specific target for ACTH-dependent Cushing disease.

Funding information:
  • Canadian Institutes of Health Research - (Canada)

Multivalent Small-Molecule Pan-RAS Inhibitors.

  • Welsch ME
  • Cell
  • 2017 Feb 23

Literature context:


Abstract:

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

Funding information:
  • NCI NIH HHS - R01 CA097061()
  • NCI NIH HHS - R01 CA161061()
  • NCRR NIH HHS - S10 RR025431()
  • NIGMS NIH HHS - P41 GM111244()
  • NIGMS NIH HHS - R01 GM085081()
  • NIGMS NIH HHS - T32 GM008281()
  • NIH HHS - S10 OD012018()

Autophagy inhibition overcomes multiple mechanisms of resistance to BRAF inhibition in brain tumors.

  • Mulcahy Levy JM
  • Elife
  • 2017 Jan 17

Literature context:


Abstract:

Kinase inhibitors are effective cancer therapies, but tumors frequently develop resistance. Current strategies to circumvent resistance target the same or parallel pathways. We report here that targeting a completely different process, autophagy, can overcome multiple BRAF inhibitor resistance mechanisms in brain tumors. BRAFV600Emutations occur in many pediatric brain tumors. We previously reported that these tumors are autophagy-dependent and a patient was successfully treated with the autophagy inhibitor chloroquine after failure of the BRAFV600E inhibitor vemurafenib, suggesting autophagy inhibition overcame the kinase inhibitor resistance. We tested this hypothesis in vemurafenib-resistant brain tumors. Genetic and pharmacological autophagy inhibition overcame molecularly distinct resistance mechanisms, inhibited tumor cell growth, and increased cell death. Patients with resistance had favorable clinical responses when chloroquine was added to vemurafenib. This provides a fundamentally different strategy to circumvent multiple mechanisms of kinase inhibitor resistance that could be rapidly tested in clinical trials in patients with BRAFV600E brain tumors.

Funding information:
  • NCI NIH HHS - K08 CA193982()
  • NCI NIH HHS - R01 CA150925()
  • NCI NIH HHS - R01 CA190170()

Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling.

  • Calcagnì A
  • Elife
  • 2016 Sep 26

Literature context:


Abstract:

TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to overexpression of the TFEB and TFE3 genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific TFEB overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT β-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway.

Funding information:
  • NIGMS NIH HHS - R01 GM064709(United States)

Obesity-Dependent Increases in Oocyte mRNAs Are Associated With Increases in Proinflammatory Signaling and Gut Microbial Abundance of Lachnospiraceae in Female Mice.

  • Xie F
  • Endocrinology
  • 2016 Aug 17

Literature context:


Abstract:

RNAs stored in the metaphase II-arrested oocyte play important roles in successful embryonic development. Their abundance is defined by transcriptional activity during oocyte growth and selective degradation of transcripts during LH-induced oocyte maturation. Our previous studies demonstrated that mRNA abundance is increased in mature ovulated oocytes collected from obese humans and mice and therefore may contribute to reduced oocyte developmental competence associated with metabolic dysfunction. In the current study mouse models of diet-induced obesity were used to determine whether obesity-dependent increases in proinflammatory signaling regulate ovarian abundance of oocyte-specific mRNAs. The abundance of oocyte-specific Bnc1, Dppa3, and Pou5f1 mRNAs as well as markers of proinflammatory signaling were significantly increased in ovaries of obese compared with lean mice which were depleted of fully grown preovulatory follicles. Chromatin-immunoprecipitation analyses also demonstrated increased association of phosphorylated signal transducer and activator of transcription 3 with the Pou5f1 promoter in ovaries of obese mice suggesting that proinflammatory signaling regulates transcription of this gene in the oocyte. The cecum microbial content of lean and obese female mice was subsequently examined to identify potential relationships between microbial composition and proinflammatory signaling in the ovary. Multivariate Association with Linear Models identified significant positive correlations between cecum abundance of the bacterial family Lachnospiraceae and ovarian abundance of Tnfa as well as Dppa3, Bnc1, and Pou5f1 mRNAs. Together, these data suggest that diet-induced changes in gut microbial composition may be contributing to ovarian inflammation which in turn alters ovarian gene expression and ultimately contributes to obesity-dependent reduction in oocyte quality and development of infertility in obese patients.

