TSSC3 suppresses fibrosis of renal tubular epithelium by regulating PI3K/Akt signaling pathway and anoikis resistance in myofibroblasts

Abstract

Objective To investigate the effects of tumor suppressing STF cDNA 3 (TSSC3) on the anoikis resistance and profibrogenic ability of renal myofibroblasts (MyoFb) and its related mechanisms. Methods Renal tubular epithelial HK-2 cells were stimulated with transforming growth factor beta 1 (TGF-β1) to induce the formation of MyoFb. The model of cell anoikis was established by culturing cells in ultra-low adhesion plate suspension or by exogenous RGD-containing peptides culture. In addition, HK-2 cells transfected with TSSC3-overexpressing lentiviral vector were treated with or without TGF-β1, and the effects of TSSC3 on the anoikis and proliferation of cells were observed using flow cytometry and cell counting kit-8 (CCK-8) assay. Finally, qRT-PCR and Western blotting were performed to detect the mRNA and protein expression of the PI3K/AKT pathway and fibrosis related molecules. Results The results demonstrated that α-SMA expression was significantly increased while that of Villin was decreased in HK-2 cells after stimulation of TGF-β1 in a time-dependent manner (P < 0.05), indicating that HK-2 cells were successfully induced to MyoFb, which showed remarkably lower anoikis rate and higher proliferation than HK-2 cells (P < 0.01), and had anoikis resistance. However, the above changes were partially reversed by PI3K/Akt pathway inhibitor LY294002. Further study suggested that overexpression of TSSC3 obviously inhibited the mRNA and protein expression of fibrosis related genes, such as typeⅠand type Ⅲ collagen, fibronectin, PAI-1, MMP-2 and MMP-9 in MyoFb and HK-2 cells. TSSC3 overexpression also up-regulated the level of cleaved-caspase 3 and attenuated the expression of p-PI3K and p-Akt in MyoFb. Conclusion TSSC3 suppresses the anoikis resistance and profibrogenic ability of renal myofibroblasts by down-regulating the PI3K/Akt pathway.

Keywords

• renal fibrosis
• tssc3
• tgf-β1
• hk-2 cells
• pi3k/akt signaling pathway