Endoplasmic reticulum stress in the intestinal epithelium and inflammatory bowel disease.
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ABSTRACT:
SUBMITTER: Kaser A
PROVIDER: S-EPMC4736746 | biostudies-literature | 2009 Jun
REPOSITORIES: biostudies-literature
Publications
Endoplasmic reticulum stress in the intestinal epithelium and inflammatory bowel disease.
Kaser Arthur A Blumberg Richard S RS
Seminars in immunology 20090223 3
The unfolded protein response as a consequence of endoplasmic reticulum (ER) stress has recently been implicated as a novel mechanism that may lead to inflammatory bowel disease (IBD). Impairment of proper ER stress resolution in highly secretory Paneth and, to a lesser extent, goblet cells within the epithelium can primarily lead to intestinal inflammation. An inability to manage ER stress may not only be a primary originator of intestinal inflammation as exemplified by genetic polymorphisms in ...[more]
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Project description:BACKGROUND: Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously. METHODOLOGY/PRINCIPAL FINDINGS: Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn's disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa. CONCLUSIONS/SIGNIFICANCE: Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.
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Project description:Background & aimsExcess and unresolved endoplasmic reticulum (ER) stress in intestinal epithelial cells (IECs) promotes intestinal inflammation. Activating transcription factor 6 (ATF6) is one of the signaling mediators of ER stress. We studied the pathways that regulate ATF6 and its role for inflammation in IECs.MethodsWe performed an RNA interference screen, using 23,349 unique small interfering RNAs targeting 7783 genes and a luciferase reporter controlled by an ATF6-dependent ERSE (ER stress-response element) promoter, to identify proteins that activate or inhibit the ATF6 signaling pathway in HEK293 cells. To validate the screening results, intestinal epithelial cell lines (Caco-2 cells) were transfected with small interfering RNAs or with a plasmid overexpressing a constitutively active form of ATF6. Caco-2 cells with a CRISPR-mediated disruption of autophagy related 16 like 1 gene (ATG16L1) were used to study the effect of ATF6 on ER stress in autophagy-deficient cells. We also studied intestinal organoids derived from mice that overexpress constitutively active ATF6, from mice with deletion of the autophagy related 16 like 1 or X-Box binding protein 1 gene in IECs (Atg16l1ΔIEC or Xbp1ΔIEC, which both develop spontaneous ileitis), from patients with Crohn's disease (CD) and healthy individuals (controls). Cells and organoids were incubated with tunicamycin to induce ER stress and/or chemical inhibitors of newly identified activator proteins of ATF6 signaling, and analyzed by real-time polymerase chain reaction and immunoblots. Atg16l1ΔIEC and control (Atg16l1fl/fl) mice were given intraperitoneal injections of tunicamycin and were treated with chemical inhibitors of ATF6 activating proteins.ResultsWe identified and validated 15 suppressors and 7 activators of the ATF6 signaling pathway; activators included the regulatory subunit of casein kinase 2 (CSNK2B) and acyl-CoA synthetase long chain family member 1 (ACSL1). Knockdown or chemical inhibition of CSNK2B and ACSL1 in Caco-2 cells reduced activity of the ATF6-dependent ERSE reporter gene, diminished transcription of the ATF6 target genes HSP90B1 and HSPA5 and reduced NF-κB reporter gene activation on tunicamycin stimulation. Atg16l1ΔIEC and or Xbp1ΔIEC organoids showed increased expression of ATF6 and its target genes. Inhibitors of ACSL1 or CSNK2B prevented activation of ATF6 and reduced CXCL1 and tumor necrosis factor (TNF) expression in these organoids on induction of ER stress with tunicamycin. Injection of mice with inhibitors of ACSL1 or CSNK2B significantly reduced tunicamycin-mediated intestinal inflammation and IEC death and expression of CXCL1 and TNF in Atg16l1ΔIEC mice. Purified ileal IECs from patients with CD had higher levels of ATF6, CSNK2B, and HSPA5 messenger RNAs than controls; early-passage organoids from patients with active CD show increased levels of activated ATF6 protein, incubation of these organoids with inhibitors of ACSL1 or CSNK2B reduced transcription of ATF6 target genes, including TNF.ConclusionsIleal IECs from patients with CD have higher levels of activated ATF6, which is regulated by CSNK2B and HSPA5. ATF6 increases expression of TNF and other inflammatory cytokines in response to ER stress in these cells and in organoids from Atg16l1ΔIEC and Xbp1ΔIEC mice. Strategies to inhibit the ATF6 signaling pathway might be developed for treatment of inflammatory bowel diseases.
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Project description:Lactobacillus paracasei is a major probiotic and is well known for its anti-inflammatory properties. Thus, we investigated the effects of L. paracasei-derived extracellular vesicles (LpEVs) on LPS-induced inflammation in HT29 human colorectal cancer cells and dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice. ER stress inhibitors (salubrinal or 4-PBA) or CHOP siRNA were utilized to investigate the relationship between LpEV-induced endoplasmic reticulum (ER) stress and the inhibitory effect of LpEVs against LPS-induced inflammation. DSS (2%) was administered to male C57BL/6 mice to induce inflammatory bowel disease, and disease activity was measured by determining colon length, disease activity index, and survival ratio. In in vitro experiments, LpEVs reduced the expression of the LPS-induced pro-inflammatory cytokines IL-1α, IL-1β, IL-2, and TNFα and increased the expression of the anti-inflammatory cytokines IL-10 and TGFβ. LpEVs reduced LPS-induced inflammation in HT29 cells and decreased the activation of inflammation-associated proteins, such as COX-2, iNOS and NFκB, as well as nitric oxide. In in vivo mouse experiments, the oral administration of LpEVs also protected against DSS-induced colitis by reducing weight loss, maintaining colon length, and decreasing the disease activity index (DAI). In addition, LpEVs induced the expression of endoplasmic reticulum (ER) stress-associated proteins, while the inhibition of these proteins blocked the anti-inflammatory effects of LpEVs in LPS-treated HT29 cells, restoring the pro-inflammatory effects of LPS. This study found that LpEVs attenuate LPS-induced inflammation in the intestine through ER stress activation. Our results suggest that LpEVs have a significant effect in maintaining colorectal homeostasis in inflammation-mediated pathogenesis.
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