Literature context: echnology Cat# 9255 RRID:AB_2307321), JNK (R and D Systems Cat# AF1
Breast cancer is the most commonly diagnosed malignancy in women. Analysis of breast cancer genomic DNA indicates frequent loss-of-function mutations in components of the cJUN NH2-terminal kinase (JNK) signaling pathway. Since JNK signaling can promote cell proliferation by activating the AP1 transcription factor, this apparent association of reduced JNK signaling with tumor development was unexpected. We examined the effect of JNK deficiency in the murine breast epithelium. Loss of JNK signaling caused genomic instability and the development of breast cancer. Moreover, JNK deficiency caused widespread early neoplasia and rapid tumor formation in a murine model of breast cancer. This tumor suppressive function was not mediated by a role of JNK in the growth of established tumors, but by a requirement of JNK to prevent tumor initiation. Together, these data identify JNK pathway defects as 'driver' mutations that promote genome instability and tumor initiation.
Literature context: 100 (IHC) Cell Signaling 9255S; RRID:AB_2307321 Rabbit anti-aPKC
Drosophila tumor suppressor genes have revealed molecular pathways that control tissue growth, but mechanisms that regulate mitogenic signaling are far from understood. Here we report that the Drosophila TSG tumorous imaginal discs (tid), whose phenotypes were previously attributed to mutations in a DnaJ-like chaperone, are in fact driven by the loss of the N-linked glycosylation pathway component ALG3. tid/alg3 imaginal discs display tissue growth and architecture defects that share characteristics of both neoplastic and hyperplastic mutants. Tumorous growth is driven by inhibited Hippo signaling, induced by excess Jun N-terminal kinase (JNK) activity. We show that ectopic JNK activation is caused by aberrant glycosylation of a single protein, the fly tumor necrosis factor (TNF) receptor homolog, which results in increased binding to the continually circulating TNF. Our results suggest that N-linked glycosylation sets the threshold of TNF receptor signaling by modifying ligand-receptor interactions and that cells may alter this modification to respond appropriately to physiological cues.
Literature context: Signaling Technology Cat#9255; RRID:AB_2307321 Rabbit polyclonal anti-Ubiquiti
Evasion of apoptosis is a hallmark of cancer. Bcl-2 and p53 represent two important nodes in apoptosis signaling pathways. We find that concomitant p53 activation and Bcl-2 inhibition overcome apoptosis resistance and markedly prolong survival in three mouse models of resistant acute myeloid leukemia (AML). Mechanistically, p53 activation negatively regulates the Ras/Raf/MEK/ERK pathway and activates GSK3 to modulate Mcl-1 phosphorylation and promote its degradation, thus overcoming AML resistance to Bcl-2 inhibition. Moreover, Bcl-2 inhibition reciprocally overcomes apoptosis resistance to p53 activation by switching cellular response from G1 arrest to apoptosis. The efficacy, together with the mechanistic findings, reveals the potential of simultaneously targeting these two apoptosis regulators and provides a rational basis for clinical testing of this therapeutic approach.
Literature context: Mouse; monoclonal 1:2000 (W) RRID:AB_2307321
Inflammation can interfere with endometrial receptivity. We examined how interleukin 1β (IL-1β) affects expression of the uterine gap junction protein connexin 43 (Cx43), which is known to be critical for embryonic implantation. We used an in vitro model of human endometrial stromal cells (ESCs), Western blotting, and a combination of validated, selective kinase inhibitors to evaluate five canonical IL-1β signaling pathways. Cx43 and two other markers of ESC differentiation (prolactin and VEGF) were inhibited predominantly via IL-1β-activated ERK1/2 and p38 MAP kinase cascades. The findings were corroborated using small interfering RNA to silence critical genes in either pathway. By contrast, upregulation of endogenous pro-IL-1α and pro-IL-1β following recombinant IL-1β treatment was mediated via the Jun N-terminal kinase pathway. The clinicopharmacological significance of our findings is that multiple signaling cascades may need to be neutralized to reverse deleterious effects of IL-1β on human endometrial function.
