Tripartite motif protein 52 (TRIM52) promoted fibrosis in LX‐2 cells through PPM1A‐mediated Smad2/3 pathway

To investigate the roles of tripartite motif containing 52 (TRIM52) in human hepatic fibrosis in vitro, human hepatic stellate cell line LX‐2 cells were transfected with hepatitis B virus (HBV) replicon to establish HBV‐induced fibrosis in LX‐2 cells, and then treated with small interfering RNA‐mediated knockdown of TRIM52 (siTRIM52). LX‐2 cells without HBV replicon transfection were treated with lentiviruses‐mediated overexpression of TRIM52 and phosphatase magnesium dependent 1A (PPM1A). Fibrosis response of LX‐2 cells were assessed by the production of hydroxyproline (Hyp) and collagen I/III, as well as protein levels of α‐smooth muscle actin (α‐SMA). PPM1A and phosphorylated (p)‐Smad2/3 were measured to assess the mechanism. The correlation between TRIM52 and PPM1A was determined using co‐immunoprecipitation, and whether and how TRIM52 regulated the degradation of PPM1A were determined by ubiquitination assay. Our data confirmed HBV‐induced fibrogenesis of LX‐2 cells, as evidenced by significant increase in Hyp and collagen I/III and α‐SMA, which was associated with reduction of PPM1A and elevation of transforming growth factor‐β (TGF‐β), p‐Smad2/3, and p‐Smad3L. However, those changes induced by HBV were significantly attenuated with additional siTRIM52 treatment. Similar to HBV, overexpression of TRIM52 exerted promoted effect in the fibrosis of LX‐2 cells. Interestingly, TRIM52 induced the fibrogenesis of LX‐2 cells and the activation of TGF‐β/Smad pathway were significantly reversed by PPM1A overexpression. Furthermore, our data confirmed TRIM52 as a deubiquitinase that influenced the accumulation of PPM1A protein, and subsequently regulated the fibrogenesis of LX‐2 cells. TRIM52 was a fibrosis promoter in hepatic fibrosis in vitro, likely through PPM1A‐mediated TGF‐β/Smad pathway.     

Chemokine (C-X-C motif) ligand 14 contributes to lipopolysaccharide-induced fibrogenesis in mouse L929 fibroblasts via modulating PPM1A

Herein, we found that serum chemokine ligand 14 (CXCL14) was significantly enhanced in patients with idiopathic pulmonary fibrosis (IPF). In our current study, mouse L929 fibroblasts were stimulated with lipopolysaccharide (LPS) (100 ng/mL). Cell proliferation, the levels of matrix metalloproteinase 2 (MMP2) and MMP9, as well as extracellular matrix (ECM) content were assessed to evaluate the fibrogenesis of L929 cells. Proliferating cell nuclear antigen and cell viability were assessed to evaluate cell proliferation. Hydroxyproline (Hyp), collagen I/III, connective tissue growth factor (CTGF), and phosphorylated Smad2/3 (p-Smad2/3) were assessed to evaluate ECM secretion and deposition. α-Smooth muscle actin (α-SMA) was used to measure the occurrence of differentiation from fibroblast toward myofibroblast. Our data suggested that knockdown of CXCL14 prevented LPS-induced fibrogenesis of L929 cells through inhibiting cell proliferation and decreasing the expression of MMP2/9, Hyp, collagen I/III, CTGF, p-Smad2/3, and α-SMA. Notably, upregulation of protein phosphatase magnesium-dependent 1A (PPM1A) was involved in this process. On the contrary, recombinant CXCL14 protein led to an opposite effect. We first suggested that overexpression of PPM1A ameliorated LPS-induced fibrogenesis. Furthermore, we substantiated that knockdown of CXCL14 exerted an antifibrotic effect in IPF in vitro probably via the upregulation of PPM1A. Besides, evidently enhanced CXCL14, yet reduced PPM1A, was found in bleomycin-induced rat pulmonary fibrosis, confirming the roles of CXCL14 and its potential association with PPM1A in IPF in vivo. In conclusion, CXCL14 could be considered as a therapeutic target for preventing fibrogenesis of mouse L929 fibroblasts.