Abstract
BackgroundTransforming growth factor-β1 (TGF-β1) plays a key role in mesothelial-to-mesenchymal transition (MMT) during peritoneal dialysis (PD). However, the role of Akt in MMT transformation in PD is not clear.ResultsIn this study, we observed that the phosphorylated form of protein kinase B (Akt), termed as pAkt, was up-regulated in the peritoneum of mice undergoing PD. It was associated with thickening of the peritoneum and up-regulation of TGF-β1. Upregulation of pAkt paralleled with the increased expression of Smad ubiquitination regulatory factor 2 (Smurf2), Vimentin and fibronectin (FN), and decreased expression of mothers against decapentaplegic homolog 7 (Smad7) and Zonula Occludens protein 1(ZO-1) in mice undergoing PD treatment and in TGF-β1 induced human peritoneal mesothelial cells (HPMCs). These changes were reversed with the treatment of a PI3K/Akt inhibitor LY294002 in vivo or in cells transfected with Akt dominant-negative (Akt-DN) plasmids in vitro. Increased Smurf2 expression in HPMCs, induced by TGF-β1 was accompanied with altered expression of Transforming growth factor receptor I (TβR-I), Smad7, ZO-1, Vimentin and FN via Akt modulation. In addition, inhibition of Ubiquitin carboxyl-terminal hydrolase 4 (USP4) decreased TGF- β1-induced expression of TβR-I and reversed the altered expression of Smad7, Smurf2, ZO-1 and Vimentin. Moreover, TGF-β1 accentuated the interactions between Smurf2 and Smad7, while reduced the association between TβR-I and Smurf2. These interactions were reversed by the treatment of Akt-DN and USP4 siRNA, respectively.ConclusionsThese data implied that Akt mediated MMT in PD via Smurf2 modulation/and or Smad7 degradation while conceivably maintaining the TβRI stability, most likely by the USP4.
Highlights
Transforming growth factor-β1 (TGF-β1) plays a key role in mesothelial-to-mesenchymal transition (MMT) during peritoneal dialysis (PD)
Expression of TGF-β1 and p-Akt, Smad7 ubiquitination regulatory factor2 (Smurf2) and Smad7 in PD mice Enzyme-Linked Immunosorbent Assay (ELISA) assay showed that the concentration of TGF-β1 increased in the peritoneal effluent of PD mice compared to control (P < 0.01), while there are no further significant changes in mice treated with the phosphatidylinositol 3-kinase kinase (PI3K)/Akt inhibitor, LY294002 (Figure 1A)
Confocal imaging with Antiphospho-Akt1 (Ser473/Tyr474) antibody showed that there was a low level of phosphorylated Akt expression in the peritoneum of control mice and it markedly increased in PD mice
Summary
Transforming growth factor-β1 (TGF-β1) plays a key role in mesothelial-to-mesenchymal transition (MMT) during peritoneal dialysis (PD). The Smurf levels have been reported to be increased in early period of fibrosis in rat liver and TGF-β1-treated LX-2 cells, and they are accompanied with reduced levels of Smad7 [15]. It seems that Smad provides a negative feedback to TβR1 by binding to Smurf and brings Smurf to the activated TβR for their polyubiquitination and degradation [16]. Overexpression of Smad inhibits Smad2/ 3 activation and the EMT related protein expression, extracellular matrix protein (ECM) and fibrosis in the peritoneal mesothelial cells and animal models of PD [17,18,19]. Whether Akt induces Smurf expression and inhibits Smad participation in MMT transformation during PD and the relevant mechanism(s) involved have not been thoroughly explored
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