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Goat Anti-Mouse Goat anti-mouse IgG-HRP Polyclonal, Hrp Conjugated antibody

RRID:AB_631736

Antibody ID

AB_631736

Target Antigen

Mouse IgG mouse

Proper Citation

(Santa Cruz Biotechnology Cat# sc-2005, RRID:AB_631736)

Clonality

polyclonal antibody

Comments

Discontinued: 2016; validation status unknown check with seller; recommendations: Flow Cytometry; Immunohistochemistry; Immunoprecipitation; Immunoprecipitation, Immunohistochemistry, Flow Cytometry

Host Organism

goat

Vendor

Santa Cruz Biotechnology

Nicotinamide Nucleotide Transhydrogenase as a Novel Treatment Target in Adrenocortical Carcinoma.

  • Chortis V
  • Endocrinology
  • 2018 Aug 1

Literature context:


Abstract:

Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. In this study, we evaluated a potential new treatment target for ACC, focusing on the mitochondrial reduced form of NAD phosphate (NADPH) generator nicotinamide nucleotide transhydrogenase (NNT). NNT has a central role within mitochondrial antioxidant pathways, protecting cells from oxidative stress. Inactivating human NNT mutations result in congenital adrenal insufficiency. We hypothesized that NNT silencing in ACC cells will induce toxic levels of oxidative stress. To explore this, we transiently knocked down NNT in NCI-H295R ACC cells. As predicted, this manipulation increased intracellular levels of oxidative stress; this resulted in a pronounced suppression of cell proliferation and higher apoptotic rates, as well as sensitization of cells to chemically induced oxidative stress. Steroidogenesis was paradoxically stimulated by NNT loss, as demonstrated by mass spectrometry-based steroid profiling. Next, we generated a stable NNT knockdown model in the same cell line to investigate the longer lasting effects of NNT silencing. After long-term culture, cells adapted metabolically to chronic NNT knockdown, restoring their redox balance and resilience to oxidative stress, although their proliferation remained suppressed. This was associated with higher rates of oxygen consumption. The molecular pathways underpinning these responses were explored in detail by RNA sequencing and nontargeted metabolome analysis, revealing major alterations in nucleotide synthesis, protein folding, and polyamine metabolism. This study provides preclinical evidence of the therapeutic merit of antioxidant targeting in ACC as well as illuminating the long-term adaptive response of cells to oxidative stress.

Funding information:
  • Biotechnology and Biological Sciences Research Council - R103963(United Kingdom)

Targeting Breast Cancer Stem Cell State Equilibrium through Modulation of Redox Signaling.

  • Luo M
  • Cell Metab.
  • 2018 Jul 3

Literature context:


Abstract:

Although breast cancer stem cells (BCSCs) display plasticity transitioning between quiescent mesenchymal-like (M) and proliferative epithelial-like (E) states, how this plasticity is regulated by metabolic or oxidative stress remains poorly understood. Here, we show that M- and E-BCSCs rely on distinct metabolic pathways and display markedly different sensitivities to inhibitors of glycolysis and redox metabolism. Metabolic or oxidative stress generated by 2DG, H2O2, or hypoxia promotes the transition of ROSlo M-BCSCs to a ROShi E-state. This transition is reversed by N-acetylcysteine and mediated by activation of the AMPK-HIF1α axis. Moreover, E-BCSCs exhibit robust NRF2-mediated antioxidant responses, rendering them vulnerable to ROS-induced differentiation and cytotoxicity following suppression of NRF2 or downstream thioredoxin (TXN) and glutathione (GSH) antioxidant pathways. Co-inhibition of glycolysis and TXN and GSH pathways suppresses tumor growth, tumor-initiating potential, and metastasis by eliminating both M- and E-BCSCs. Exploiting metabolic vulnerabilities of distinct BCSC states provides a novel therapeutic approach targeting this critical tumor cell population.

Funding information:
  • NCI NIH HHS - P30 CA086862()
  • NCI NIH HHS - R01 CA101860()
  • NCI NIH HHS - R01 CA182804()
  • NCI NIH HHS - R01 CA196018()
  • NCI NIH HHS - R35 CA197585()
  • NCI NIH HHS - U01 CA141455(United States)
  • NCI NIH HHS - U01 CA210152()

PTBP1-Mediated Alternative Splicing Regulates the Inflammatory Secretome and the Pro-tumorigenic Effects of Senescent Cells.

  • Georgilis A
  • Cancer Cell
  • 2018 Jul 9

Literature context:


Abstract:

Oncogene-induced senescence is a potent tumor-suppressive response. Paradoxically, senescence also induces an inflammatory secretome that promotes carcinogenesis and age-related pathologies. Consequently, the senescence-associated secretory phenotype (SASP) is a potential therapeutic target. Here, we describe an RNAi screen for SASP regulators. We identified 50 druggable targets whose knockdown suppresses the inflammatory secretome and differentially affects other SASP components. Among the screen candidates was PTBP1. PTBP1 regulates the alternative splicing of genes involved in intracellular trafficking, such as EXOC7, to control the SASP. Inhibition of PTBP1 prevents the pro-tumorigenic effects of the SASP and impairs immune surveillance without increasing the risk of tumorigenesis. In conclusion, our study identifies SASP inhibition as a powerful and safe therapy against inflammation-driven cancer.

Funding information:
  • Wellcome Trust - U117588499(88499)(United Kingdom)

CAPTURE: In Situ Analysis of Chromatin Composition of Endogenous Genomic Loci by Biotinylated dCas9.

  • Liu X
  • Curr Protoc Mol Biol
  • 2018 Jun 22

Literature context:


Abstract:

Cis-regulatory elements (CREs) play a pivotal role in spatiotemporal control of tissue-specific gene expression, yet the molecular composition of the vast majority of CREs in native chromatin remains unknown. In this article, we describe the clustered regularly interspaced short palindromic repeats (CRISPR) affinity purification in situ of regulatory elements (CAPTURE) approach to simultaneously identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 (dCas9) protein and programmable single guide RNAs (sgRNAs), this approach allows for high-resolution and locus-specific isolation of protein complexes and long-range chromatin looping associated with single copy CREs in mammalian cells. Unbiased analysis of the compositional structure of developmentally regulated or disease-associated CREs identifies new features of transcriptional regulation. Hence, CAPTURE provides a versatile platform to study genomic locus-regulating chromatin composition in a mammalian genome. © 2018 by John Wiley & Sons, Inc.

Funding information:
  • Medical Research Council - MC_U105161047(United Kingdom)
  • NIDDK NIH HHS - K01 DK093543()
  • NIDDK NIH HHS - R01 DK111430()
  • NIDDK NIH HHS - R03 DK101665()
  • NIMH NIH HHS - R01 MH102616()

mfat-1 transgene protects cultured adult neural stem cells against cobalt chloride-mediated hypoxic injury by activating Nrf2/ARE pathways.

  • Yu J
  • J. Neurosci. Res.
  • 2018 Jun 22

Literature context:


Abstract:

Ischemic stroke is a devastating neurological disorder and one of the leading causes of death and serious disability in adults. Adult neural stem cell (NSC) replacement therapy is a promising treatment for both structural and functional neurological recovery. However, for the treatment to work, adult NSCs must be protected against hypoxic-ischemic damage in the ischemic penumbra. In the present study, we aimed to investigate the neuroprotective effects of the mfat-1 transgene on cobalt chloride (CoCl2 )-induced hypoxic-ischemic injury in cultured adult NSCs as well as its underlying mechanisms. The results show that in the CoCl2 -induced hypoxic-ischemic injury model, the mfat-1 transgene enhanced the viability of adult NSCs and suppressed CoCl2 -mediated apoptosis of adult NSCs. Additionally, the mfat-1 transgene promoted the proliferation of NSCs as shown by increased bromodeoxyuridine labeling of adult NSCs. This process was related to the reduction of reactive oxygen species. Quantitative real-time polymerase chain reaction and Western blot analysis revealed a much higher expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream genes (HO-1, NQO-1, GCLC). Taken together, our findings show that the mfat-1 transgene restored the CoCl2 -inhibited viability and proliferation of NSCs by activating nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) signal pathway to inhibit oxidative stress injury. Further investigation of the function of the mfat-1 transgene in adult protective mechanisms may accelerate the development of adult NSC replacement therapy for ischemic stroke.

Biochemical properties of bacterial reverse transcriptase-related (rvt) gene products: multimerization, protein priming, and nucleotide preference.

  • Yushenova IA
  • Curr. Genet.
  • 2018 May 14

Literature context:


Abstract:

Cellular reverse transcriptase-related (rvt) genes represent a novel class of reverse transcriptases (RTs), which are only distantly related to RTs of retrotransposons and retroviruses, but, similarly to telomerase RTs, are immobilized in the genome as single-copy genes. They have been preserved by natural selection throughout the evolutionary history of large taxonomic groups, including most fungi, a few plants and invertebrates, and even certain bacteria, being the only RTs present across different domains of life. Bacterial rvt genes are exceptionally rare but phylogenetically related, consistent with common origin of bacterial rvt genes rather than eukaryote-to-bacteria transfer. To investigate biochemical properties of bacterial RVTs, we conducted in vitro studies of recombinant HaRVT protein from the filamentous gliding bacterium Herpetosiphon aurantiacus (Chloroflexi). Although HaRVT does not utilize externally added standard primer-template combinations, in the presence of divalent manganese, it can polymerize very short products, using dNTPs rather than NTPs, with a strong preference for dCTP incorporation. Furthermore, we investigated the highly conserved N- and C-terminal domains, which distinguish RVT proteins from other RTs. We show that the N-terminal coiled-coil motif, which is present in nearly all RVTs, is responsible for the ability of HaRVT to multimerize in solution, forming up to octamers. The C-terminal domain may be capable of protein priming, which is abolished by site-directed mutagenesis of the catalytic aspartate and greatly reduced in the absence of the conserved tyrosine residues near the C-terminus. The unusual biochemical properties displayed by RVT in vitro will provide the basis for understanding its biological function in vivo.

