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MCL-1 is modulated in Crohn's disease fibrosis by miR-29b via IL-6 and IL-8.

ABSTRACT: The miR-29 family is involved in fibrosis in multiple organs, including the intestine where miR-29b facilitates TGF-β-mediated up-regulation of collagen in mucosal fibroblasts from Crohn's disease (CD) patients. Myeloid cell leukemia-1 (MCL-1), a member of the B-cell CLL/Lymphoma 2 (BCL-2) apoptosis family, is involved in liver fibrosis and is targeted by miR-29b via its 3'-UTR in cultured cell lines. We investigate the role of MCL-1 and miR-29b in primary intestinal fibroblasts and tissue from stricturing CD patients. Transfection of CD intestinal fibroblasts with pre-miR-29b resulted in a significant increase in the mRNA expression of MCL-1 isoforms [MCL-1Long (L)/Extra Short (ES) and MCL-1Short (S)], although MCL-1S was expressed at significantly lower levels. Western blotting predominantly detected the anti-apoptotic MCL-1L isoform, and immunofluorescence showed that staining was localised in discrete nuclear foci. Transfection with pre-miR-29b or anti-miR-29b resulted in a significant increase or decrease, respectively, in MCL-1L foci. CD fibroblasts treated with IL-6 and IL-8, inflammatory cytokines upstream of MCL-1, increased the total mass of MCL-1L-positive foci. Furthermore, transfection of intestinal fibroblasts with pre-miR-29b resulted in an increase in mRNA and protein levels of IL-6 and IL-8. Finally, immunohistochemistry showed reduced MCL-1 protein expression in fibrotic CD samples compared to non-stricturing controls. Together, our findings suggest that induction of MCL-1 by IL-6/IL-8 may surmount any direct down-regulation by miR-29b via its 3'-UTR. We propose that an anti-fibrotic miR-29b/IL-6 IL-8/MCL-1L axis may influence intestinal fibrosis in CD. In the future, therapeutic modulation of this pathway might contribute to the management of fibrosis in CD.

SUBMITTER: Nijhuis A

PROVIDER: S-EPMC5397660 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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MCL-1 is modulated in Crohn's disease fibrosis by miR-29b via IL-6 and IL-8.

Nijhuis Anke A Curciarello Renata R Mehta Shameer S Feakins Roger R Bishop Cleo L CL Lindsay James O JO Silver Andrew A

Cell and tissue research 20170211 2

The miR-29 family is involved in fibrosis in multiple organs, including the intestine where miR-29b facilitates TGF-β-mediated up-regulation of collagen in mucosal fibroblasts from Crohn's disease (CD) patients. Myeloid cell leukemia-1 (MCL-1), a member of the B-cell CLL/Lymphoma 2 (BCL-2) apoptosis family, is involved in liver fibrosis and is targeted by miR-29b via its 3'-UTR in cultured cell lines. We investigate the role of MCL-1 and miR-29b in primary intestinal fibroblasts and tissue from ...[more]

PMID: 28190086

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Project description:The Na/K-ATPase is an important membrane ion transporter and a signaling receptor that is essential for maintaining normal cell function. The current study examined the role of Na/K-ATPase signaling in regulating miR-29b-3p, an anti-fibrotic microRNA, in a mouse chronic kidney disease (CKD) model (5/6th partial nephrectomy or PNx). The results showed that CKD induced significant reduction of miR-29b-3p expression in the heart tissue by activation of Src and NFκB signaling in these animals. To demonstrate the role of Na/K-ATPase signaling, we also performed the PNx surgery on Na/K-ATPase α1 heterozygous (α1+/-) mice, which expresses ~40% less Na/K-ATPase α1 compared to their wild type littermates (WT) and exhibits deficiency in Na/K-ATPase signaling. We found that CKD did not significantly change the miR-29b-3p expression in heart tissue from the α1+/- animals. We also found that CKD failed to activate Src and NFκB signaling in these animals. Using isolated cardiac fibroblasts from α1+/- mice and their WT littermates, we showed that ouabain, a specific Na/K-ATPase ligand, induces decreased miR-29b-3p expression in fibroblasts isolated from WT mice, but had no effect in cells from α1+/- mice. Inhibition of NFκB by Bay11-7082 prevented ouabain-induced miR-29b-3p reduction in WT fibroblasts. To further confirm the in vivo effect of Na/K-ATPase signaling in regulation of miR-29b-3p and cardiac fibrosis in CKD animals, we used pNaKtide, a Src inhibiting peptide derived from the sequence of Na/K-ATPase, to block the activation of Na/K-ATPase signaling. The result showed that pNaKtide injection significantly increased miR-29b-3p expression and mitigated the CKD-induced cardiac fibrosis in these animals. These results clearly demonstrated that Na/K-ATPase signaling is an important mediator in CKD that regulates miR-29b-3p expression and cardiac fibrosis, which provides a novel target for regulation of miR-29b-3p in CKD. We also demonstrate that antagonizing Na/K-ATPase signaling by pNaKtide can reduce organ fibrosis through the stimulation of tissue miR-29b-3p expression.

Dataset Information

MCL-1 is modulated in Crohn's disease fibrosis by miR-29b via IL-6 and IL-8.

Publications

MCL-1 is modulated in Crohn's disease fibrosis by miR-29b via IL-6 and IL-8.

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