NDR1 protein kinase promotes IL‐17‐ and TNF‐α‐mediated inflammation by competitively binding TRAF3

Interleukin 17 (IL‐17) is an important inducer of tissue inflammation and is involved in numerous autoimmune diseases. However, how its signal transduction is regulated is not well understood. Here, we report that nuclear Dbf2‐related kinase 1 (NDR1) functions as a positive regulator of IL‐17 signal transduction and IL‐17‐induced inflammation. NDR1 deficiency or knockdown inhibits the IL‐17‐induced phosphorylation of p38, ERK1/2, and p65 and the expression of chemokines and cytokines, whereas the overexpression of NDR1 promotes IL‐17‐induced signaling independent of its kinase activity. Mechanistically, NDR1 interacts with TRAF3 and prevents its binding to IL‐17R, which promotes the formation of an IL‐17R‐Act1‐TRAF6 complex and downstream signaling. Consistent with this, IL‐17‐induced inflammation is significantly reduced in NDR1‐deficient mice, and NDR1 deficiency significantly protects mice from MOG‐induced experimental autoimmune encephalomyelitis (EAE) and 2,4,6‐trinitrobenzenesulfonic acid (TNBS)‐induced colitis likely by its inhibition of IL‐17‐mediated signaling pathway. NDR1 expression is increased in the colons of ulcerative colitis (UC) patients. Taken together, these findings suggest that NDR1 is involved in the development of autoimmune diseases.     

The TLR4‐IRE1α pathway activation contributes to palmitate‐elicited lipotoxicity in hepatocytes

Lipotoxicity induced by saturated fatty acids (SFAs) plays a pathological role in the development of non‐alcoholic fatty liver disease (NAFLD); however, the exact mechanism(s) remain to be clearly elucidated. Toll‐like receptor (TLR) 4 plays a fundamental role in activating the innate immune system. Intriguingly, hepatocytes express TLR4 and machinery for TLR4 signalling pathway. That liver‐specific TLR4 knockout mice are protective against diet‐induced NAFLD suggests that hepatocyte TLR4 signalling pathway plays an important role in NAFLD pathogenesis. Herein, using cultured hepatocytes, we sought to directly examine the role of TLR4 signalling pathway in palmitate‐elicited hepatotoxicity and to elucidate underlying mechanism(s). Our data reveal that palmitate exposure up‐regulates TLR4 expression at both mRNA and protein levels in hepatocytes, which are associated with NF‐κB activation. The inhibition of TLR4 signalling pathway through both pharmacological and genetic approaches abolished palmitate‐induced cell death, suggesting that TLR4 signalling pathway activation contributes to palmitate‐induced hepatotoxicity. Mechanistic investigations demonstrate that inositol‐requiring enzyme 1α (IRE1α), one of three major signal transduction pathways activated during endoplasmic reticulum (ER) stress, is the downstream target of palmitate‐elicited TLR4 activation and mechanistically implicated in TLR4 activation‐triggered cell death in response to palmitate exposure. Collectively, our data identify that the TLR4‐IRE1α pathway activation contributes to palmitate‐elicited lipotoxicity in hepatocytes. Our findings suggest that targeting TLR4‐IRE1α pathway can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism.     

Synoviolin promotes IRE1 ubiquitination and degradation in synovial fibroblasts from mice with collagen-induced arthritis

The E3 ubiquitin ligase synoviolin (SYVN1) functions as an anti-apoptotic factor that is responsible for the outgrowth of synovial cells during the development of rheumatoid arthritis. The molecular mechanisms underlying SYVN1 regulation of cell death are largely unknown. Here, we report that elevated SYVN1 expression correlates with decreased levels of the protein inositol-requiring enzyme 1 (IRE1)-a pro-apoptotic factor in the endoplasmic reticulum (ER)-stress-induced apoptosis pathway-in synovial fibroblasts from mice with collagen-induced arthritis (CIA). SYVN1 interacts with and catalyses IRE1 ubiquitination and consequently promotes IRE1 degradation. Suppression of SYVN1 expression in synovial fibroblasts from CIA mice restores IRE1 protein expression and reverses the resistance of ER-stress-induced apoptosis of CIA synovial fibroblasts. These results show that SYVN1 causes the overgrowth of synovial cells by degrading IRE1, and therefore antagonizes ER-stress-induced cell death.     

MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule dynamics and intracellular transport

MGRN1‐mediated ubiquitination of α‐tubulin regulates microtubule stability and mitotic spindle positioning in mitotic cells. This study elucidates the effect of MGRN1‐mediated ubiquitination of α‐tubulin in interphase cells. Here, we show that MGRN1‐mediated ubiquitination regulates dynamics of EB1‐labeled plus ends of microtubules. Intracellular transport of mitochondria and endosomes are affected in cultured cells where functional MGRN1 is depleted. Defects in microtubule‐dependent organellar transport are evident in cells where noncanonical K6‐mediated ubiquitination of α‐tubulin by MGRN1 is compromised. Loss of MGRN1 has been previously correlated with late‐onset spongiform neurodegeneration. Mislocalised cytosolically exposed PrP (^CtmPrP) interacts with MGRN1 leading to its loss of function. Expression of ^CtmPrP generating mutants of PrP[PrP(A117V) and PrP(KHII)] lead to decrease in MGRN1‐mediated ubiquitination of α‐tubulin and intracellular transport defects. Brain lysates from PrP(A117V) transgenic mice also indicate loss of tubulin polymerization as compared to non‐transgenic controls. Depletion of MGRN1 activity may hamper physiologically important processes like mitochondrial movement in neuronal processes and intracellular transport of ligands through the endosomal pathway thereby contributing to the pathogenesis of neurodegeneration in certain types of prion diseases.      

PELP1 is a reader of histone H3 methylation that facilitates oestrogen receptor‐α target gene activation by regulating lysine demethylase 1 specificity

Histone methylation has a key role in oestrogen receptor (ERα)‐mediated transactivation of genes. Proline glutamic acid and leucine‐rich protein 1 (PELP1) is a new proto‐oncogene that functions as an ERα co‐regulator. In this study, we identified histone lysine demethylase, KDM1, as a new PELP1‐interacting protein. These proteins, PELP1 and KDM1, were both recruited to ERα target genes, and PELP1 depletion affected the dimethyl histone modifications at ERα target genes. Dimethyl‐modified histones H3K4 and H3K9 are recognized by PELP1, and PELP1 alters the substrate specificity of KDM1 from H3K4 to H3K9. Effective demethylation of dimethyl H3K9 by KDM1 requires a KDM1–ERα–PELP1 functional complex. These results suggest that PELP1 is a reader of H3 methylation marks and has a crucial role in modulating the histone code at the ERα target genes.     

Reactive oxygen species‐mediated endoplasmic reticulum stress response induces apoptosis of Mycobacterium avium‐infected macrophages by activating regulated IRE1‐dependent decay pathway

Mycobacterium avium, a slow‐growing nontuberculous mycobacterium, causes fever, diarrhoea, loss of appetite, and weight loss in immunocompromised people. We have proposed that endoplasmic reticulum (ER) stress‐mediated apoptosis plays a critical role in removing intracellular mycobacteria. In the present study, we investigated the role of the regulated IRE1‐dependent decay (RIDD) pathway in macrophages during M. avium infection based on its role in the regulation of gene expression. The inositol‐requiring enzyme 1 (IRE1)/apoptosis signal‐regulating kinase 1 (ASK1)/c‐Jun N‐terminal kinase (JNK) signalling pathway was activated in macrophages after infection with M. avium. The expression of RIDD‐associated genes, such as Bloc1s1 and St3gal5, was decreased in M. avium‐infected macrophages. Interestingly, M. avium‐induced apoptosis was significantly suppressed by pretreatment with irestatin (inhibitor of IRE1α) and 4μ8c (RIDD blocker). Macrophages pretreated with N‐acetyl cysteine (NAC) showed decreased levels of reactive oxygen species (ROS), IRE1α, and apoptosis after M. avium infection. The expression of Bloc1s1 and St3gal5 was increased in NAC‐pretreated macrophages following infection with M. avium. Growth of M. avium was significantly increased in irestatin‐, 4μ8c‐, and NAC‐treated macrophages compared with the control. The data indicate that the ROS‐mediated ER stress response induces apoptosis of M. avium‐infected macrophages by activating IRE1α‐RIDD. Thus, activation of IRE1α suppresses the intracellular survival of M. avium in macrophages.     

