Dataset Information

Negative regulation of TGF-?1-induced MKK6-p38 and MEK-ERK signalling and epithelial-mesenchymal transition by Rac1b.

ABSTRACT: Prompted by earlier findings that the Rac1-related isoform Rac1b inhibits transforming growth factor (TGF)-?1-induced canonical Smad signalling, we studied here whether Rac1b also impacts TGF-?1-dependent non-Smad signalling such as the MKK6-p38 and MEK-ERK mitogen-activated protein kinase (MAPK) pathways and epithelial-mesenchymal transition (EMT). Transient depletion of Rac1b protein in pancreatic cancer cells by RNA interference increased the extent and duration of TGF-?1-induced phosphorylation of p38 MAPK in a Smad4-independent manner. Rac1b depletion also strongly increased basal ERK activation - independent of the kinase function of the TGF-? type I receptor ALK5 - and sensitised cells towards further upregulation of phospho-ERK levels by TGF-?1, while ectopic overexpression of Rac1b had the reverse effect. Rac1b depletion increased an EMT phenotype as evidenced by cell morphology, gene expression of EMT markers, cell migration and growth inhibition. Inhibition of MKK6-p38 or MEK-ERK signalling partially relieved the Rac1b depletion-dependent increase in TGF-?1-induced gene expression and cell migration. Rac1b depletion also enhanced TGF-?1 autoinduction of crucial TGF-? pathway components and decreased that of TGF-? pathway inhibitors. Our results show that Rac1b antagonises TGF-?1-dependent EMT by inhibiting MKK6-p38 and MEK-ERK signalling and by controlling gene expression in a way that favors attenuation of TGF-? signalling.

SUBMITTER: Witte D

PROVIDER: S-EPMC5725500 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Negative regulation of TGF-β1-induced MKK6-p38 and MEK-ERK signalling and epithelial-mesenchymal transition by Rac1b.

Witte David D Otterbein Hannah H Förster Maria M Giehl Klaudia K Zeiser Robert R Lehnert Hendrik H Ungefroren Hendrik H

Scientific reports 20171211 1

Prompted by earlier findings that the Rac1-related isoform Rac1b inhibits transforming growth factor (TGF)-β1-induced canonical Smad signalling, we studied here whether Rac1b also impacts TGF-β1-dependent non-Smad signalling such as the MKK6-p38 and MEK-ERK mitogen-activated protein kinase (MAPK) pathways and epithelial-mesenchymal transition (EMT). Transient depletion of Rac1b protein in pancreatic cancer cells by RNA interference increased the extent and duration of TGF-β1-induced phosphorylat ...[more]

PMID: 29229918

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Project description:Background and purposeExcessive fructose consumption is a risk factor for liver fibrosis. Pterostilbene protects against liver fibrosis. Here, we investigated the potential role and the mechanisms underlying the hepatocyte epithelial-mesenchymal transition (EMT) in fructose-induced liver fibrosis and protection by pterostilbene.Experimental approachCharacteristic features of liver fibrosis in 10% fructose-fed rats and EMT in 5 mM fructose-exposed BRL-3A cells with or without pterostilbene and the change of miR-34a/Sirt1/p53 and transforming growth factor-β1 (TGF-β1)/Smads signalling were examined. MiR-34a inhibitor, miR-34a minic, or p53 siRNA were used to explore the role of miR-34a/Sirt1/p53 signalling in fructose-induced EMT and the action of pterostilbene.Key resultsPterostilbene prevented fructose-induced liver injury with fibrosis in rats. Fructose caused hepatocyte undergoing EMT, gaining fibroblast-specific protein 1 and vimentin, and losing E-cadherin, effects attenuated by pterostilbene. Moreover, fructose induced miR-34a overexpression in hepatocytes with down-regulated Sirt1, increased p53 and ac-p53, and activated TGF-β1/Smads signalling, whereas these disturbances were suppressed by miR-34a inhibitor. Additionally, miR-34a inhibitor and p53 siRNA prevented TGF-β1-driven hepatocyte EMT under fructose exposure. Pterostilbene down-regulated miR-34a, up-regulated Sirt1, and suppressed p53 activation and TGF-β1/Smads signalling in fructose-stimulated animals and cells but showed no additional effects with miR-34a inhibitor on miR-34a/Sirt1/p53 signalling in fructose-exposed hepatocytes.Conclusions and implicationsThese results strongly suggest that activation of miR-34a/Sirt1/p53 signalling is required for fructose-induced hepatocyte EMT mediated by TGF-β1/Smads signalling, contributing to liver fibrosis in rats. Pterostilbene exhibits a protective effect against liver fibrosis at least partly through inhibiting miR-34a/Sirt1/p53 signalling activation.

Project description:Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies regarding their therapeutic value in the lung EMT process are lacking. The human bronchial epithelial cell line (BEAS-2B) and primary human airway epithelial (pHAE) cells were exposed to TGF-β1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers (collagen Ӏ, α-SMA, fibronectin) were analyzed (mRNA, protein). ELISA measured TGF-β1 release. TGF-β1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced while using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-β1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, and forskolin were used to elevate intracellular cAMP. TGF-β1 induced morphological changes, decreased E-cadherin, but increased collagen Ӏ and cell migration, a process that was reversed by the inhibitor of δ/epsilon casein kinase I, PF-670462. TGF-β1 altered (mRNA, protein) expression of Ezrin, AKAP95, and Yotiao. St-Ht31, the AKAP antagonist, decreased E-cadherin (mRNA, protein), but counteracted TGF-β1-induced collagen Ӏ upregulation. Cigarette smoke (CS) increased TGF-β1 release, activated TGF signaling, augmented cell migration, and reduced E-cadherin expression, a process that was blocked by TGF-β1 neutralizing antibody. The silencing of Ezrin, AKAP95, and Yotiao diminished TGF-β1-induced collagen Ӏ expression, as well as TGF-β1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells, pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95, and Yotiao promote TGF-β1-mediated EMT, linked to a TGF-β1 release by CS. AKAP members might define the ability of fenoterol, rolipram, and cilostamide to modulate the EMT process, and they might represent potential relevant targets in the treatment of COPD.

Dataset Information

Negative regulation of TGF-?1-induced MKK6-p38 and MEK-ERK signalling and epithelial-mesenchymal transition by Rac1b.

Publications

Negative regulation of TGF-β1-induced MKK6-p38 and MEK-ERK signalling and epithelial-mesenchymal transition by Rac1b.

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