Phlda3 regulates beta cell survival during stress.
ABSTRACT: The loss of functional beta cell mass characterises all forms of diabetes. Beta cells are highly susceptible to stress, including cytokine, endoplasmic reticulum (ER) and oxidative stress. This study examined the role of pleckstrin homology-like, domain family A, member 3 (Phlda3) in beta cell survival under stress conditions and the regulatory basis. We found that the mRNA levels of Phlda3 were markedly upregulated in vivo in the islets of diabetic humans and mice. In vitro, exposure of MIN6 cells or islets to cytokines, palmitate, thapsigargin or ribose upregulated Phlda3 mRNA and protein levels, concurrent with the induction of ER stress (Ddit3 and Trb3) and antioxidant (Hmox1) genes. Furthermore, H2O2 treatment markedly increased PHLDA3 immunostaining in human islets. Phlda3 expression was differentially regulated by adaptive (Xbp1) and apoptotic (Ddit3) unfolded protein response (UPR) mediators. siRNA-mediated knockdown of Xbp1 inhibited the induction of Phlda3 by cytokines and palmitate, whereas knockdown of Ddit3 upregulated Phlda3. Moreover, knockdown of Phlda3 potentiated cytokine-induced apoptosis in association with upregulation of inflammatory genes (iNos, IL1? and I?B?) and NF?B phosphorylation and downregulation of antioxidant (Gpx1 and Srxn1) and adaptive UPR (Xbp1, Hspa5 and Fkbp11) genes. Knockdown of Phlda3 also potentiated apoptosis under oxidative stress conditions induced by ribose treatment. These findings suggest that Phlda3 is crucial for beta cell survival under stress conditions. Phlda3 regulates the cytokine, oxidative and ER stress responses in beta cells via the repression of inflammatory gene expression and the maintenance of antioxidant and adaptive UPR gene expression. Phlda3 may promote beta cell survival in diabetes.
Project description:Endoplasmic reticulum (ER) stress is involved in liver injury, but molecular determinants are largely unknown. This study investigated the role of pleckstrin homology-like domain, family A, member-3 (PHLDA3), in hepatocyte death caused by ER stress and the regulatory basis.Hepatic PHLDA3 expression was assessed in HCV patients with hepatitis and in several animal models with ER stress. Immunoblottings, PCR, reporter gene, chromatin immunoprecipitation (ChIP) and mutation analyses were done to explore gene regulation. The functional effect of PHLDA3 on liver injury was validated using lentiviral delivery of shRNA.PHLDA3 was overexpressed in relation to hepatocyte injury in patients with acute liver failure or liver cirrhosis or in toxicant-treated mice. In HCV patients with liver injury, PHLDA3 was upregulated in parallel with the induction of ER stress marker. Treatment of mice with tunicamycin (Tm) (an ER stress inducer) increased PHLDA3 expression in the liver. X box-binding protein-1 (Xbp1) was newly identified as a transcription factor responsible for PHLDA3 expression. Inositol-requiring enzyme 1 (IRE1) (an upstream regulator of Xbp1) was required for PHLDA3 induction by Tm, whereas other pathways (c-Jun N-terminal kinase (JNK), protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6)) were not. PHLDA3 overexpression correlated with the severity of hepatocyte injury in animal or cell model of ER stress. In p53-deficient cells, ER stress inducers transactivated PHLDA3 with a decrease in cell viability. ER stress-induced hepatocyte death depended on serine/threonine protein kinase B (Akt) inhibition by PHLDA3. Lentiviral delivery of PHLDA3 shRNA to mice abrogated p-Akt inhibition in the liver by Tm, attenuating hepatocyte injury.ER stress in hepatocytes induces PHLDA3 via IRE1-Xbp1s pathway, which facilitates liver injury by inhibiting Akt.
Project description:Coronaviruses have emerged as alarming pathogens owing to their inherent ability of genetic variation and cross-species transmission. Coronavirus infection burdens the endoplasmic reticulum (ER.), causes reactive oxygen species production and induces host stress responses, including unfolded protein response (UPR) and antioxidant system. In this study, we have employed a neurotropic murine β-coronavirus (M-CoV) infection in the Central Nervous System (CNS) of experimental mice model to study the role of host stress responses mediated by interplay of DJ-1 and XBP1. DJ-1 is an antioxidant molecule with established functions in neurodegeneration. However, its regulation in virus-induced cellular stress response is less explored. Our study showed that M-CoV infection activated the glial cells and induced antioxidant and UPR genes during the acute stage when the viral titer peaks. As the virus particles decreased and acute neuroinflammation diminished at day ten p.i., a significant up-regulation in UPR responsive XBP1, antioxidant DJ-1, and downstream signaling molecules, including Nrf2, was recorded in the brain tissues. Additionally, preliminary in silico analysis of the binding between the DJ-1 promoter and a positively charged groove of XBP1 is also investigated, thus hinting at a mechanism behind the upregulation of DJ-1 during MHV-infection. The current study thus attempts to elucidate a novel interplay between the antioxidant system and UPR in the outcome of coronavirus infection.
