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Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.

ABSTRACT: Saturated fatty acids (SFAs) have been shown to induce endoplasmic reticulum (ER) stress and chronic inflammatory responses, as well as alter sphingolipid metabolism. Disruptions in ER stress and sphingolipid metabolism have also been implicated in intestinal inflammation. Therefore, to elucidate the roles of SFAs in ER stress and inflammation in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, induced ER stress signaling, including activation of inositol-requiring enzyme 1 (IRE1) and X-box binding protein 1 (XBP1) signaling. Myristate significantly increased C14-ceramide levels, whereas palmitate increased several long-chain ceramides. To define the role of ceramide synthases (CerSs) in myristate-induced ER stress, we used the pharmacologic inhibitor, fumonisin B1 (FB1), and small interfering RNA (siRNA) for CerS5 and 6, the primary isoforms that are involved in C14-ceramide generation. FB1 and siRNA for CerS5 or 6 suppressed myristate-induced C14-ceramide generation and XBP1 splicing (XBP1s). Moreover, increased XBP1s induced the downstream expression of IL-6 in a CerS5/6-dependent manner. In addition, a myristate-enriched milk fat-based diet, but not a lard-based diet, increased C14-ceramide, XBP1s, and IL-6 expression in vivo. Taken together, our data suggest that myristate modulates ER stress and cytokine production in the intestinal epithelium via CerS5/6 and C14-ceramide generation.-Choi, S., Snider, J. M., Olakkengil, N., Lambert, J. M., Anderson, A. K., Ross-Evans, J. S., Cowart, L. A., Snider, A. J. Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.

SUBMITTER: Choi S

PROVIDER: S-EPMC6133702 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.

Choi Songhwa S Snider Justin M JM Olakkengil Nicole N Lambert Johana M JM Anderson Andrea K AK Ross-Evans Jessica S JS Cowart L Ashley LA Snider Ashley J AJ

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20180516 10

Saturated fatty acids (SFAs) have been shown to induce endoplasmic reticulum (ER) stress and chronic inflammatory responses, as well as alter sphingolipid metabolism. Disruptions in ER stress and sphingolipid metabolism have also been implicated in intestinal inflammation. Therefore, to elucidate the roles of SFAs in ER stress and inflammation in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, induced ER stress signaling, including ...[more]

PMID: 29768040

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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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Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.

Publications

Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.

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