Funding information:
  • Division of Molecular and Cellular Biosciences - MCB-1121334()
  • Wellcome Trust - BB/G023123/1(United Kingdom)

Treatment with antioxidants ameliorates oxidative damage in a mouse model of propionic acidemia.

  • Rivera-Barahona A
  • Mol. Genet. Metab.
  • 2018 May 18

Literature context:


Abstract:

Oxidative stress contributes to the pathogenesis of propionic acidemia (PA), a life threatening disease caused by the deficiency of propionyl CoA-carboxylase, in the catabolic pathway of branched-chain amino acids, odd-number chain fatty acids and cholesterol. Patients develop multisystemic complications including seizures, extrapyramidal symptoms, basal ganglia deterioration, pancreatitis and cardiomyopathy. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species and oxidative damage, all of which have been documented in patients' samples and in a hypomorphic mouse model. Here we set out to investigate whether treatment with a mitochondria-targeted antioxidant, MitoQ, or with the natural polyphenol resveratrol, which is reported to have antioxidant and mitochondrial activation properties, could ameliorate the altered redox status and its functional consequences in the PA mouse model. The results show that oral treatment with MitoQ or resveratrol decreases lipid peroxidation and the expression levels of DNA repair enzyme OGG1 in PA mouse liver, as well as inducing tissue-specific changes in the expression of antioxidant enzymes. Notably, treatment decreased the cardiac hypertrophy marker BNP that is found upregulated in the PA mouse heart. Overall, the results provide in vivo evidence to justify more in depth investigations of antioxidants as adjuvant therapy in PA.

Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.

  • Huang C
  • Immunity
  • 2018 May 15

Literature context:


Abstract:

Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis and reduces animal survival, at least in part, through suppression of cytosolic phospholipase A2 (cPla2), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish.

Funding information:
  • NIEHS NIH HHS - 2P42 ES04699(United States)

Adipocyte-derived Lysophosphatidylcholine Activates Adipocyte and Adipose Tissue Macrophage Nod-Like Receptor Protein 3 Inflammasomes Mediating Homocysteine-Induced Insulin Resistance.

  • Zhang SY
  • EBioMedicine
  • 2018 May 8

Literature context:


Abstract:

The adipose Nod-like receptor protein 3 (NLRP3) inflammasome senses danger-associated molecular patterns (DAMPs) and initiates insulin resistance, but the mechanisms of adipose inflammasome activation remains elusive. In this study, Homocysteine (Hcy) is revealed to be a DAMP that activates adipocyte NLRP3 inflammasomes, participating in insulin resistance. Hcy-induced activation of NLRP3 inflammasomes were observed in both adipocytes and adipose tissue macrophages (ATMs) and mediated insulin resistance. Lysophosphatidylcholine (lyso-PC) acted as a second signal activator, mediating Hcy-induced adipocyte NLRP3 inflammasome activation. Hcy elevated adipocyte lyso-PC generation in a hypoxia-inducible factor 1 (HIF1)-phospholipase A2 group 16 (PLA2G16) axis-dependent manner. Lyso-PC derived from the Hcy-induced adipocyte also activated ATM NLRP3 inflammasomes in a paracrine manner. This study demonstrated that Hcy activates adipose NLRP3 inflammasomes in an adipocyte lyso-PC-dependent manner and highlights the importance of the adipocyte NLRP3 inflammasome in insulin resistance.

Funding information:
  • NCCIH NIH HHS - P50AT006268(United States)

The arterial calcification is regulated via a miR-204/DNMT3a regulatory circuit both in vitro and in female mice.

  • Lin X
  • Endocrinology
  • 2018 May 29

Literature context:


Abstract:

Arterial calcification is a common cardiovascular disease, which initiates from a process of osteoblastic differentiation of VSMCs. Accumulating evidence has demonstrated that microRNAs play an important role in regulating arterial calcification. Our results showed that miR-204 was significantly down-regulated in calcified human renal arteries from uraemia patients, mice calcified arteries due to 5/6 nephrectomy with a high phosphate diet (5/6 NTP) and VSMCs induced by high phosphate. The overexpression of miR-204 alleviated the osteoblastic differentiation of VSMCs. Bisulphite sequencing PCR (BSP) revealed that CpG sites upstream of miR-204 DNA were hypermethylated in calcified VSMCs, and in mice calcified arteries due to 5/6 NTP, as well as calcified renal artery tissues from uraemia patients. Moreover, increased DNMT3a resulted in the hypermethylation of miR-204 in high phosphate-induced VSMCs, while 5-aza-2'-deoxycytidine could restore the expression of miR-204 in high phosphate-induced VSMCs. Moreover, we found that DNMT3a was the target of miR-204, and the methylation ratio of miR-204 was decreased significantly, meaning that the expression of miR-204 was restored when DNMT3a was knocked down by using DNMT3a siRNA; which resulted in the effect of high phosphate on the calcification of VSMCs being abrogated. Our findings revealed that the progress of arterial calcification is regulated by the miR-204/DNMT3a regulatory circuit.

Funding information:
  • Intramural NIH HHS - HHSN261200800001E(United States)

Calcium-binding protein and some neuropeptides in the retina of Octopus vulgaris: A morpho-histochemical study.

  • Altobelli GG
  • J. Cell. Physiol.
  • 2018 Apr 24

Literature context:


Abstract:

The existence of both calcium-binding proteins (CBPs) and neuropeptides in the retina and brain of various species of vertebrates and invertebrates is well documented. Octopus retina is particularly interesting because it represents a case of convergent evolution. The aim of this study was to characterize the distribution of two CBPs, calretinin and calbindin, in Octopus retina using morphology, in situ hybridization, immunocytochemistry and Western blot. Calretinin-like immunoreactivity was found in the photoreceptor cells, but unexpectedly also in the supporting cells. In situ hybridization and Western blot analysis confirmed these results. No immunoreactivity was found for calbindin. Two neuropeptides, Substance P and calcitonin gene-related peptide (CGRP), as well as neurofilament protein and glial fibrillary acidic protein were also localized in the Octopus retina by immunocytochemistry. Our work provides new insights about calcium-binding proteins and neuropeptide distribution in Octopus retina and suggests a functional role for calretinin, a highly conserved protein, in visual signal transduction of cephalopods.

Funding information:
  • NIAID NIH HHS - T32-AI007419(United States)

Single HIV-1 Imaging Reveals Progression of Infection through CA-Dependent Steps of Docking at the Nuclear Pore, Uncoating, and Nuclear Transport.

  • Francis AC
  • Cell Host Microbe
  • 2018 Apr 11

Literature context:


Abstract:

The HIV-1 core consists of capsid proteins (CA) surrounding viral genomic RNA. After virus-cell fusion, the core enters the cytoplasm and the capsid shell is lost through uncoating. CA loss precedes nuclear import and HIV integration into the host genome, but the timing and location of uncoating remain unclear. By visualizing single HIV-1 infection, we find that CA is required for core docking at the nuclear envelope (NE), whereas early uncoating in the cytoplasm promotes proteasomal degradation of viral complexes. Only docked cores exhibiting accelerated loss of CA at the NE enter the nucleus. Interestingly, a CA mutation (N74D) altering virus engagement of host factors involved in nuclear transport does not alter the uncoating site at the NE but reduces the nuclear penetration depth. Thus, CA protects HIV-1 complexes from degradation, mediates docking at the nuclear pore before uncoating, and determines the depth of nuclear penetration en route to integration.

Funding information:
  • NCI NIH HHS - P30 CA008748(United States)
  • NIAID NIH HHS - R01 AI129862()
  • NIGMS NIH HHS - R01 GM054787()

Participation of the dentate-rubral pathway in the kindling model of epilepsy.

  • Hernández-Cerón M
  • J. Neurosci. Res.
  • 2018 Mar 12

Literature context:


Abstract:

Lesions of the cerebellar dentate nucleus (DN) reduce the after-discharge duration induced by repetitive kindling stimulation and decrease seizures to a lower rank according to Racine's scale. The DN sends cholinergic and glutamatergic fibers to the red nucleus (RN), which is composed of glutamatergic and GABAergic cells. To test the participation of these neurotransmitters in seizures, we compared the levels of glutamate and gamma-aminobutyric acid (GABA) at the RN in a control condition, a kindled stage, and a kindled stage followed by DN lesions. We found that the kindled stage was associated with significant reductions in glutamate and GABA in the RN and that the lesions of the DN in kindled rats reversed the severity of seizures and restored the GABA levels. GAD65 , a GABA-synthesizing enzyme, was increased in kindled rats and decreased after DN lesions. GAD65 commonly appears localized at nerve terminals and synapses, and it is only activated when GABA neurotransmission occurs. Thus, it is possible that the increased expression of GAD65 found in kindled rats could be due to an exacerbated demand for GABA due to kindled seizures. It is known that GABA maintains the inhibitory tone that counterbalances neuronal excitation. The decreased expression of GAD65 found after the DN lesions indicated that the GABA-synthesizing enzyme was no longer required once it eliminated the excitatory glutamate input to the RN. We thus conclude that DN lesions and their consequent biochemical changes are capable of decreasing the generalized seizures induced by kindling stimulation. © 2016 Wiley Periodicals, Inc.

Funding information:
  • NINDS NIH HHS - T32 NS007220(United States)

Reshaping of the Dendritic Cell Chromatin Landscape and Interferon Pathways during HIV Infection.

  • Johnson JS
  • Cell Host Microbe
  • 2018 Mar 14

Literature context:


Abstract:

Myeloid dendritic cells (DCs) have the innate capacity to sense pathogens and orchestrate immune responses. However, DCs do not mount efficient immune responses to HIV-1, primarily due to restriction of virus reverse transcription, which prevents accumulation of viral cDNA and limits its detection through the cGAS-STING pathway. By allowing reverse transcription to proceed, we find that DCs detect HIV-1 in distinct phases, before and after virus integration. Blocking integration suppresses, but does not abolish, activation of the transcription factor IRF3, downstream interferon (IFN) responses, and DC maturation. Consistent with two stages of detection, HIV-1 "primes" chromatin accessibility of innate immune genes before and after integration. Once primed, robust IFN responses can be unmasked by agonists of the innate adaptor protein, MyD88, through a process that requires cGAS, STING, IRF3, and nuclear factor κB. Thus, HIV-1 replication increases material available for sensing, and discrete inflammatory inputs tune cGAS signaling to drive DC maturation.