Funding information:
  • NEI NIH HHS - R01 EY018660(United States)

The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding.

  • Woodford MR
  • Nat Commun
  • 2016 Jun 29

Literature context:


Abstract:

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors.

Funding information:
  • NIGMS NIH HHS - R01 GM050291(United States)

FSH Regulates mRNA Translation in Mouse Oocytes and Promotes Developmental Competence.

  • Franciosi F
  • Endocrinology
  • 2016 Feb 2

Literature context:


Abstract:

A major challenge in assisted reproductive technology is to develop conditions for in vitro oocyte maturation yielding high-quality eggs. Efforts are underway to assess whether known hormonal and local factors play a role in oocyte developmental competence and to identify the molecular mechanism involved. Here we have tested the hypothesis that FSH improves oocyte developmental competence by regulating the translational program in the oocyte. Accumulation of oocyte proteins (targeting protein for the Xenopus kinesin xklp2 and IL-7) associated with improved oocyte quality is increased when cumulus-oocyte complexes are incubated with FSH. This increase is due to enhanced translation of the corresponding mRNAs, as indicated by microinjection of constructs in which the 3' untranslated region of the Tpx2 or Il7 transcripts is fused to the luciferase reporter. A transient activation of the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte preceded the increase in translation. When the epidermal growth factor (EGF) receptor is down-regulated in follicular cells, the FSH-induced rate of maternal mRNA translation and AKT activation were lost, demonstrating that the effects of FSH are indirect and require EGF receptor signaling in the somatic compartment. Using Pten(fl/fl):Zp3cre oocytes in which the AKT is constitutively activated, translation of reporters was increased and was no longer sensitive to FSH stimulation. More importantly, the oocytes lacking the phosphate and tensin homolog gene showed increased developmental competence, even when cultured in the absence of FSH or growth factors. Thus, we demonstrate that FSH intersects with the follicular EGF network to activate the phosphatidyl-inositol 3-phosphate/AKT cascade in the oocyte to control translation and developmental competence. These findings provide a molecular rationale for the use of FSH to improve egg quality.

Funding information:
  • Wellcome Trust - 102645(United Kingdom)

Deficiency of FcϵR1 Increases Body Weight Gain but Improves Glucose Tolerance in Diet-Induced Obese Mice.

  • Lee YJ
  • Endocrinology
  • 2015 Nov 17

Literature context:


Abstract:

Prior studies demonstrated increased plasma IgE in diabetic patients, but the direct participation of IgE in diabetes or obesity remains unknown. This study found that plasma IgE levels correlated inversely with body weight, body mass index, and body fat mass among a population of randomly selected obese women. IgE receptor FcϵR1-deficient (Fcer1a(-/-)) mice and diet-induced obesity (DIO) mice demonstrated that FcϵR1 deficiency in DIO mice increased food intake, reduced energy expenditure, and increased body weight gain but improved glucose tolerance and glucose-induced insulin secretion. White adipose tissue from Fcer1a(-/-) mice showed an increased expression of phospho-AKT, CCAAT/enhancer binding protein-α, peroxisome proliferator-activated receptor-γ, glucose transporter-4 (Glut4), and B-cell lymphoma 2 (Bcl2) but reduced uncoupling protein 1 (UCP1) and phosphorylated c-Jun N-terminal kinase (JNK) expression, tissue macrophage accumulation, and apoptosis, suggesting that IgE reduces adipogenesis and glucose uptake but induces energy expenditure, adipocyte apoptosis, and white adipose tissue inflammation. In 3T3-L1 cells, IgE inhibited the expression of CCAAT/enhancer binding protein-α and peroxisome proliferator-activated receptor-γ, and preadipocyte adipogenesis and induced adipocyte apoptosis. IgE reduced the 3T3-L1 cell expression of Glut4, phospho-AKT, and glucose uptake, which concurred with improved glucose tolerance in Fcer1a(-/-) mice. This study established two novel pathways of IgE in reducing body weight gain in DIO mice by suppressing adipogenesis and inducing adipocyte apoptosis while worsening glucose tolerance by reducing Glut4 expression, glucose uptake, and insulin secretion.