Literature context: anti-pJNK Cell Signaling 9255; RRID:AB_2307321 Rabbit polyclonal anti-JNK Cell
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.
Literature context: (RRID:AB_2307321), p-p38 (R
The ubiquitin ligase TRAF6 is a key regulator of canonical IκB kinase (IKK)/NF-κB signaling in response to interleukin-1 (IL-1) stimulation. Here, we identified the deubiquitinating enzyme YOD1 (OTUD2) as a novel interactor of TRAF6 in human cells. YOD1 binds to the C-terminal TRAF homology domain of TRAF6 that also serves as the interaction surface for the adaptor p62/Sequestosome-1, which is required for IL-1 signaling to NF-κB. We show that YOD1 competes with p62 for TRAF6 association and abolishes the sequestration of TRAF6 to cytosolic p62 aggregates by a non-catalytic mechanism. YOD1 associates with TRAF6 in unstimulated cells but is released upon IL-1β stimulation, thereby facilitating TRAF6 auto-ubiquitination as well as NEMO/IKKγ substrate ubiquitination. Further, IL-1 triggered IKK/NF-κB signaling and induction of target genes is decreased by YOD1 overexpression and augmented after YOD1 depletion. Hence, our data define that YOD1 antagonizes TRAF6/p62-dependent IL-1 signaling to NF-κB.
Literature context: at# 9255; RRID:AB_2307321 Anti-SAPK/
Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2, ApoE3, and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), ApoE3 is neutral, and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis, however, remains unclear. Using ES-cell-derived human neurons, we show that ApoE secreted by glia stimulates neuronal Aβ production with an ApoE4 > ApoE3 > ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK), a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation, stimulating the transcription factor AP-1, which in turn enhances transcription of amyloid-β precursor protein (APP) and thereby increases amyloid-β levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-β synthesis.
We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3-1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3-1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca(2+) influx via the L-type Ca(2+) channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca(2+) influx through L-type Ca(2+) channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca(2+) influx via L-type channels to facilitate ERK phosphorylation.
Excessive hepatic lipid accumulation promotes macrophages/Kupffer cells activation, resulting in exacerbation of insulin resistance and progression of nonalcoholic steatohepatitis (NASH). However, few promising treatment modalities target lipotoxicity-mediated hepatic activation/polarization of macrophages for NASH. Recent epidemiological surveys showed that serum β-cryptoxanthin, an antioxidant carotenoid, was inversely associated with the risks of insulin resistance and liver dysfunction. In the present study, we first showed that β-cryptoxanthin administration ameliorated hepatic steatosis in high-fat diet-induced obese mice. Next, we investigated the preventative and therapeutic effects of β-cryptoxanthin using a lipotoxic model of NASH: mice fed a high-cholesterol and high-fat (CL) diet. After 12 weeks of CL diet feeding, β-cryptoxanthin administration attenuated insulin resistance and excessive hepatic lipid accumulation and peroxidation, with increases in M1-type macrophages/Kupffer cells and activated stellate cells, and fibrosis in CL diet-induced NASH. Comprehensive gene expression analysis showed that β-cryptoxanthin down-regulated macrophage activation signal-related genes significantly without affecting most lipid metabolism-related genes in the liver. Importantly, flow cytometry analysis revealed that, on a CL diet, β-cryptoxanthin caused a predominance of M2 over M1 macrophage populations, in addition to reducing total hepatic macrophage and T-cell contents. In parallel, β-cryptoxanthin decreased lipopolysaccharide-induced M1 marker mRNA expression in peritoneal macrophages, whereas it augmented IL-4-induced M2 marker mRNA expression, in a dose-dependent manner. Moreover, β-cryptoxanthin reversed steatosis, inflammation, and fibrosis progression in preexisting NASH in mice. In conclusion, β-cryptoxanthin prevents and reverses insulin resistance and steatohepatitis, at least in part, through an M2-dominant shift in macrophages/Kupffer cells in a lipotoxic model of NASH.
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.