Funding information:
  • NIAID NIH HHS - R21 AI084633()
  • NINDS NIH HHS - R21 NS050463(United States)

Losmapimod Overcomes Gefitinib Resistance in Non-small Cell Lung Cancer by Preventing Tetraploidization.

  • Yeung YT
  • EBioMedicine
  • 2018 Feb 6

Literature context:


Abstract:

The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer (NSCLC). Constitutively active EGFR mutations, including in-frame deletion in exon 19 and L858R point mutation in exon 21, contribute about 90% of all EGFR-activating mutations in NSCLC. Although oral EGFR-tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, show dramatic clinical efficacy with significantly prolonged progression-free survival in patients harboring these EGFR-activating mutations, most of these patients will eventually develop acquired resistance. Researchers have recently named genomic instability as one of the hallmarks of cancer. Genomic instability usually involves a transient phase of polyploidization, in particular tetraploidization. Tetraploid cells can undergo asymmetric cell division or chromosome loss, leading to tumor heterogeneity and multidrug resistance. Therefore, identification of signaling pathways involved in tetraploidization is crucial in overcoming drug resistance. In our present study, we found that gefitinib could activate YAP-MKK3/6-p38 MAPK-STAT3 signaling and induce tetraploidization in gefitinib-resistance cells. Using p38 MAPK inhibitors, SB203580 and losmapimod, we could eliminate gefitinib-induced tetraploidization and overcome gefitinib-resistance. In addition, shRNA approach to knockdown p38α MAPK could prevent tetraploidy formation and showed significant inhibition of cancer cell growth. Finally, in an in vivo study, losmapimod could successfully overcome gefitinib resistance using an in-house established patient-derived xenograft (PDX) mouse model. Overall, these findings suggest that losmapimod could be a potential clinical agent to overcome gefitinib resistance in NSCLC.

Funding information:
  • NIA NIH HHS - R01 AG010668(United States)

Induction of Stress Signaling In Vitro and Suppression of Gonadotropin Secretion by Free Fatty Acids in Female Mouse Gonadotropes.

  • Li S
  • Endocrinology
  • 2018 Feb 1

Literature context:


Abstract:

An emerging body of evidence supports the concept that the pituitary is a site for integration of multiple physiological and metabolic signals that inform and modulate endocrine pathways. Multiple endocrine mediators of energy balance and adiposity are known to impinge on the neuroendocrine axis regulating reproduction. Observations in humans show that obesity is correlated with decreased gonadotropin secretion, and studies have also suggested that pituitary sensitivity to stimulation by gonadotropin-releasing hormone (GnRH) is decreased in obese individuals. Free fatty acids are a potential mediator of adiposity and energy balance, but their impact as an endocrine modulator of pituitary function has not been closely examined. We evaluated the impact of free fatty acids on a pituitary gonadotrope cell line and in primary pituitary cultures of female mice. We show that increasing physiologically relevant doses of the monounsaturated ω-9 fatty acid oleate induces cellular stress and increases production of reactive oxygen species in a mouse gonadotrope cell line. In contrast, the unsaturated ω-3 α-linolenic and ω-6 linoleic fatty acids do not have this effect. Additionally, oleate can activate immediate-early gene expression independent of GnRH stimulation but has a negative impact on GnRH induction and expression of the gonadotropin subunit gene Lhb. Further, oleate suppresses gonadotropin secretion in response to pulsatile stimulation by GnRH. These results indicate that free fatty acids can directly alter gonadotropin gene expression and secretion in response to GnRH and may provide a link between energy sensing and reproduction.

Funding information:
  • Intramural NIH HHS - 2R01AI041699-12(United States)
  • NICHD NIH HHS - P50 HD012303()
  • NICHD NIH HHS - R01 HD037568()
  • NICHD NIH HHS - R01 HD082567()
  • NICHD NIH HHS - T32 HD007203()

N6-Methyladenosines Modulate A-to-I RNA Editing.

  • Xiang JF
  • Mol. Cell
  • 2018 Jan 4

Literature context:


Abstract:

N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) editing are two of the most abundant RNA modifications, both at adenosines. Yet, the interaction of these two types of adenosine modifications is largely unknown. Here we show a global A-to-I difference between m6A-positive and m6A-negative RNA populations. Both the presence and extent of A-to-I sites in m6A-negative RNA transcripts suggest a negative correlation between m6A and A-to-I. Suppression of m6A-catalyzing enzymes results in global A-to-I RNA editing changes. Further depletion of m6A modification increases the association of m6A-depleted transcripts with adenosine deaminase acting on RNA (ADAR) enzymes, resulting in upregulated A-to-I editing on the same m6A-depleted transcripts. Collectively, the effect of m6A on A-to-I suggests a previously underappreciated interplay between two distinct and abundant RNA modifications, highlighting a complex epitranscriptomic landscape.

Replication Stress Shapes a Protective Chromatin Environment across Fragile Genomic Regions.

  • Kim J
  • Mol. Cell
  • 2018 Jan 4

Literature context:


Abstract:

Recent integrative epigenome analyses highlight the importance of functionally distinct chromatin states for accurate cell function. How these states are established and maintained is a matter of intense investigation. Here, we present evidence for DNA damage as an unexpected means to shape a protective chromatin environment at regions of recurrent replication stress (RS). Upon aberrant fork stalling, DNA damage signaling and concomitant H2AX phosphorylation coordinate the FACT-dependent deposition of macroH2A1.2, a histone variant that promotes DNA repair by homologous recombination (HR). MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from RS-induced DNA damage. Consequently, replicating primary cells steadily accrue macroH2A1.2 at fragile regions, whereas macroH2A1.2 loss in these cells triggers DNA damage signaling-dependent senescence, a hallmark of RS. Altogether, our findings demonstrate that recurrent DNA damage contributes to the chromatin landscape to ensure the epigenomic integrity of dividing cells.

Funding information:
  • Intramural NIH HHS - ZIA BC011282-01()
  • NIGMS NIH HHS - R01 GM073046(United States)

The Histamine H1 Receptor Participates in the Increased Dorsal Telencephalic Neurogenesis in Embryos from Diabetic Rats.

  • Solís KH
  • Front Neurosci
  • 2018 Jan 10

Literature context:


Abstract:

Increased neuron telencephalic differentiation during deep cortical layer formation has been reported in embryos from diabetic mice. Transitory histaminergic neurons within the mesencephalon/rhombencephalon are responsible for fetal histamine synthesis during development, fibers from this system arrives to the frontal and parietal cortex at embryo day (E) 15. Histamine is a neurogenic factor for cortical neural stem cells in vitro through H1 receptor (H1R) which is highly expressed during corticogenesis in rats and mice. Furthermore, in utero administration of an H1R antagonist, chlorpheniramine, decreases the neuron markers microtubuline associated protein 2 (MAP2) and forkhead box protein 2. Interestingly, in the diabetic mouse model of diabetes induced with streptozotocin, an increase in fetal neurogenesis in terms of MAP2 expression in the telencephalon is reported at E11.5. Because of the reported effects on cortical neuron differentiation of maternal diabetes in one hand and of histamine in the other, here the participation of histamine and H1R on the increased dorsal telencephalic neurogenesis was explored. First, the increased neurogenesis in the dorsal telencephalon at E14 in diabetic rats was corroborated by immunohistochemistry and Western blot. Then, changes during corticogenesis in the level of histamine was analyzed by ELISA and in H1R expression by qRT-PCR and Western blot and, finally, we tested H1R participation in the increased dorsal telencephalic neurogenesis by the systemic administration of chlorpheniramine. Our results showed a significant increase of histamine at E14 and in the expression of the receptor at E12. The administration of chlorpheniramine to diabetic rats at E12 prevented the increased expression of βIII-tubulin and MAP2 mRNAs (neuron markers) and partially reverted the increased level of MAP2 protein at E14, concluding that H1R have an important role in the increased neurogenesis within the dorsal telencephalon of embryos from diabetic rats. This study opens new perspective on the participation of HA and H1R receptor in early corticogenesis in health and disease.

Funding information:
  • NIGMS NIH HHS - GM082014(United States)

Runt-Related Transcription Factor 2 (Runx2) Is Responsible for Galectin-3 Overexpression in Human Thyroid Carcinoma.

  • Kaptan E
  • J. Cell. Biochem.
  • 2018 Jan 2

Literature context:


Abstract:

Runx2 promotes metastatic ability of cancer cells by directly activating some of the mediators regarding malignancy. Galectin-3 (Gal-3) extensively expressed in normal and transformed cells and it is responsible for many cellular processes. In this study, we aimed to investigate whether there is any relationship between runx2 transcription factor and regulation of galectin-3 expression in different human thyroid carcinoma cell lines. To show effects of runx2 transcription factor on gal-3 expression, we developed runx2 knockdown model in the thyroid carcinoma cell lines; anaplastic 8505C and 8305C and, papillary TPC-1 and follicular FTC-133 by using siRNA transfection. We analyzed the protein expressions and mRNA levels of gal-3 and MMP2/9 in the runx2-silenced cell lines using Western blotting, qPCR, and fluorescent microscopy. Our results showed that mRNA expression levels of gal-3 and MMP2/9 were downregulated in runx2-silenced cell lines. In this investigation, we revealed that regulation of gal-3 expression was strongly correlated with runx2 transcription factor in human thyroid carcinoma. Considering the contribution of human gal-3 in collaboration with MMP2/9 to the malignant characters of many cancers, regulation of their expressions through runx2 seems like one of the key regulatory mechanism for malignant potential of human thyroid carcinoma. Accordingly, runx2 transcription factor inhibitors can be a potential target in order to prevent gal-3 mediated malignancy of human thyroid carcinoma. J. Cell. Biochem. 118: 3911-3919, 2017. © 2017 Wiley Periodicals, Inc.