Hydrogen sulphide decreases IL‐1β‐induced activation of fibroblast‐like synoviocytes from patients with osteoarthritis

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H₂S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H₂S on fibroblast‐like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro‐inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL‐1β and treated with the H₂S donor NaHS. Cellular responses were analysed by ELISA, quantitative real‐time PCR, phospho‐MAPkinase array and Western blotting. Treatment‐induced effects on cellular structure and synovial architecture were investigated in three‐dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL‐1β‐induced secretion of IL‐6, IL‐8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo‐proteinases MMP‐2 and MMP‐14. IL‐1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL‐1β‐induced activation of several MAPK whereas it increased phosphorylation of pro‐survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer‐like structure; stimulation with IL‐1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H₂S partially antagonizes IL‐1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.     

ARRB1 ameliorates liver ischaemia/reperfusion injury via antagonizing TRAF6‐mediated Lysine 6‐linked polyubiquitination of ASK1 in hepatocytes

Hepatic ischaemia/reperfusion (I/R) injury is a major clinical problem during liver surgical procedures, which usually lead to early transplantation failure and higher organ rejection rate, and current effective therapeutic strategies are still limited. Therefore, in‐depth exploring of the molecular mechanisms underlying liver I/R injury is key to the development of new therapeutic methods. β‐arrestins are multifunctional proteins serving as important signalling scaffolds in numerous physiopathological processes, including liver‐specific diseases. However, the role and underlying mechanism of β‐arrestins in hepatic I/R injury remain largely unknown. Here, we showed that only ARRB1, but not ARRB2, was down‐regulated during liver I/R injury. Hepatocyte‐specific overexpression of ARRB1 significantly ameliorated liver damage, as demonstrated by decreases in serum aminotransferases, hepatocellular necrosis and apoptosis, infiltrating inflammatory cells and secretion of pro‐inflammatory cytokines relative to control mice, whereas experiments with ARRB1 knockout mice gotten opposite effects. Mechanistically, ARRB1 directly interacts with ASK1 in hepatocytes and inhibits its TRAF6‐mediated Lysine 6‐linked polyubiquitination, which then prevents the activation of ASK1 and its downstream signalling pathway during hepatic I/R injury. In addition, inhibition of ASK1 remarkably abolished the disruptive effect result from ARRB1 deficiency in liver I/R injury in vivo, indicating that ASK1 was required for ARRB1 function in hepatic I/R injury. In conclusion, we proposed that ARRB1 is a novel protective regulator during liver I/R injury, and modulation of the regulatory axis between ARRB1 and ASK1 could be a novel therapeutic strategy to prevent this pathological process.     

Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA‐mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis‐inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR‐A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma.     

IL‐1α and IL‐1β have different effects on formation and activity of large osteoclasts

Interleukin 1 (IL‐1) is a proinflammatory cytokine upregulated in conditions such as rheumatoid arthritis and periodontal disease. Both isoforms, IL‐1α and IL‐1β, have been shown to activate osteoclasts (OCs), the cells responsible for resorbing bone. Inflammatory conditions are also characterized by increased bone loss and by the presence of large OCs (10+ nuclei). We and others have previously shown that large OCs are more likely to be resorbing compared to small OCs (2–5 nuclei). Moreover, large OCs express higher levels of the IL‐1 activating receptor IL‐1RI, integrins αv and β3, RANK, and TNFR1, while small OCs have higher levels of the decoy receptor IL‐1RII. We hypothesized that IL‐1 would have different effects on large and small OCs due to these distinct receptor expression patterns. To test this hypothesis, RAW 264.7 cells were differentiated into populations of small and large OCs and treated with IL‐1α or IL‐1β (1 and 10 ng/ml). In the presence of sRANKL, both IL‐1α and IL‐1β increased total OC number and resorptive activity of large OCs. IL‐1α stimulated formation of large OCs and increased the number of resorption pits, while IL‐1β changed the morphology of large OCs and integrin‐β3 phosphorylation. No effects were seen in small OCs in response to either IL‐1 isoform. These results demonstrate that IL‐1 predominantly affects large OCs. The dissimilarity of responses to IL‐1α and IL‐1β suggests that these isoforms activate different signaling pathways within the two OC populations. J. Cell. Biochem. 109: 975–982, 2010. © 2010 Wiley‐Liss, Inc.     

Phosphorylation at Ser724 of the ER stress sensor IRE1α governs its activation state and limits ER stress–induced hepatosteatosis

Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here, we generated a mouse model harboring a S724A knock-in mutation (Ern1^S724A/S724A) and investigated the importance of phosphorylation at Ser⁷²⁴ within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast cells and in primary hepatocytes, S724A mutation resulted in markedly reduced IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze X-box binding protein 1 (Xbp1) mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser⁷²⁴ exacerbated ER stress–induced hepatic steatosis in tunicamycin-treated Ern1^S724A/S724A mice. This was accompanied by significantly decreased hepatic production of spliced XBP1 protein but increased CCAAT-enhancer–binding protein homologous protein (CHOP) level, along with suppressed expression of key metabolic regulators of fatty acid β-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser⁷²⁴ of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions.     