Project description:Success or failure of pancreatic beta cell adaptation to ER stress is a determinant of diabetes susceptibility. The ATF6 and IRE1/XBP1 pathways are separate ER stress-response effectors important to beta cell health and function. ATF6α. and XBP1 direct overlapping transcriptional responses in some cell types. However, the signaling dynamics and interdependence of ATF6α and XBP1 in pancreatic beta cells have not been explored. To assess pathway-specific signal onset, we performed timed exposures of primary mouse islet cells to ER stressors and measured the early transcriptional response. Comparing the time course of induction of ATF6 and XBP1 targets suggested that the two pathways have similar response dynamics. The role of ATF6α in target induction was assessed by acute knockdown using islet cells from <i>Atf6</i>α <i><sup>flox/flox</sup></i> mice transduced with adenovirus expressing Cre recombinase. Surprisingly, given the mild impact of chronic deletion in mice, acute ATF6α knockdown markedly reduced ATF6-pathway target gene expression under both basal and stressed conditions. Intriguingly, although ATF6α knockdown did not alter <i>Xbp1</i> splicing dynamics or intensity, it did reduce induction of XBP1 targets. Inhibition of <i>Xbp1</i> splicing did not decrease induction of ATF6α targets. Taken together, these data suggest that the XBP1 and ATF6 pathways are simultaneously activated in islet cells in response to acute stress and that ATF6α is required for full activation of XBP1 targets, but XBP1 is not required for activation of ATF6α targets. These observations improve understanding of the ER stress transcriptional response in pancreatic islets.
Project description:HCV infection induces autophagy, but how this occurs is unclear. Here, we report the induction of autophagy by the structural HCV core protein and subsequent endoplasmic reticular (ER) stress in Huh7 hepatoma cells. During ER stress, both the EIF2AK3 and ATF6 pathways of the unfolded protein response (UPR) were activated by HCV core protein. Then, these pathways upregulated transcription factors ATF4 and DDIT3. The ERN1-XBP1 pathway was not activated. Through ATF4 in the EIF2AK3 pathway, the autophagy gene ATG12 was upregulated. DDIT3 upregulated the transcription of autophagy gene MAP1LC3B (LC3B) by directly binding to the -253 to -99 base region of the LC3B promoter, contributing to the development of autophagy. Collectively, these data suggest not only a novel role for the HCV core protein in autophagy but also offer new insight into detailed molecular mechanisms with respect to HCV-induced autophagy, specifically how downstream UPR molecules regulate key autophagic gene expression.
Project description:Autophagy dysfunction is a potential toxic effect of nanoparticles. Previous studies have indicated that silica nanoparticles (SiNPs) induce macroautophagy/autophagy dysfunction, while the precise mechanisms remain uncertain. Hence, the present study investigated the molecular mechanisms by which SiNPs enhanced autophagosome synthesis, which then contributed to autophagy dysfunction. First, the effects of SiNPs on autophagy and autophagic flux were verified using transmission electron microscopy, laser scanning confocal microscopy, and western blot assays. Then, the activation of endoplasmic reticular (ER) stress was validated to be through the EIF2AK3 and ATF6 UPR pathways but not the ERN1-XBP1 pathway, along with the upregulation of downstream ATF4 and DDIT3. Thereafter, the ER stress inhibitor 4-phenylbutyrate (4-PBA) was used to verify that SiNP-induced autophagy could be influenced by ER stress. Furthermore, specialized lentiviral shRNA were employed to determine that autophagy was induced via specific activation of the EIF2AK3 and ATF6 UPR pathways. Finally, the 2 autophagic genes LC3B and ATG12 were found to be transcriptionally upregulated by downstream ATF4 and DDIT3 in ER stress, which contributed to the SiNP-enhanced autophagosome synthesis. Taken together, these data suggest that SiNPs induced autophagosome accumulation via the activation of the EIF2AK3 and ATF6 UPR pathways in hepatocytes, which offers a new insight into detailed molecular mechanisms underlying SiNP-induced autophagy dysfunction, and specifically how UPR pathways regulate key autophagic genes. This work provides novel evidence for the study of toxic effects and risk assessment of SiNPs.
Project description:Major intrinsic protein (MIP) is a functional water-channel (AQP0) that also plays a key role in establishing lens fiber cell architecture. Genetic variants of MIP have been associated with inherited and age-related forms of cataract; however, the underlying pathogenic mechanisms are unclear. Here we have used lens transcriptome profiling by microarray-hybridization and qPCR to identify pathogenic changes during cataract development in Mip-mutant (Lop/+) mice. In postnatal Lop/+ lenses (P7) 99 genes were up-regulated and 75 were down-regulated (>2-fold, p=<0.05) when compared with wild-type. A pathway analysis of up-regulated genes in the Lop/+ lens (P7) was consistent with endoplasmic reticulum (ER)-stress and activation of the unfolded protein response (UPR). The most up-regulated UPR genes (>4-fold) in the Lop/+ lens included Chac1>Ddit3>Atf3>Trib3>Xbp1 and the most down-regulated genes (>5-fold) included two anti-oxidant genes, Hspb1 and Hmox1. Lop/+ lenses were further characterized by abundant TUNEL-positive nuclei within central degenerating fiber cells, glutathione depletion, free-radical overproduction, and calpain hyper-activation. These data suggest that Lop/+ lenses undergo proteotoxic ER-stress induced cell-death resulting from prolonged activation of the Eif2ak3/Perk-Atf4-Ddit3-Chac1 branch of the UPR coupled with severe oxidative-stress.