Intra-arterial transplantation of human bone marrow mesenchymal stem cells (hBMMSCs) improves behavioral deficits and alters gene expression in rodent stroke model.

  • Vibhuti
  • J. Neurochem.
  • 2017 Dec 20

Literature context:


Abstract:

Stroke is a multi-factorial polygenic disease and is a major cause of death and adult disability. Administration of bone marrow stem cells protects ischemic rat brain by facilitating recovery of neurological functions. But the molecular mechanism of stem cells action and their effect on gene expression is not well explored. In this study, we have transplanted 1 × 106 human bone marrow mesenchymal stem cells (hBMMSCs) in middle cerebral artery occluded (MCAo) adult male Wistar rats through intracarotid artery route at 24 h after surgery. Motor behavioral tests (rotarod and open field) were performed to assess the changes in motor functions at day 0 and day1, 4, 8 and 14. The expression of studied genes at mRNA and protein level was quantified by using Q-PCR and western blotting, respectively. Further, we have assessed the methylation pattern of promoter of these genes by using methylation-specific PCR. Data were analyzed statistically and correlated. A significant improvement in behavioral deficits was observed in stem cells treated group after 14th day post stroke. Significantly (p < 0.05) increased mRNA and protein levels of brain derived neurotrophic factor and ANP genes in hBMMSCs treated group along with decrease in methylation level at their promoter was observed. On the other hand, significantly decreased mRNA and protein level of TSP1 and WNK1 in hBMMSCs treated group was observed. In conclusion, hBMMSCs administration significantly improves the behavioral deficits by improving motor and locomotor coordination. The promoter of TSP1 and WNK1 genes was found to be hyper-methylated in hBMMSCs group resulting in their decreased expression while the promoter of ANP and brain derived neurotrophic factor was found to be hypo-methylated. This study might shed a light on how hBMMSCs affect the gene expression by modulating methylation status.

Cystic Fibrosis Transmembrane Conductance Regulator Attaches Tumor Suppressor PTEN to the Membrane and Promotes Anti Pseudomonas aeruginosa Immunity.

  • Riquelme SA
  • Immunity
  • 2017 Dec 19

Literature context:


Abstract:

The tumor suppressor PTEN controls cell proliferation by regulating phosphatidylinositol-3-kinase (PI3K) activity, but the participation of PTEN in host defense against bacterial infection is less well understood. Anti-inflammatory PI3K-Akt signaling is suppressed in patients with cystic fibrosis (CF), a disease characterized by hyper-inflammatory responses to airway infection. We found that Ptenl-/- mice, which lack the NH2-amino terminal splice variant of PTEN, were unable to eradicate Pseudomonas aeruginosa from the airways and could not generate sufficient anti-inflammatory PI3K activity, similar to what is observed in CF. PTEN and the CF transmembrane conductance regulator (CFTR) interacted directly and this interaction was necessary to position PTEN at the membrane. CF patients under corrector-potentiator therapy, which enhances CFTR transport to the membrane, have increased PTEN amounts. These findings suggest that improved CFTR trafficking could enhance P. aeruginosa clearance from the CF airway by activating PTEN-mediated anti-bacterial responses and might represent a therapeutic strategy.

Funding information:
  • NCI NIH HHS - R01 CA184016()
  • NCRR NIH HHS - S10 RR027050()
  • NHLBI NIH HHS - R35 HL135800()
  • NIDDK NIH HHS - R01 DK054890-11(United States)

EPA blocks TNF-α-induced inhibition of sugar uptake in Caco-2 cells via GPR120 and AMPK.

  • Castilla-Madrigal R
  • J. Cell. Physiol.
  • 2017 Dec 12

Literature context:


Abstract:

The aim of the present work was to investigate in Caco-2 cells whether eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid, could block the inhibitory effect of tumor necrosis factor-α (TNF-α) on sugar transport, and identify the intracellular signaling pathways involved. After pre-incubation of the Caco-2 cells with TNF-α and EPA for 1 hr, EPA prevented the inhibitory effect of the cytokine on α-methyl-d-glucose (αMG) uptake (15 min) and on SGLT1 expression at the brush border membrane, measured by Western blot. The ERK1/2 inhibitor PD98059 and the AMPK activator AICAR also prevented the inhibitory effect of TNF-α on both αMG uptake and SGLT1 expression. Interestingly, the AMPK inhibitor, Compound C, abolished the ability of EPA to prevent TNF-α-induced reduction of sugar uptake and transporter expression. The GPR120 antagonist, AH7614, also blocked the preventive effect of EPA on TNF-α-induced decrease of αMG uptake and AMPK phosphorylation. In summary, TNF-α inhibits αMG uptake by decreasing SGLT1 expression in the brush border membrane through the activation of ERK1/2 pathway. EPA prevents the inhibitory effect of TNF-α through the involvement of GPR120 and AMPK activation.

The ameliorative effect of fluoxetine on neuroinflammation induced by sleep deprivation.

  • Xia M
  • J. Neurochem.
  • 2017 Dec 9

Literature context:


Abstract:

It is well known that sleep disorders are harmful to people's health and performance, and growing evidence suggests that sleep deprivation (SD) can trigger neuroinflammation in the brain. The nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome is reported to be relevant to the neuroinflammation induced by SD, but the regulatory signaling that governs the NLRP3 inflammasome in SD is still unknown. Meanwhile, whether the regulatory action of antidepressants in astrocytes could affect the neuroinflammation induced by SD also remains obscure. In this study, we were the first to discover that the antidepressant fluoxetine, a type of specific serotonin reuptake inhibitor widely used in clinical practice, could suppress the neuroinflammation and neuronal apoptosis induced by SD. The main findings from this study are as follows: (i) SD stimulated the expression of activated NLRP3 inflammasomes and the maturation of IL-1β/18 via suppressing the phosphorylation of STAT3 in astrocytes; (ii) SD decreased the activation of AKT and stimulated the phosphorylation of GSK-3β, which inhibited the phosphorylation of STAT3; (iii) the NLRP3 inflammasome expression stimulated by SD was partly mediated by the P2X7 receptor; (iv) an agonist of STAT3 could significantly abolish the expression of NLRP3 inflammasomes induced by an agonist of the P2X7 receptor in primary cultured astrocytes; (v) the administration of fluoxetine could reverse the stimulation of NLRP3 inflammasome expression and function by SD through elevating the activation of STAT3. In conclusion, our present research suggests the promising possibility that fluoxetine could ameliorate the neuronal impairment induced by SD.

Funding information:
  • Canadian Institutes of Health Research - 201592(Canada)

Neuroplastin and Basigin Are Essential Auxiliary Subunits of Plasma Membrane Ca2+-ATPases and Key Regulators of Ca2+ Clearance.

  • Schmidt N
  • Neuron
  • 2017 Nov 15

Literature context:


Abstract:

Plasma membrane Ca2+-ATPases (PMCAs), a family of P-type ATPases, extrude Ca2+ ions from the cytosol to the extracellular space and are considered to be key regulators of Ca2+ signaling. Here we show by functional proteomics that native PMCAs are heteromeric complexes that are assembled from two pore-forming PMCA1-4 subunits and two of the single-span membrane proteins, either neuroplastin or basigin. Contribution of the two Ig domain-containing proteins varies among different types of cells and along postnatal development. Complex formation of neuroplastin or basigin with PMCAs1-4 occurs in the endoplasmic reticulum and is obligatory for stability of the PMCA proteins and for delivery of PMCA complexes to the surface membrane. Knockout and (over)-expression of both neuroplastin and basigin profoundly affect the time course of PMCA-mediated Ca2+ transport, as well as submembraneous Ca2+ concentrations under steady-state conditions. Together, these results establish neuroplastin and basigin as obligatory auxiliary subunits of native PMCAs and key regulators of intracellular Ca2+ concentration.

Transgenic Expression of the Vitamin D Receptor Restricted to the Ileum, Cecum, and Colon of Vitamin D Receptor Knockout Mice Rescues Vitamin D Receptor-Dependent Rickets.

  • Dhawan P
  • Endocrinology
  • 2017 Nov 1

Literature context:


Abstract:

Although the intestine plays the major role in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action on calcium homeostasis, the mechanisms involved remain incompletely understood. The established model of 1,25(OH)2D3-regulated intestinal calcium absorption postulates a critical role for the duodenum. However, the distal intestine is where 70% to 80% of ingested calcium is absorbed. To test directly the role of 1,25(OH)2D3 and the vitamin D receptor (VDR) in the distal intestine, three independent knockout (KO)/transgenic (TG) lines expressing VDR exclusively in the ileum, cecum, and colon were generated by breeding VDR KO mice with TG mice expressing human VDR (hVDR) under the control of the 9.5-kb caudal type homeobox 2 promoter. Mice from one TG line (KO/TG3) showed low VDR expression in the distal intestine (<50% of the levels observed in KO/TG1, KO/TG2, and wild-type mice). In the KO/TG mice, hVDR was not expressed in the duodenum, jejunum, kidney, or other tissues. Growth arrest, elevated parathyroid hormone level, and hypocalcemia of the VDR KO mice were prevented in mice from KO/TG lines 1 and 2. Microcomputed tomography analysis revealed that the expression of hVDR in the distal intestine of KO/TG1 and KO/TG2 mice rescued the bone defects associated with systemic VDR deficiency, including growth plate abnormalities and altered trabecular and cortical parameters. KO/TG3 mice showed rickets, but less severely than VDR KO mice. These findings show that expression of VDR exclusively in the distal intestine can prevent abnormalities in calcium homeostasis and bone mineralization associated with systemic VDR deficiency.

Funding information:
  • NIGMS NIH HHS - SC2 GM095428(United States)

Metabolic, Reproductive, and Neurologic Abnormalities in Agpat1-Null Mice.

