Kindlin-2 regulates hepatic stellate cells activation and liver fibrogenesis

Abstract

Liver fibrosis, the common response associated with chronic liver diseases, ultimately leads to cirrhosis, a major public health problem worldwide. Activation of hepatic stellate cells (HSCs) by transforming growth factor (TGF)-β1 is a key step in liver fibrosis. Here we report that Kindlin-2 expression is elevated in the livers of mice with experimental liver fibrosis and also in the livers of patients with liver fibrosis. TGF-β1 increases Kindlin-2 expression in cultured HSCs in a p38 and ERK mitogen-activated protein kinase (MAPK)-dependent manner, partly. More importantly, Kindlin-2 deficiency significantly attenuated mouse liver fibrosis and HSC activation. Mechanistically, Kindlin-2 promotes TGF-β signaling through upregulation of Smad2 and Smad3 phosphorylation. Our work demonstrates an important role for Kindlin-2 in liver fibrosis, and inhibiting Kindlin-2 in the livers may represent a novel strategy to treat liver fibrosis.

Fig. 1. Expression of Kindlin-2 is upregulated in human and mouse liver fibrosis.

a HE, Masson staining and double immunofluorescence staining for Kindlin-2 (green) and a-SMA (red) in normal (n = 5) and fibrotic human livers (n = 8). Scale bars = 200 μm. b Dual immunofluorescence was used to identify the expression of Kindlin-2 (green) and a-SMA (red) in liver biopsy samples from WT mice with or without injection of CCl₄ (n = 6 for WT and n = 6 for CCl₄). Scale bars = 200 μm. c Representative western blot analysis of Kindlin-2 in mouse livers from WT mice with or without injection of CCl₄. Quantitative analyses of Kindlin-2 and Fn are shown in the lower panel. **p < 0.01

Fig. 2. TGF-β1 increases Kindlin-2 expression in HSCs via p38 and ERK MAPK.

a Representative western blot analysis of Kindlin-2 from LX-2 cells treated with TGF-β1 (10 ng/ml) for the indicated time period. The right panel shows quantitative analyses of Kindlin-2 after normalization with tubulin. b Representative western blot analysis of Kindlin-2 from LX-2 cells treated with TGF-β1 for 24 h for indicated concentrations. c Quantitative PCR analyses of Kindlin-2 mRNA from LX-2 cells treated with indicated concentrations of TGF-β1 for 24 h (left panel) or 10 ng/ml of TGF-β1 for indicated time intervals (right panel). Western blot (d) and immunofluorescence analysis (e) of Kindlin-2 from LX-2 cells were treated with or without SP600125 (10 μM), U0126 (1 μM), or SB203580 (1 μM), following TGF-β1 treatment for 24 h. The data are representative of three independent experiments. Scale bars = 200 μm. *p < 0.05. **p < 0.01

Fig. 3. Inhibition of Kindlin-2 diminishes the pro-fibrogenic activities of TGF-β1.

a LX-2 cells are transfected with control siRNA or Kindlin-2 siRNA followed by TGF-β1 (10 ng/ml) treatment for 48 h. The expressions of Kindlin-2, Col1A1, and Fn were determined by western blot. b–d After transfection with control siRNA or Kindlin-2 siRNA, LX-2 cells were treated with or without TGF-β1 (10 ng/ml) treatment for 24 h. Real-time PCR analyses of Fn, Col1A1, and α-SMA mRNA were performed. e Immunofluorescence analysis of Fn and α-SMA from LX-2 cells that were transfected with control siRNA or Kindlin-2 siRNA and then treated with or without 10 ng/ml TGF-β1 for 48 h. The data are representative of three independent experiments. Scale bars = 200 μm. *p < 0.05

Fig. 4. Overexpression of Kindlin-2 promotes TGF-β1-induced HSC activation.

a LX-2 cells were transfected with control vector or Kindlin-2 vector followed by TGF-β1 (10 ng/ml) treatment for 48 h. The levels of Kindlin-2, Col1A1, and Fn were determined by western blot analysis. b LX-2 cells were transfected and treated as in a–c. Immunofluorescence assays were performed with anti-Fn and anti-α-SMA antibodies. The data are representative of three independent experiments. Scale bars = 200 μm. *p < 0.05

Fig. 5. Kindlin-2 promotes Smad2/3 phosphorylation.

a LX-2 cells transfected with control siRNA or Kindlin-2 siRNA and further treated with 10 ng/ml TGF-β1 for the indicated time period. P-Smad2/3 and total Smad2/3 levels were detected by western blot. b LX-2 cells transfected with control siRNA or Kindlin-2 siRNA and further treated with 10 ng/ml TGF-β1 for 30 min. The expressions of P-Smad2/3 and total Smad2/3 were determined by immunofluorescence assays. c LX-2 cells were transfected with control vector or Kindlin-2 vector, followed by TGF-β1 (10 ng/ml) treatment for the indicated time period. P-Smad2/3 and total Smad2/3 levels were detected by western blot. d LX-2 cells were transfected with control vector or Kindlin-2 vector, followed by TGF-β1 (10 ng/ml) treatment for 30 min. The expressions of P-Smad2/3 and total Smad2/3 were determined by immunofluorescence assays. The data are representative of three independent experiments. Scale bars = 200 μm

Fig. 6. Kindlin-2 deficiency attenuates mouse liver fibrosis following CCl₄ treatment.

WT and Kindlin-2^+/− mice (n = 6–8 in each group) underwent CCl₄ injection for 6 weeks. a Liver fibrosis was evaluated by H&E, Sirius Red, and Masson’s trichrome staining. Scale bars = 200 μm. b Western blot analysis of Kindlin-2 in livers from WT and Kindlin-2^+/− mice. The right panels show quantitative analyses of Kindlin-2 expression after normalization with β-actin. c Representative western blot analysis of α-SMA expressions in the livers from WT and Kindlin-2^+/− mice. The right panels show quantitative analyses of α-SMA expression after normalization with β-actin. d The mRNA expressions of col1a1, col1a2, and col3a1 were determined by real-time PCR. *p < 0.05, **p < 0.01, ***p < 0.001