Funding information:
  • NHLBI NIH HHS - P01 HL096571(United States)

Chronic Exposure to TNFα Impairs Secretion of Glucagon-Like Peptide-1.

  • Gagnon J
  • Endocrinology
  • 2015 Nov 17

Literature context:


Abstract:

Obesity is associated with systemic inflammation and elevated levels of TNFα, leading to impaired glucose tolerance. In humans, obesity is also associated with reduced nutrient-stimulated secretion of the intestinal incretin hormone, glucagon-like peptide-1 (GLP-1). We hypothesized that TNFα plays a direct role in the impairment of GLP-1 secretion from the enteroendocrine L-cell and that blocking TNFα can restore both GLP-1 secretion and glucose homeostasis. Expression of the TNFα receptor subytpe-1 was detected in the human NCI-H716 and murine GLUTag L-cell models and in mouse ileal sections. Although TNFα acutely increased GLP-1 release from NCI-H716 cells (P < .05-.001), preincubation with TNFα for 24 hours reduced proglucagon mRNA (P < .05) and GLP-1 cellular (P < .05) levels without affecting cell viability. Furthermore, both NCI-H716 and GLUTag cells pretreated with TNFα for 24 hours no longer responded to known GLP-1 secretagogues, an effect that was reversed by coincubation with the Nuclear Factor Kappa B inhibitor, 5-aminosalicylic acid, in the NCI-H716 cells. Mice given a high-fat diet (HFD) for 12 weeks developed impaired glucose tolerance, hyperinsulinemia, and increased TNFα mRNA expression in fat and ileal tissue. Hyperglycemia and hyperinsulinemia were reduced in HFD mice treated with the anti-TNFα biological, etanercept, for 2 weeks. In primary intestinal cultures from these animals, HFD control mice had impaired GLP-1 secretion, and this was not observed in the HFD etanercept-derived cultures (P < .05). In conclusion, chronic exposure to TNFα directly impairs GLP-1 secretion at the level of the intestinal L-cell, an effect that is reversed by anti-TNFα therapy in association with improved glucose tolerance.

Funding information:
  • NHLBI NIH HHS - P01 HL095491(United States)

Adiponectin-mediated antilipotoxic effects in regenerating pancreatic islets.

  • Ye R
  • Endocrinology
  • 2015 Jun 18

Literature context:


Abstract:

Pathways that stimulate β-cell regeneration remain of great clinical interest, yet effective therapeutic avenues that promote survival or reconstitution of β-cell mass remain elusive. Using a mouse model with inducible β-cell apoptosis followed by adiponectin-mediated regeneration, we aimed to identify key molecules boosting β-cell viability. In the regenerating pancreatic islets, we examined changes within the transcriptome and observed an extensive up-regulation of genes encoding proteins involved in lipid transport and metabolism. The most prominent targets were further confirmed by quantitative PCR and immunofluorescence. Among the upstream regulators predicted by pathway analysis of the transcriptome, we detected enhanced levels of 2 key transcription factors, Hepatocyte Nuclear Factor 4α and Peroxisome Proliferator-Activated Receptorα. Our data suggest that improving pancreatic islet lipid metabolism as an important antilipotoxic phenomenon to boost β-cell regeneration. This is primarily mediated by the adipokine adiponectin that exerts its action on both the beta-cell directly as well as on the adipocyte. Adiponectin induces lipid metabolism gene expression in regenerating islets through Hepatocyte Nuclear Factor 4α and Peroxisome Proliferator-Activated Receptorα. Adiponectin also modulates leptin levels via preserving adipose tissue mass in the insulinopenic state.

Funding information:
  • Wellcome Trust - 101253/Z/13/Z(United Kingdom)

Vitamin d deficiency attenuates high-fat diet-induced hyperinsulinemia and hepatic lipid accumulation in male mice.