  • Agarwal AK
  • Endocrinology
  • 2017 Nov 1

Literature context:


Abstract:

Defects in the biosynthesis of phospholipids and neutral lipids are associated with cell membrane dysfunction, disrupted energy metabolism, and diseases including lipodystrophy. In these pathways, the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) enzymes transfer a fatty acid to the sn-2 carbon of sn-1-acylglycerol-3-phosphate (lysophosphatidic acid) to form sn-1, 2-acylglycerol-3-phosphate [phosphatidic acid (PA)]. PA is a precursor for key phospholipids and diacylglycerol. AGPAT1 and AGPAT2 are highly homologous isoenzymes that are both expressed in adipocytes. Genetic defects in AGPAT2 cause congenital generalized lipodystrophy, indicating that AGPAT1 cannot compensate for loss of AGPAT2 in adipocytes. To further explore the physiology of AGPAT1, we characterized a loss-of-function mouse model (Agpat1-/-). The majority of Agpat1-/- mice died before weaning and had low body weight and low plasma glucose levels, independent of plasma insulin and glucagon levels, with reduced percentage of body fat but not generalized lipodystrophy. These mice also had decreased hepatic messenger RNA expression of Igf-1 and Foxo1, suggesting a decrease in gluconeogenesis. In male mice, sperm development was impaired, with a late meiotic arrest near the onset of round spermatid production, and gonadotropins were elevated. Female mice showed oligoanovulation yet retained responsiveness to gonadotropins. Agpat1-/- mice also demonstrated abnormal hippocampal neuron development and developed audiogenic seizures. In summary, Agpat1-/- mice developed widespread disturbances of metabolism, sperm development, and neurologic function resulting from disrupted phospholipid homeostasis. AGPAT1 appears to serve important functions in the physiology of multiple organ systems. The Agpat1-deficient mouse provides an important model in which to study the contribution of phospholipid and triacylglycerol synthesis to physiology and diseases.

Funding information:
  • Canadian Institutes of Health Research - (Canada)
  • NICHD NIH HHS - R01 HD053889()
  • NIDDK NIH HHS - R01 DK054387()
  • NIH HHS - R24 OD011108()

Enhanced apoptosis from early physical exercise rehabilitation following ischemic stroke.

  • Li F
  • J. Neurosci. Res.
  • 2017 Nov 16

Literature context:


Abstract:

The effectiveness of the rehabilitative benefits of physical exercise appears to be contingent upon when the exercise is initiated after stroke. The present study assessed the hypothesis that very early exercise increases the extent of apoptotic cell death via increased expression of proapoptotic proteins in a rat stroke model. Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 2 hr using an intraluminal filament and assigned to four nonexercise and three exercise groups. Exercise on a Rota-Rod was initiated for 30 min at 6 hr (considered very early), at 24 hr (early), and at 3 days (relatively late) after reperfusion. At 24 hr after exercise, apoptotic cell death was determined. At 3 and 24 hr after exercise, the expression of pro- and antiapoptotic proteins was evaluated through Western blotting. As expected, ischemic stroke significantly increased the levels of apoptotic cell death. Compared with the stroke group without exercise, apoptotic cell death was further increased (P < 0.05) at 6 hr but not at 24 hr or 3 days with exercise. This exacerbated cell injury was associated with increased expression of proapoptotic proteins (BAX and caspase-3). The expression of Bcl-2, an antiapoptotic protein, was not affected by exercise. In ischemic stroke, apoptotic cell death was enhanced by very early exercise in association with increased expression of proapoptotic proteins. These results shed light on the time-sensitive effect of exercise in poststroke rehabilitation. © 2016 Wiley Periodicals, Inc.

NRF1 Is an ER Membrane Sensor that Is Central to Cholesterol Homeostasis.

  • Widenmaier SB
  • Cell
  • 2017 Nov 16

Literature context:


Abstract:

Cholesterol is a critical nutrient requiring tight constraint in the endoplasmic reticulum (ER) due to its uniquely challenging biophysical properties. While the mechanisms by which the ER defends against cholesterol insufficiency are well described, it remains unclear how the ER senses and effectively defends against cholesterol excess. Here, we identify the ER-bound transcription factor nuclear factor erythroid 2 related factor-1, Nrf1/Nfe2L1, as a critical mediator of this process. We show that Nrf1 directly binds to and specifically senses cholesterol in the ER through a defined domain and that cholesterol regulates Nrf1 turnover, processing, localization, and activity. In Nrf1 deficiency, in vivo cholesterol challenges induce massive hepatic cholesterol accumulation and damage, which is rescued by replacing Nrf1 exogenously. This Nrf1-mediated mechanism involves the suppression of CD36-driven inflammatory signaling and derepression of liver X receptor activity. These findings reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excess cellular cholesterol.

Funding information:
  • NINDS NIH HHS - F31NS077750(United States)

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

  • Bar-Peled L
  • Cell
  • 2017 Oct 19

Literature context:


Abstract:

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.

Rac1 Dosage Is Crucial for Normal Endochondral Bone Growth.

  • Suzuki D
  • Endocrinology
  • 2017 Oct 1

Literature context:


Abstract:

Rac1, a member of the small Rho GTPase family, plays multiple cellular roles. Studies of mice conditionally lacking Rac1 have revealed essential roles for Rac1 in various tissues, including cartilage and limb mesenchyme, where Rac1 loss produces dwarfism and long bone shortening. To gain further insight into the role of Rac1 in skeletal development, we have used transgenic mouse lines to express a constitutively active (ca) Rac1 mutant protein in a Cre recombinase-dependent manner. Overexpression of caRac1 in limb bud mesenchyme or chondrocytes leads to reduced body weight and shorter bones compared with control mice. Histological analysis of growth plates showed that caRac1;Col2-Cre mice displayed ectopic hypertrophic chondrocytes in the proliferative zone and enlarged hypertrophic zones. These mice also displayed a reduced proportion of proliferating cell nuclear antigen-positive cells in the proliferative zone and nuclear β-catenin localization in the ectopic hypertrophic chondrocytes. Importantly, overexpression of caRac1 partially rescued the phenotypes of Rac1fl/fl;Col2-Cre and Rac1fl/fl;Prx1-Cre conditional knockout mice, including body weight, bone length, and growth plate disorganization. These results suggest that tight regulation of Rac1 activity is necessary for normal cartilage development.

Connexin 43-Mediated Astroglial Metabolic Networks Contribute to the Regulation of the Sleep-Wake Cycle.

  • Clasadonte J
  • Neuron
  • 2017 Sep 13

Literature context:


Abstract:

Astrocytes produce and supply metabolic substrates to neurons through gap junction-mediated astroglial networks. However, the role of astroglial metabolic networks in behavior is unclear. Here, we demonstrate that perturbation of astroglial networks impairs the sleep-wake cycle. Using a conditional Cre-Lox system in mice, we show that knockout of the gap junction subunit connexin 43 in astrocytes throughout the brain causes excessive sleepiness and fragmented wakefulness during the nocturnal active phase. This astrocyte-specific genetic manipulation silenced the wake-promoting orexin neurons located in the lateral hypothalamic area (LHA) by impairing glucose and lactate trafficking through astrocytic networks. This global wakefulness instability was mimicked with viral delivery of Cre recombinase to astrocytes in the LHA and rescued by in vivo injections of lactate. Our findings propose a novel regulatory mechanism critical for maintaining normal daily cycle of wakefulness and involving astrocyte-neuron metabolic interactions.

Hyperandrogenemia Induced by Letrozole Treatment of Pubertal Female Mice Results in Hyperinsulinemia Prior to Weight Gain and Insulin Resistance.

  • Skarra DV
  • Endocrinology
  • 2017 Sep 1

Literature context:


Abstract:

Women with polycystic ovary syndrome (PCOS) diagnosed with hyperandrogenism and ovulatory dysfunction have an increased risk of developing metabolic disorders, including type 2 diabetes and cardiovascular disease. We previously developed a model that uses letrozole to elevate endogenous testosterone levels in female mice. This model has hallmarks of PCOS, including hyperandrogenism, anovulation, and polycystic ovaries, as well as increased abdominal adiposity and glucose intolerance. In the current study, we further characterized the metabolic dysfunction that occurs after letrozole treatment to determine whether this model represents a PCOS-like metabolic phenotype. We focused on whether letrozole treatment results in altered pancreatic or liver function as well as insulin resistance. We also investigated whether hyperinsulinemia occurs secondary to weight gain and insulin resistance in this model or if it can occur independently. Our study demonstrated that letrozole-treated mice developed hyperinsulinemia after 1 week of treatment and without evidence of insulin resistance. After 2 weeks of letrozole treatment, mice became significantly heavier than placebo mice, demonstrating that weight gain was not required to develop hyperinsulinemia. After 5 weeks of letrozole treatment, mice exhibited blunted glucose-stimulated insulin secretion, insulin resistance, and impaired insulin-induced phosphorylation of AKT in skeletal muscle. Moreover, letrozole-treated mice exhibited dyslipidemia after 5 weeks of treatment but no evidence of hepatic disease. Our study demonstrated that the letrozole-induced PCOS mouse model exhibits multiple features of the metabolic dysregulation observed in obese, hyperandrogenic women with PCOS. This model will be useful for mechanistic studies investigating how hyperandrogenemia affects metabolism in females.

Funding information:
  • NICHD NIH HHS - F32 HD074414()
  • NICHD NIH HHS - P50 HD012303()
  • NICHD NIH HHS - P50 HD028934()
  • NICHD NIH HHS - R01 HD067448()
  • NICHD NIH HHS - T32 HD007203()
  • NIGMS NIH HHS - K12 GM068524()

The Short Isoform of BRD4 Promotes HIV-1 Latency by Engaging Repressive SWI/SNF Chromatin-Remodeling Complexes.