Human placenta‐derived mesenchymal stem cells inhibit apoptosis of granulosa cells induced by IRE1α pathway in autoimmune POF mice

Previous studies have shown that the ovarian failure in autoimmune‐induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta‐derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol‐requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress‐induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X‐box binding protein 1 (XBP1), up‐regulated 78 kDa glucose‐regulated protein (GRP78) and caspase‐12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis‐induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation‐mediating ovarian function recovery.     

Inhibition of TRAF6 ubiquitin-ligase activity by PRDX1 leads to inhibition of NFKB activation and autophagy activation

TRAF6 (TNF receptor associated factor 6) plays a pivotal role in NFKB activation and macroautphagy/autophagy activation induced by TLR4 (toll like receptor 4) signaling. The objective of this study was to determine the functional role of PRDX1 (peroxiredoxin 1) in NFKB activation and autophagy activation. PRDX1 interacted with the ring finger domain of TRAF6 and inhibited its ubiquitin-ligase activity. The inhibition on TRAF6 ubiquitin-ligase activity by PRDX1 induced the suppression of ubiquitination of an evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) essential for NFKB activation and BECN1 (beclin 1) required for autophagy activation. An inhibitory effect of PRDX1 on TRAF6 was clearly evidenced in PRDX1-knockdown (PRDX1KD) THP-1, PRDX1KD MDA-MB-231, and PRDX1KD SK-HEP-1 cells. PRDX1KD THP-1 cells showed increases of NFKB activation, pro-inflammatory cytokine production, NFKB-dependent gene expression induced by TLR4 stimulation, and resistance against Salmonella typhimurium infection. Additionally, migration and invasion abilities of PRDX1KD MDA-MB-231 and PRDX1KD SK-HEP-1 cancer cells were significantly enhanced compared to those of control cancer cells. Taken together, these results suggest that PRDX1 negatively regulates TLR4 signaling for NFKB activation and autophagy functions such as bactericidal activity, cancer cell migration, and cancer cell invasion by inhibiting TRAF6 ubiquitin-ligase activity.

Abbreviations: 3-MA: 3-methyladenine; BECN1: beclin 1; CHUK/IKKA: conserved helix-loop-helix ubiquitous kinase; ECSIT: ECSIT signalling integrator; ELISA: enzyme-linked immunosorbent assay; NFKB: nuclear factor kappa-light-chain-enhancer of activated B cells; IB: immunoblotting; IKBKB/IKKB: inhibitor of nuclear factor kappa B kinase subunit beta; IL1B: interleukin 1 beta; IL6: interleukin 6; IP: immunoprecipitation; LPS: lipopolysaccharide; MAP1LC3/LC3: microtuble associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK14/p38: mitogen-activated protein kinase 14; mROS: mitochondrial reactive oxygen species; PRDX1: peroxiredoxin 1; PRDX6: peroxiredoxin 6; RELA/p65: RELA proto-oncogene, NF-kB subunit; TRAF6 TNF: receptor associated factor 6.     

Tumor necrosis factor α‐mediated activation of c‐Jun NH2‐terminal kinase as a mechanism for fumonisin B1 induced apoptosis in murine primary hepatocytes

Fumonisin B₁ is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species‐specific and organ‐specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B₁ perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B₁ caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B₁ induced hepatic apoptosis in mice. The role of tumor necrosis factor α (TNFα) in fumonisin B₁ mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B₁ induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B₁ caused accumulation of sphingoid bases and led to increase in TNFα expression. Phosphorylated and total c‐Jun NH₂‐terminal kinase (JNK) activities were increased after 24 h fumonisin B₁ treatment. JNK inhibitor (SP600125) and anti‐TNFα reduced the apoptosis induced by fumonisin B₁. The role of JNK signaling in fumonisin B₁ induced apoptosis is downstream of TNFα production, as fumonisin B₁‐mediated activation of JNK was reduced by the presence of anti‐TNFα in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B₁ induced TNFα expression. Results of this study imply that generation of fumonisin B₁ induced TNFα results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:359‐367, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20102