  • Liu XJ
  • Endocrinology
  • 2015 Jun 18

Literature context:


Abstract:

It is increasingly recognized that vitamin D deficiency is associated with increased risks of metabolic disorders among overweight children. A recent study showed that vitamin D deficiency exacerbated inflammation in nonalcoholic fatty liver disease through activating toll-like receptor 4 in a high-fat diet (HFD) rat model. The present study aimed to further investigate the effects of vitamin D deficiency on HFD-induced insulin resistance and hepatic lipid accumulation. Male ICR mice (35 d old) were randomly assigned into 4 groups as follows. In control diet and vitamin D deficiency diet (VDD) groups, mice were fed with purified diets. In HFD and VDD+HFD groups, mice were fed with HFD. In VDD and VDD+HFD groups, vitamin D in feed was depleted. Feeding mice with vitamin D deficiency diet did not induce obesity, insulin resistance, and hepatic lipid accumulation. By contrary, vitamin D deficiency markedly alleviated HFD-induced overweight, hyperinsulinemia, and hepatic lipid accumulation. Moreover, vitamin D deficiency significantly attenuated HFD-induced up-regulation of hepatic peroxisome proliferator-activated receptor γ, which promoted hepatic lipid uptake and lipid droplet formation, and its target gene cluster of differentiation 36. In addition, vitamin D deficiency up-regulated carnitine palmitoyltrans 2, the key enzyme for fatty acid β-oxidation, and uncoupling protein 3, which separated oxidative phosphorylation from ATP production, in adipose tissue. These data suggest that vitamin D deficiency is not a direct risk factor for obesity, insulin resistance, and hepatic lipid accumulation. Vitamin D deficiency alleviates HFD-induced overweight, hyperinsulinemia, and hepatic lipid accumulation through promoting fatty acid β-oxidation and elevating energy expenditure in adipose tissue.

Funding information:
  • Canadian Institutes of Health Research - (Canada)
  • NIMH NIH HHS - R37 MH049428(United States)

Muscle-specific deletion of comparative gene identification-58 (CGI-58) causes muscle steatosis but improves insulin sensitivity in male mice.

  • Xie P
  • Endocrinology
  • 2015 May 18

Literature context:


Abstract:

Intramyocellular accumulation of lipids is often associated with insulin resistance. Deficiency of comparative gene identification-58 (CGI-58) causes cytosolic deposition of triglyceride (TG)-rich lipid droplets in most cell types, including muscle due to defective TG hydrolysis. It was unclear, however, whether CGI-58 deficiency-induced lipid accumulation in muscle influences insulin sensitivity. Here we show that muscle-specific CGI-58 knockout mice relative to their controls have increased glucose tolerance and insulin sensitivity on a Western-type high-fat diet, despite TG accumulation in both heart and oxidative skeletal muscle and cholesterol deposition in heart. Although the intracardiomyocellular lipid deposition results in cardiac ventricular fibrosis and systolic dysfunction, muscle-specific CGI-58 knockout mice show increased glucose uptake in heart and soleus muscle, improved insulin signaling in insulin-sensitive tissues, and reduced plasma concentrations of glucose, insulin, and cholesterol. Hepatic contents of TG and cholesterol are also decreased in these animals. Cardiac steatosis is attributable, at least in part, to decreases in cardiac TG hydrolase activity and peroxisome proliferator-activated receptor-α/peroxisome proliferator-activated receptor-γ coactivator-1-dependent mitochondrial fatty acid oxidation. In conclusion, muscle CGI-58 deficiency causes cardiac dysfunction and fat deposition in oxidative muscles but induces a series of favorable metabolic changes in mice fed a high-fat diet.

Funding information:
  • NEI NIH HHS - R01 EY015128(United States)

rpS6 regulates blood-testis barrier dynamics through Arp3-mediated actin microfilament organization in rat sertoli cells. An in vitro study.

  • Mok KW
  • Endocrinology
  • 2015 May 18

Literature context:


Abstract:

In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers.

Funding information:
  • NINDS NIH HHS - NS-23805(United States)

The role of hypothalamic mTORC1 signaling in insulin regulation of food intake, body weight, and sympathetic nerve activity in male mice.