  • Conrad RJ
  • Mol. Cell
  • 2017 Sep 21

Literature context:


Abstract:

BET proteins commonly activate cellular gene expression, yet inhibiting their recruitment paradoxically reactivates latent HIV-1 transcription. Here we identify the short isoform of BET family member BRD4 (BRD4S) as a corepressor of HIV-1 transcription. We found that BRD4S was enriched in chromatin fractions of latently infected T cells, and it was more rapidly displaced from chromatin upon BET inhibition than the long isoform. BET inhibition induced marked nucleosome remodeling at the latent HIV-1 promoter, which was dependent on the activity of BRG1-associated factors (BAF), an SWI/SNF chromatin-remodeling complex with known repressive functions in HIV-1 transcription. BRD4S directly bound BRG1, a catalytic subunit of BAF, via its bromodomain and extraterminal (ET) domain, and this isoform was necessary for BRG1 recruitment to latent HIV-1 chromatin. Using chromatin immunoprecipitation sequencing (ChIP-seq) combined with assay for transposase-accessible chromatin coupled to high-throughput sequencing (ATAC-seq) data, we found that the latent HIV-1 promoter phenotypically resembles endogenous long terminal repeat (LTR) sequences, pointing to a select role of BRD4S-BRG1 complexes in genomic silencing of invasive retroelements.

Funding information:
  • NINDS NIH HHS - R01 NS083898(United States)

Cell-Intrinsic Glycogen Metabolism Supports Early Glycolytic Reprogramming Required for Dendritic Cell Immune Responses.

  • Thwe PM
  • Cell Metab.
  • 2017 Sep 5

Literature context:


Abstract:

Dendritic cell (DC) activation by Toll-like receptor (TLR) agonists causes rapid glycolytic reprogramming that is required to meet the metabolic demands of their immune activation. Recent efforts in the field have identified an important role for extracellular glucose sourcing to support DC activation. However, the contributions of intracellular glucose stores to these processes have not been well characterized. We demonstrate that DCs possess intracellular glycogen stores and that cell-intrinsic glycogen metabolism supports the early effector functions of TLR-activated DCs. Inhibition of glycogenolysis significantly attenuates TLR-mediated DC maturation and impairs their ability to initiate lymphocyte activation. We further report that DCs exhibit functional compartmentalization of glucose- and glycogen-derived carbons, where these substrates preferentially contribute to distinct metabolic pathways. This work provides novel insights into nutrient homeostasis in DCs, demonstrating that differential utilization of glycogen and glucose metabolism regulates their optimal immune function.

Funding information:
  • NIGMS NIH HHS - P30 GM118228()

Ubiquitination-dependent control of sexual differentiation in fission yeast.

  • Simonetti F
  • Elife
  • 2017 Aug 25

Literature context:


Abstract:

In fission yeast, meiosis-specific transcripts are selectively eliminated during vegetative growth by the combined action of the YTH-family RNA-binding protein Mmi1 and the nuclear exosome. Upon nutritional starvation, the master regulator of meiosis Mei2 inactivates Mmi1, thereby allowing expression of the meiotic program. Here, we show that the E3 ubiquitin ligase subunit Not4/Mot2 of the evolutionarily conserved Ccr4-Not complex, which associates with Mmi1, promotes suppression of meiotic transcripts expression in mitotic cells. Our analyses suggest that Mot2 directs ubiquitination of Mei2 to preserve the activity of Mmi1 during vegetative growth. Importantly, Mot2 is not involved in the constitutive pathway of Mei2 turnover, but rather plays a regulatory role to limit its accumulation or inhibit its function. We propose that Mmi1 recruits the Ccr4-Not complex to counteract its own inhibitor Mei2, thereby locking the system in a stable state that ensures the repression of the meiotic program by Mmi1.

Dendritic Cells but Not Macrophages Sense Tumor Mitochondrial DNA for Cross-priming through Signal Regulatory Protein α Signaling.

  • Xu MM
  • Immunity
  • 2017 Aug 15

Literature context:


Abstract:

Inhibition of cytosolic DNA sensing represents a strategy that tumor cells use for immune evasion, but the underlying mechanisms are unclear. Here we have shown that CD47-signal regulatory protein α (SIRPα) axis dictates the fate of ingested DNA in DCs for immune evasion. Although macrophages were more potent in uptaking tumor DNA, increase of DNA sensing by blocking the interaction of SIRPα with CD47 preferentially occurred in dendritic cells (DCs) but not in macrophages. Mechanistically, CD47 blockade enabled the activation of NADPH oxidase NOX2 in DCs, which in turn inhibited phagosomal acidification and reduced the degradation of tumor mitochondrial DNA (mtDNA) in DCs. mtDNA was recognized by cyclic-GMP-AMP synthase (cGAS) in the DC cytosol, contributing to type I interferon (IFN) production and antitumor adaptive immunity. Thus, our findings have demonstrated how tumor cells inhibit innate sensing in DCs and suggested that the CD47-SIRPα axis is critical for DC-driven antitumor immunity.

Funding information:
  • NCI NIH HHS - R01 CA134563()
  • NCI NIH HHS - R01 CA141975()

GATA Factor-Regulated Samd14 Enhancer Confers Red Blood Cell Regeneration and Survival in Severe Anemia.

  • Hewitt KJ
  • Dev. Cell
  • 2017 Aug 7

Literature context:


Abstract:

An enhancer with amalgamated E-box and GATA motifs (+9.5) controls expression of the regulator of hematopoiesis GATA-2. While similar GATA-2-occupied elements are common in the genome, occupancy does not predict function, and GATA-2-dependent genetic networks are incompletely defined. A "+9.5-like" element resides in an intron of Samd14 (Samd14-Enh) encoding a sterile alpha motif (SAM) domain protein. Deletion of Samd14-Enh in mice strongly decreased Samd14 expression in bone marrow and spleen. Although steady-state hematopoiesis was normal, Samd14-Enh-/- mice died in response to severe anemia. Samd14-Enh stimulated stem cell factor/c-Kit signaling, which promotes erythrocyte regeneration. Anemia activated Samd14-Enh by inducing enhancer components and enhancer chromatin accessibility. Thus, a GATA-2/anemia-regulated enhancer controls expression of an SAM domain protein that confers survival in anemia. We propose that Samd14-Enh and an ensemble of anemia-responsive enhancers are essential for erythrocyte regeneration in stress erythropoiesis, a vital process in pathologies, including β-thalassemia, myelodysplastic syndrome, and viral infection.

Funding information:
  • NIDDK NIH HHS - R01 DK050107()
  • NIDDK NIH HHS - R01 DK068634()

Wnt-Dependent Inactivation of the Groucho/TLE Co-repressor by the HECT E3 Ubiquitin Ligase Hyd/UBR5.

  • Flack JE
  • Mol. Cell
  • 2017 Jul 20

Literature context:


Abstract:

Extracellular signals are transduced to the cell nucleus by effectors that bind to enhancer complexes to operate transcriptional switches. For example, the Wnt enhanceosome is a multiprotein complex associated with Wnt-responsive enhancers through T cell factors (TCF) and kept silent by Groucho/TLE co-repressors. Wnt-activated β-catenin binds to TCF to overcome this repression, but how it achieves this is unknown. Here, we discover that this process depends on the HECT E3 ubiquitin ligase Hyd/UBR5, which is required for Wnt signal responses in Drosophila and human cell lines downstream of activated Armadillo/β-catenin. We identify Groucho/TLE as a functionally relevant substrate, whose ubiquitylation by UBR5 is induced by Wnt signaling and conferred by β-catenin. Inactivation of TLE by UBR5-dependent ubiquitylation also involves VCP/p97, an AAA ATPase regulating the folding of various cellular substrates including ubiquitylated chromatin proteins. Thus, Groucho/TLE ubiquitylation by Hyd/UBR5 is a key prerequisite that enables Armadillo/β-catenin to activate transcription.

In vitro analysis of RQC activities provides insights into the mechanism and function of CAT tailing.

  • Osuna BA
  • Elife
  • 2017 Jul 18

Literature context:


Abstract:

Ribosomes can stall during translation due to defects in the mRNA template or translation machinery, leading to the production of incomplete proteins. The Ribosome-associated Quality control Complex (RQC) engages stalled ribosomes and targets nascent polypeptides for proteasomal degradation. However, how each RQC component contributes to this process remains unclear. Here we demonstrate that key RQC activities-Ltn1p-dependent ubiquitination and Rqc2p-mediated Carboxy-terminal Alanine and Threonine (CAT) tail elongation-can be recapitulated in vitro with a yeast cell-free system. Using this approach, we determined that CAT tailing is mechanistically distinct from canonical translation, that Ltn1p-mediated ubiquitination depends on the poorly characterized RQC component Rqc1p, and that the process of CAT tailing enables robust ubiquitination of the nascent polypeptide. These findings establish a novel system to study the RQC and provide a framework for understanding how RQC factors coordinate their activities to facilitate clearance of incompletely synthesized proteins.

Funding information:
  • NIGMS NIH HHS - DP2 GM110772()
  • NIH HHS - DP5 OD017895()

Nicotinamide Ameliorates Disease Phenotypes in a Human iPSC Model of Age-Related Macular Degeneration.

  • Saini JS
  • Cell Stem Cell
  • 2017 May 4

Literature context:


Abstract:

Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE), a cell monolayer essential for photoreceptor survival, and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD, which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD, including two donors with the rare ARMS2/HTRA1 homozygous genotype. The hiPSC-derived RPE cells produce several AMD/drusen-related proteins, and those from the AMD donors show significantly increased complement and inflammatory factors, which are most exaggerated in the ARMS2/HTRA1 lines. Using a panel of AMD biomarkers and candidate drug screening, combined with transcriptome analysis, we discover that nicotinamide (NAM) ameliorated disease-related phenotypes by inhibiting drusen proteins and inflammatory and complement factors while upregulating nucleosome, ribosome, and chromatin-modifying genes. Thus, targeting NAM-regulated pathways is a promising avenue for developing therapeutics to combat AMD.

Funding information:
  • NEI NIH HHS - F32 EY025931()
  • NEI NIH HHS - R01 EY022079()
  • NIA NIH HHS - RF1 AG042932()

A Brain-Region-Specific Neural Pathway Regulating Germinal Matrix Angiogenesis.