Project description:Endoplasmic reticulum (ER) stress and inappropriate adaptation through the unfolded protein response (UPR) are predominant features of pathological processes. However, little is known about the link between ER stress and endovascular injury. We investigated the involvement of ER stress in neointima hyperplasia after vascular injury. The femoral arteries of 7-8-week-old male mice were subjected to wire-induced vascular injury. After 4 weeks, immunohistological analysis showed that ER stress markers were upregulated in the hyperplastic neointima. Neointima formation was increased by 54.8% in X-box binding protein-1 (XBP1) heterozygous mice, a model of compromised UPR. Knockdown of Xbp1 in human coronary artery smooth muscle cells (CASMC) in vitro promoted cell proliferation and migration. Furthermore, treatment with ER stress reducers, 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA), decreased the intima-to-media ratio after wire injury by 50.0% and 72.8%, respectively. Chronic stimulation of CASMC with PDGF-BB activated the UPR, and treatment with 4-PBA and TUDCA significantly suppressed the PDGF-BB-induced ER stress markers in CASMC and the proliferation and migration of CASMC. In conclusion, increased ER stress contributes to neointima formation after vascular injury, while UPR signaling downstream of XBP1 plays a suppressive role. Suppression of ER stress would be a novel strategy against post-angioplasty vascular restenosis.
Project description:Elevated plasma concentration of the pro-atherogenic oxidized low density lipoprotein cholesterol (LDL) triggers adverse effects in pancreatic beta-cells and is associated with type 2 diabetes. Here, we investigated whether the endoplasmic reticulum (ER) stress is a key player coupling oxidative stress to beta-cell dysfunction and death elicited by human oxidized LDL. We found that human oxidized LDL activates ER stress as evidenced by the activation of the inositol requiring 1?, and the elevated expression of both DDIT3 (also called CHOP) and DNAJC3 (also called P58IPK) ER stress markers in isolated human islets and the mouse insulin secreting MIN6 cells. Silencing of Chop and inhibition of ER stress markers by the chemical chaperone phenyl butyric acid (PBA) prevented cell death caused by oxidized LDL. Finally, we found that oxidative stress accounts for activation of ER stress markers induced by oxidized LDL. Induction of Chop/CHOP and p58IPK/P58IPK by oxidized LDL was mimicked by hydrogen peroxide and was blocked by co-treatment with the N-acetylcystein antioxidant. As a conclusion, the harmful effects of oxidized LDL in beta-cells requires ER stress activation in a manner that involves oxidative stress. This mechanism may account for impaired beta-cell function in diabetes and can be reversed by antioxidant treatment.
Project description:ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased <i>DDIT3</i> and <i>TRIB3</i> mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer.<b>Abbreviations:</b> 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ<sup>9</sup>-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.
Project description:Loss of protein homeostasis (proteostasis) in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR), restoring correct protein folding. Sustained ER stress exacerbates activation of the major UPR branches (IRE1α/XBP1, PERK/ATF4, ATF6), inducing expression of numerous genes involved in inflammation, cell death, autophagy, and oxidative stress. We investigated whether epigenetic dynamics mediated by histone H3K9 and H3K27 methylation might help to reduce or inhibit the exacerbated and maladaptive UPR triggered in tubular epithelial cells. Epigenetic treatments, specific silencing, and chromatin immunoprecipitation assays were performed in human proximal tubular cells subjected to ER stress. Pharmacological blockage of KDM4C and JMJD3 histone demethylases with SD-70 and GSKJ4, respectively, enhanced trimethylation of H3K9 and H3K27 in the <i>ATF4</i> and <i>XBP1</i> genes, inhibiting their expression and that of downstream genes. Conversely, specific G9a and EZH2 knockdown revealed increases in ATF4 and XBP1 expression. This is a consequence of the reduced recruitment of G9a and EZH2 histone methylases, diminished H3K9me3 and H3K27me3 levels, and enhanced histone acetylation at the <i>ATF4</i> and <i>XBP1</i> promoter region. G9a and EZH2 cooperate to maintain the repressive chromatin structure in both UPR-induced genes, ATF4 and XBP1. Therefore, preserving histone H3K9 and H3K27 methylation could ameliorate the ER stress, and consequently the oxidative stress and the triggered pathological processes that aggravate renal damage.