  • Muta K
  • Endocrinology
  • 2015 Apr 21

Literature context:


Abstract:

Insulin action in the brain particularly the hypothalamus is critically involved in the regulation of several physiological processes, including energy homeostasis and sympathetic nerve activity, but the underlying mechanisms are poorly understood. The mechanistic target of rapamycin complex 1 (mTORC1) is implicated in the control of diverse cellular functions, including sensing nutrients and energy status. Here, we examined the role of hypothalamic mTORC1 in mediating the anorectic, weight-reducing, and sympathetic effects of central insulin action. In a mouse hypothalamic cell line (GT1-7), insulin treatment increased mTORC1 activity in a time-dependent manner. In addition, intracerebroventricular (ICV) administration of insulin to mice activated mTORC1 pathway in the hypothalamic arcuate nucleus, a key site of central action of insulin. Interestingly, inhibition of hypothalamic mTORC1 with rapamycin reversed the food intake- and body weight-lowering effects of ICV insulin. Rapamycin also abolished the ability of ICV insulin to cause lumbar sympathetic nerve activation. In GT1-7 cells, we found that insulin activation of mTORC1 pathway requires phosphatidylinositol 3-kinase (PI3K). Consistent with this, genetic disruption of PI3K in mice abolished insulin stimulation of hypothalamic mTORC1 signaling as well as the lumbar sympathetic nerve activation evoked by insulin. These results demonstrate the importance of mTORC1 pathway in the hypothalamus in mediating the action of insulin to regulate energy homeostasis and sympathetic nerve traffic. Our data also highlight the key role of PI3K as a link between insulin receptor and mTORC1 signaling in the hypothalamus.

Funding information:
  • NHLBI NIH HHS - R01 HL122494(United States)

Registered report: Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion.

  • Blum D
  • Elife
  • 2014 Dec 10

Literature context:


Abstract:

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered Report describes the proposed replication plan of key experiments from "Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion" by Straussman and colleagues, published in Nature in 2012 (Straussman et al., 2012). The key experiments being replicated in this study are from Figure 2A, C, and D (and Supplemental Figure 11) and Figure 4C (and Supplemental Figure 19) (Straussman et al., 2012). Figure 2 demonstrates resistance to drug sensitivity conferred by co-culture with some stromal cell lines and identifies the secreted factor responsible as HGF. In Figure 4, Straussman and colleagues show that blocking the HGF receptor MET abrogates HGF’s rescue of drug sensitivity. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

Funding information:
  • NINDS NIH HHS - R03 NS071442(United States)

Comparative analysis of Tsc1 and Tsc2 single and double radial glial cell mutants.

  • Mietzsch U
  • J. Comp. Neurol.
  • 2013 Nov 30

Literature context:


Abstract:

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with variable expressivity. Heterozygous mutations in either of two genes, TSC1 (hamartin) or TSC2 (tuberin), are responsible for most cases. Hamartin and tuberin form a heterodimer that functions as a major cellular inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) kinase. Genotype-phenotype studies suggest that TSC2 mutations are associated with a more severe neurologic phenotype, although the biologic basis for the difference between TSC1- and TSC2-based disease is unclear. Here we performed a study to compare and contrast the brain phenotypes of Tsc1 and Tsc2 single and double mutants. Using Tsc1 and Tsc2 floxed alleles and a radial glial transgenic Cre driver (FVB-Tg(GFAP-cre)25Mes/J), we deleted Tsc1 and/or Tsc2 in radial glial progenitor cells. Single and double mutants had remarkably similar phenotypes: early postnatal mortality, brain overgrowth, laminar disruption, astrogliosis, a paucity of oligodendroglia, and myelination defects. Double Tsc1/Tsc2 mutants died earlier than single mutants, and single mutants showed differences in the location of heterotopias and the organization of the hippocampal stratum pyramidale. The differences were not due to differential mTORC1 activation or feedback inhibition on Akt. These data provide further genetic evidence for individual hamartin and tuberin functions that may explain some of the genotype-phenotype differences seen in the human disease.

Funding information:
  • Telethon - GSP04289(Italy)