  • Ma S
  • Dev. Cell
  • 2017 May 22

Literature context:


Abstract:

Intimate communication between neural and vascular cells is critical for normal brain development and function. Germinal matrix (GM), a key primordium for the brain reward circuitry, is unique among brain regions for its distinct pace of angiogenesis and selective vulnerability to hemorrhage during development. A major neonatal condition, GM hemorrhage can lead to cerebral palsy, hydrocephalus, and mental retardation. Here we identify a brain-region-specific neural progenitor-based signaling pathway dedicated to regulating GM vessel development. This pathway consists of cell-surface sphingosine-1-phosphate receptors, an intracellular cascade including Gα co-factor Ric8a and p38 MAPK, and target gene integrin β8, which in turn regulates vascular TGF-β signaling. These findings provide insights into region-specific specialization of neurovascular communication, with special implications for deciphering potent early-life endocrine, as well as potential gut microbiota impacts on brain reward circuitry. They also identify tissue-specific molecular targets for GM hemorrhage intervention.

Funding information:
  • NINDS NIH HHS - R01 NS076729()

Thermo-Induced Maintenance of Photo-oxidoreductases Underlies Plant Autotrophic Development.

  • Ha JH
  • Dev. Cell
  • 2017 Apr 24

Literature context:


Abstract:

Chlorophyll biosynthesis enables autotrophic development of developing seedlings. Upon light exposure, the chlorophyll precursor protochlorophyllide produces reactive oxygen species (ROS). Developing seedlings acquire photosynthetic competence through the action of protochlorophyllide oxidoreductases (PORs) that convert protochlorophyllide to chlorophyllide, reducing ROS production that would otherwise induce cellular damage and chlorophyll bleaching. Here, we show that FCA mediates the thermostabilization of PORs to trigger the conversion of protochlorophyllide to chlorophyllide in developing seedlings. FCA also facilitates the thermal induction of POR genes through histone acetylation that promotes the accessibility of RNA polymerases to the gene promoters. The combined action of FCA maintains PORs at warm temperatures, shifting the chlorophyll-ROS balance toward autotrophic development. We propose that the FCA-mediated thermal adaptation of autotrophic development allows developing seedlings to cope with the heat-absorbing soil surface layer under natural conditions. The thermal adaptive mechanism would provide a potential basis for studying crop performance at warm temperatures.

HIV-1 Tat Primes and Activates Microglial NLRP3 Inflammasome-Mediated Neuroinflammation.

  • Chivero ET
  • J. Neurosci.
  • 2017 Mar 29

Literature context:


Abstract:

Neuroinflammation associated with HIV-1 infection is a problem affecting ∼50% of HIV-infected individuals. NLR family pyrin domain containing 3 (NLRP3) inflammasome has been implicated in HIV-induced microglial activation, but the mechanism(s) remain unclear. Because HIV-1 Transactivator of Transcription (Tat) protein continues to be present despite antiretroviral therapy and activates NF-kB, we hypothesized that Tat could prime the NLRP3 inflammasome. We found a dose- and time-dependent induction of NLRP3 expression in microglia exposed to Tat compared with control. Tat exposure also time-dependently increased the mature caspase-1 and IL-1β levels and enhanced the IL-1β secretion. These in vitro findings were validated in archival brain tissues from Simian Immunodeficiency Virus (SIV)-infected and uninfected rhesus macaques. Further validation of NLRP3 priming in vivo involved administration of lipopolysaccharide (LPS) to HIV transgenic (Tg) rats followed by assessment of IL-1β mRNA expression and inflammasome activation (ASC oligomers and mature IL-1β). Intriguingly, LPS potentiated upregulation of IL-1β mRNA and inflammasome activation in HIV-Tg rats compared with the wild-type controls. Interestingly, we found an inverse relationship in the expression of NLRP3 and its negative regulator, miR-223, suggesting a miR-223-mediated mechanism for Tat-induced NLRP3 priming. Furthermore, blockade of NLRP3 resulted in decreased IL-1β secretion. Collectively, these findings suggest a novel role of Tat in priming and activating the NLRP3 inflammasome. Therefore, NLRP3 can be envisioned as a therapeutic target for ameliorating Tat-mediated neuroinflammation.SIGNIFICANCE STATEMENT Despite successful suppression of viremia with increased longevity in the era of combined antiretroviral therapy, chronic inflammation with underlying neurocognitive impairment continues to afflict almost 50% of infected individuals. Viral, bacterial, and cellular products have all been implicated in promoting the chronic inflammation found in these individuals. Understanding the molecular mechanism(s) by which viral proteins such as HIV-1 Transactivator of Transcription (Tat) protein can activate microglia is thus of paramount importance. Herein, we demonstrate a novel role of Tat in priming and activating NLR family pyrin domain containing 3 (NLRP3) inflammasomes in microglial cells and in HIV-Tg rats administered lipopolysaccharide. Targeting NLRP3 inflammasome pathway mediators could thus be developed as therapeutic interventions to alleviate or prevent neuroinflammation and subsequent cognitive impairment in HIV-positive patients.

MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging.

  • Ripa R
  • BMC Biol.
  • 2017 Feb 13

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Abstract:

BACKGROUND: A widespread modulation of gene expression occurs in the aging brain, but little is known as to the upstream drivers of these changes. MicroRNAs emerged as fine regulators of gene expression in many biological contexts and they are modulated by age. MicroRNAs may therefore be part of the upstream drivers of the global gene expression modulation correlated with aging and aging-related phenotypes. RESULTS: Here, we show that microRNA-29 (miR-29) is induced during aging in short-lived turquoise killifish brain and genetic antagonism of its function induces a gene-expression signature typical of aging. Mechanicistically, we identified Ireb2 (a master gene for intracellular iron delivery that encodes for IRP2 protein), as a novel miR-29 target. MiR-29 is induced by iron loading and, in turn, it reduces IRP2 expression in vivo, therefore limiting intracellular iron delivery in neurons. Genetically modified fish with neuro-specific miR-29 deficiency exhibit increased levels of IRP2 and transferrin receptor, increased iron content, and oxidative stress. CONCLUSIONS: Our results demonstrate that age-dependent miR-29 upregulation is an adaptive mechanism that counteracts the expression of some aging-related phenotypes and its anti-aging activity is primarily exerted by regulating intracellular iron homeostasis limiting excessive iron-exposure in neurons.

Ribosomal mutations promote the evolution of antibiotic resistance in a multidrug environment.

  • Gomez JE
  • Elife
  • 2017 Feb 21

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Abstract:

Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance.

Regulation of morphine-induced synaptic alterations: Role of oxidative stress, ER stress, and autophagy.

  • Cai Y
  • J. Cell Biol.
  • 2016 Oct 24

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Abstract:

Our findings suggest that morphine dysregulates synaptic balance in the hippocampus, a key center for learning and memory, via a novel signaling pathway involving reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy. We demonstrate in this study that exposure of morphine to hippocampal neurons leads to a reduction in excitatory synapse densities with a concomitant enhancement of inhibitory synapse densities via activation of the μ opioid receptor. Furthermore, these effects of morphine are mediated by up-regulation of intracellular ROS from NADPH oxidase, leading, in turn, to sequential induction of ER stress and autophagy. The detrimental effects of morphine on synaptic densities were shown to be reversed by platelet-derived growth factor (PDGF), a pleiotropic growth factor that has been implicated in neuroprotection. These results identify a novel cellular mechanism involved in morphine-mediated synaptic alterations with implications for therapeutic interventions by PDGF.

Funding information:
  • NIDA NIH HHS - DA14241(United States)

Region 4 of Rhizobium etli Primary Sigma Factor (SigA) Confers Transcriptional Laxity in Escherichia coli.

  • Santillán O
  • Front Microbiol
  • 2016 Jul 29

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Abstract:

Sigma factors are RNA polymerase subunits engaged in promoter recognition and DNA strand separation during transcription initiation in bacteria. Primary sigma factors are responsible for the expression of housekeeping genes and are essential for survival. RpoD, the primary sigma factor of Escherichia coli, a γ-proteobacteria, recognizes consensus promoter sequences highly similar to those of some α-proteobacteria species. Despite this resemblance, RpoD is unable to sustain transcription from most of the α-proteobacterial promoters tested so far. In contrast, we have found that SigA, the primary sigma factor of Rhizobium etli, an α-proteobacteria, is able to transcribe E. coli promoters, although it exhibits only 48% identity (98% coverage) to RpoD. We have called this the transcriptional laxity phenomenon. Here, we show that SigA partially complements the thermo-sensitive deficiency of RpoD285 from E. coli strain UQ285 and that the SigA region σ4 is responsible for this phenotype. Sixteen out of 74 residues (21.6%) within region σ4 are variable between RpoD and SigA. Mutating these residues significantly improves SigA ability to complement E. coli UQ285. Only six of these residues fall into positions already known to interact with promoter DNA and to comprise a helix-turn-helix motif. The remaining variable positions are located on previously unexplored sites inside region σ4, specifically into the first two α-helices of the region. Neither of the variable positions confined to these helices seem to interact directly with promoter sequence; instead, we adduce that these residues participate allosterically by contributing to correct region folding and/or positioning of the HTH motif. We propose that transcriptional laxity is a mechanism for ensuring transcription in spite of naturally occurring mutations from endogenous promoters and/or horizontally transferred DNA sequences, allowing survival and fast environmental adaptation of α-proteobacteria.

Funding information:
  • NICHD NIH HHS - R01 HD069560(United States)

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

  • Woodford MR
  • Nat Commun
  • 2016 Jun 29

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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)

Ghrelin Promotes Functional Angiogenesis in a Mouse Model of Critical Limb Ischemia Through Activation of Proangiogenic MicroRNAs.

  • Katare R
  • Endocrinology
  • 2016 Feb 2

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Abstract:

Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limited success. Recent in vitro evidence in the literature, using cell lines, proposes that the peptide hormone ghrelin may have angiogenic properties. In this study, we aim to investigate if ghrelin could promote postischemic angiogenesis in a mouse model of CLI and, further, identify the mechanistic pathway(s) that underpin ghrelin's proangiogenic properties. CLI was induced in male CD1 mice by femoral artery ligation. Animals were then randomized to receive either vehicle or acylated ghrelin (150 μg/kg sc) for 14 consecutive days. Subsequently, synchrotron radiation microangiography was used to assess hindlimb perfusion. Subsequent tissue samples were collected for molecular and histological analysis. Ghrelin treatment markedly improved limb perfusion by promoting the generation of new capillaries and arterioles (internal diameter less than 50 μm) within the ischemic hindlimb that were both structurally and functionally normal; evident by robust endothelium-dependent vasodilatory responses to acetylcholine. Molecular analysis revealed that ghrelin's angiogenic properties were linked to activation of prosurvival Akt/vascular endothelial growth factor/Bcl-2 signaling cascade, thus reducing the apoptotic cell death and subsequent fibrosis. Further, ghrelin treatment activated proangiogenic (miR-126 and miR-132) and antifibrotic (miR-30a) microRNAs (miRs) while inhibiting antiangiogenic (miR-92a and miR-206) miRs. Importantly, in vitro knockdown of key proangiogenic miRs (miR-126 and miR-132) inhibited the angiogenic potential of ghrelin. These results therefore suggest that clinical use of ghrelin for the early treatment of CLI may be a promising and potent inducer of reparative vascularization through modulation of key molecular factors.

Funding information:
  • BLRD VA - I01 BX001062(United States)
  • NICHD NIH HHS - R03 HD057380(United States)

GnRH Regulates Gonadotropin Gene Expression Through NADPH/Dual Oxidase-Derived Reactive Oxygen Species.

  • Kim T
  • Endocrinology
  • 2015 Jun 18

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Abstract:

The appropriate control of synthesis and secretion of the gonadotropin hormones LH and FSH by pituitary gonadotropes is essential for the regulation of reproduction. The hypothalamic neuropeptide GnRH is the central regulator of both processes, coordinating secretion with transcription and translation of the gonadotropin hormone subunit genes. The MAPK family of second messengers is strongly induced in gonadotropes upon GnRH stimulation, and multiple pathways activate these kinases. Intracellular reactive oxygen species participate in signaling cascades that target MAPKs, but also participate in signaling events indicative of cell stress. The NADPH oxidase (NOX)/dual oxidase (DUOX) family is a major enzymatic source of intracellular reactive oxygen, and we show that GnRH stimulation of mouse primary pituitary cells and the LβT2 gonadotrope cell line elevates intracellular reactive oxygen via NOX/DUOX activity. Mouse pituitary and LβT2 cells abundantly express NOX/DUOX and cofactor mRNAs. Pharmacological inhibition of NOX/DUOX activity diminishes GnRH-stimulated activation of MAPKs, immediate-early gene expression, and gonadotropin subunit gene expression. Inhibitor studies implicate the calcium-activated DUOX family as a major, but not exclusive, participant in GnRH signaling. Knockdown of DUOX2 in LβT2 cells reduces GnRH-induced Fshb, but not Lhb mRNA levels, suggesting differential sensitivity to DUOX activity. Finally, GnRH pulse-stimulated FSH and LH secretion are suppressed by inhibition of NOX/DUOX activity. These results indicate that reactive oxygen is a potent signaling intermediate produced in response to GnRH stimulation and further suggest that reactive oxygen derived from other sources may influence the gonadotrope response to GnRH stimulation.

Funding information:
  • NIGMS NIH HHS - T32 GM007616(United States)

Endothelin-1 stimulates resistin gene expression.

  • Tang YC
  • Endocrinology
  • 2014 Mar 25

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Abstract:

Resistin and endothelin (ET)-1 have been reported to inhibit adipogenesis and regulate adipocyte insulin resistance, respectively. Although both hormones interact with each other, the exact signaling pathway of ET-1 to act on resistin gene expression is still unknown. Using 3T3-L1 adipocytes, we investigated the signaling pathways involved in ET-1-stimulated resistin gene expression. The up-regulation of resistin mRNA expression by ET-1 depends on concentration and timing. The concentration of ET-1 that increased resistin mRNA levels by 100%-250% was approximately 100 nM for a range of 0.25-12 hours of treatment. Treatment with actinomycin D blocked ET-1-increased resistin mRNA levels, suggesting that the effect of ET-1 requires new mRNA synthesis. Treatment with an inhibitor of the ET type-A receptor, such as N-[1-Formyl-N-[N-[(hexahydro-1H-azepin-1-yl)carbonyl]-L-leucyl]-D-tryptophyl]-D-tryptophan (BQ610), but not with the ET type-B receptor antagonist N-[(cis-2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl-1-(methoxycarbonyl)-D-tryptophyl-D-norleucine (BQ788), blocked ET-1, increased the levels of resistin mRNA, and phosphorylated levels of downstream signaling molecules, such as ERK1/2, c-Jun N-terminal kinases (JNKs), protein kinase B (AKT), and signal transducer and activator of transcription 3 (STAT3). Moreover, pretreatment of specific inhibitors of either ERK1/2 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene [U0126] and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one [PD98059], two inhibitors of MEK1), JNKs (SP600125), phosphatidylinositol 3-kinase/AKT (LY294002 and Wortmannin), or Janus kinase 2 (JAK2)/STAT3 ((E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide, AG490) prevented ET-1-increased levels of resistin mRNA and reduced the ET-1-stimulated phosphorylation of ERK1/2, JNKs, AKT, and STAT3, respectively. However, the p38 kinase antagonist 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine (SB203580) did not alter the effect of ET-1. These results imply that ET type-A receptor, ERK1/2, JNKs, AKT, and JAK2, but not ET type-B receptor or p38, are necessary for the ET-1 stimulation of resistin gene expression. In vivo observations that ET-1 increased resistin mRNA and protein levels in sc and epididymal adipose tissues support the in vitro findings.

Funding information:
  • NHLBI NIH HHS - HL107147(United States)
  • NICHD NIH HHS - U54 HD029990(United States)

Steroidogenic factor 1 promotes aggressive growth of castration-resistant prostate cancer cells by stimulating steroid synthesis and cell proliferation.

  • Lewis SR
  • Endocrinology
  • 2014 Feb 22

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Abstract:

The dependence of prostate cancer on androgens provides a targeted means of treating advanced disease. Unfortunately, androgen deprivation therapies eventually become ineffective, leading to deadly castration-resistant prostate cancer (CRPC). One of many factors implicated in the transition to CRPC is the onset of de novo steroidogenesis. Although reactivation of steroid receptors likely plays a pivotal role in aggressive CRPC, little is understood regarding the mechanisms whereby prostate cancer cells initiate and maintain steroidogenesis. We hypothesize that steroidogenic factor 1 (SF1, NR5A1, AD4BP), a key regulator of steroidogenesis in normal endocrine tissues, is expressed in CRPC where it stimulates aberrant steroidogenesis and fuels aggressive growth. Notably, SF1 is not expressed in normal prostate tissue. Our results indicated that SF1 was absent in benign cells but present in aggressive prostate cancer cell lines. Introduction of ectopic SF1 expression in benign human prostate epithelial cells (BPH-1) stimulated increased steroidogenic enzyme expression, steroid synthesis, and cell proliferation. In contrast, data from an aggressive human prostate cancer cell line (BCaPT10) demonstrated that SF1 was required for steroid-mediated cell growth because BCaPT10 cell growth was diminished by abiraterone treatment and short hairpin RNA-mediated knockdown of SF1 (shSF1). SF1-depleted cells also exhibited defective centrosome homeostasis. Finally, whereas xenograft experiments in castrated hosts with BCaPT10 control transplants grew large, invasive tumors, BCaPT10-shSF1 knockdown transplants failed to grow. Therefore, we conclude that SF1 stimulates steroid accumulation and controls centrosome homeostasis to mediate aggressive prostate cancer cell growth within a castrate environment. These findings present a new molecular mechanism and therapeutic target for deadly CRPC.

Funding information:
  • NIH HHS - P40 OD010939(United States)

Testosterone protects against glucotoxicity-induced apoptosis of pancreatic β-cells (INS-1) and male mouse pancreatic islets.

  • Hanchang W
  • Endocrinology
  • 2013 Nov 21

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Abstract:

Male hypogonadism associates with type 2 diabetes, and T can protect pancreatic β-cells from glucotoxicity. However, the protective mechanism is still unclear. This study thus aims to examine the antiapoptotic mechanism of T in pancreatic β cells cultured in high-glucose medium. T (0.0005-2 μg/mL) was added to INS-1 cells cultured in basal glucose or high-glucose media. Then cellular apoptosis, oxidative stress, and cell viability were measured. Endoplasmic reticulum (ER) stress markers and sensors and the antiapoptotic protein (B-cell lymphoma 2) were investigated by real-time PCR and Western blot analysis. ER stress markers were also measured in male mouse pancreatic islet cultured in similar conditions. T (0.05 and 0.5 μg/mL) did not have any effect on apoptosis and viability of INS-1 cells cultured in basal glucose medium, but it could reduce apoptosis and increase viability of INS-1 cells cultured in high-glucose medium. The protective effect of T is diminished by androgen receptor inhibitor. T (0.05 μg/mL) could significantly reduce nitrotyrosine levels, mRNA, and protein levels of the ER stress markers and sensor those that were induced when INS-1 cells were cultured in high-glucose medium. It could also significantly increase the survival proteins, sarco/endoplasmic reticulum Ca(2+) ATPase-2, and B-cell lymphoma 2 in INS-1 cells cultured in the same conditions. Similarly, it could reduce ER stress markers and increase sarco/endoplasmic reticulum Ca(2+) ATPase protein levels in male mouse pancreatic islets cultured in high-glucose medium. T can protect against male pancreatic β-cell apoptosis from glucotoxicity via the reduction of both oxidative stress and ER stress.

Funding information:
  • Canadian Institutes of Health Research - MOP-93583(Canada)
  • NIGMS NIH HHS - SC3 GM109817(United States)