Murine cirrhosis induces hepatocyte epithelial mesenchymal transition and alterations in survival signaling pathways

Abstract

Hepatocytes that reside in a chronically-injured liver have altered growth responses compared to hepatocytes in normal liver. Transforming growth factor beta (TGFbeta) is upregulated in the cirrhotic liver, and cirrhotic hepatocytes, unlike normal hepatocytes exposed to this cytokine, exhibit decreased apoptosis. In fetal hepatocytes, TGFbeta also induces epithelial-mesenchymal transition (EMT) and signaling changes in cell survival pathways. Here, chronic murine liver injury was induced by twice-weekly carbon tetrachloride administration for 8 weeks. Normal liver-derived hepatocytes (NLDH) and cirrhotic liver-derived hepatocytes (CLDH) were examined for EMT and the small mothers against decapentaplegic homolog (Smad), phosphatidylinositol-3-kinase (PI3K/Akt), and mitogen activated protein kinase (MAPK) pathways were investigated. Immunofluorescence imaging of cirrhotic livers demonstrated increased vimentin expression, which was confirmed by immunoblot analysis. In vitro, CLDH exhibited increased vimentin and type 1 collagen expression within cellular extensions consistent with EMT. Treatment with TGFbeta augmented the EMT response in CLDH. In contrast, untreated NLDH did not display features of EMT but responded to TGFbeta with increased vimentin expression and EMT characteristics. In response to PI3K/Akt inhibition, CLDH had decreased basal and insulin-stimulated p-Akt expression and decreased apoptosis compared to NLDH. In both NLDH and CLDH, vimentin expression was dependent on PI3K/Akt activity. CLDH demonstrated increased basal p-extracellular signal-regulated kinase expression that was independent of Smad and PI3K/Akt signaling. Inhibition of the MAPK pathway produced a marked increase in CLDH apoptosis.

Conclusion: CLDH have increased vimentin and type 1 collagen expression and morphologic features consistent with EMT. In addition, compared to NLDH, the cellular signaling phenotype of CLDH changes from a MAPK-independent pathway to a MAPK-dependent cell survival pathway. These findings may have clinical implications for chemoprevention of hepatocellular carcinoma in the cirrhotic liver.

Fig. 1

Vimentin in cirrhotic livers. (A) Vimentin in normal and cirrhotic mouse livers was visualized by immunofluorescence staining. Each panel represents a separate experiment. Note the characteristic fibrous staining of vimentin in hepatocytes in cirrhotic liver. In contrast, little staining was observed in hepatocytes in control liver. (B) Western blot analysis of liver lysates from control (n = 5) and cirrhotic mice (n = 5). All lysates are from five separate experiments and exhibit overexpression of vimentin in cirrhotic livers.

Fig. 2

Vimentin in primary cultured hepatocytes. (A) Time lapse imaging of hepatocyte morphology changes. Note a fibroblast-like appearance of CLDH in the presence or absence of TGFβ. (B) Western blot analysis of lysates from primary cultured hepatocytes with or without TGFβ. CLDH exhibit higher vimentin expression than NLDH before and after TGFβ treatment. (C) Immunofluorescence microscopy of vimentin. Hepatocytes were costained with vimentin (green), F-actin (red), and Hoechst 33342 (blue) and confocal images were collected. TGFβ treatment induced the expression of vimentin and EMT morphologically in NLDH, whereas untreated NLDH did not show mesenchymal characteristics. However, even untreated CLDH demonstrated features of EMT. These characteristic were augmented by TGFβ.

Fig. 3

Dependence on PI3K/Akt pathway. (A) NLDH and CLDH were incubated 16–18 hours in medium with varying insulin concentrations. In NLDH, p-Akt expression is increased compared to CLDH. Even in the absence of insulin, NLDH maintained higher expression of p-Akt than CLDH. (B) In contrast to NLDH, addition of 100 nM insulin to CLDH did not show sustained expression of p-Akt. (C) Morphologic apoptosis was determined 24 hours after TGFβ stimulation in the presence of PI3K/Akt pathway inhibition. Cell death was evaluated using Hoechst 33342 and propidium iodide (PI). (D) Quantification of apoptosis. Apoptosis was expressed as the percentage of apoptotic cells over total cells. NLDH exhibited a robust increase in apoptotic death by TGFβ treatment, compared to the CLDH (P bold> 0.001). Note that CLDH treated with both LY294002 and TGFβ showed a further development of apoptosis, compared to LY294002 or TGFβ alone.

Fig. 4

The effect of inhibition of the PI3K/Akt and MEK/ERK pathways on the TGFβ Smad signaling pathway. (A) The PI3K inhibitor, LY294002, decreased p-Akt in both normal and cirrhotic hepatocytes in a dose-dependent manner during 1 hour of treatment. MEK1/2 inhibitor, U0126, decreased p-ERK1/2 in both NLDH and CLDH in a dose-dependent manner. PI3K/Akt and MAPK/ERK signaling demonstrated no interdependence. (B) Time course of Smad signaling response to TGFβ. Phosphorylation of Smad2/3 induced by TGFβ was sustained for 24 hours in NLDH. In contrast, CLDH demonstrated decreased p-Smad2 after 2 hours. With LY294002 pretreatment, p-Smad2 was substantially decreased after TGFβ treatment in both NLDH and CLDH. The initial response of Smad2/3 signal transduction, however, was not affected by LY294002. (C) P-Smad2/3 decreased at 6 hours after TGFβ1 treatment in NLDH and CLDH in a dose-dependent manner. (D) NLDH and CLDH were infected with adenoviruses expressing Smad7 (Ad.Smad7) or a control protein, luciferase (Ad.Luc), at a multiplicity of infection (MOI) of 10 for 24 hours prior to treatment with TGFβ. Smad7 over-expression inhibited expression of p-Smad2 and p-Smad3 in both NLDH and CLDH. In addition, p-Akt expression was decreased markedly in NLDH and modestly in CLDH in response to Smad7 treatment.

Fig. 5

The effect of inhibition of the PI3K/Akt and ERK1/2 pathways on EMT after TGFβ stimulation. (A) NLDH and CLDH were pretreated with LY294002 (25 μM) or U0126 (25 μM) and stimulated with TGFβ. CLDH showed higher basal expression of vimentin than NLDH, which was further increased after 24 hours of stimulation. NLDH exhibited higher occludin and E-cadherin expression and lower vimentin expression compared to the CLDH consistent with an EMT response. (B) LY294002 (LY) pretreatment suppressed expression of vimentin in both NLDH and CLDH. However, U0126 (U) did not inhibit the expression of vimentin.

Fig. 6

Type 1 collagen in primary cultured hepatocytes. (A) Western blot analysis of type 1 collagen showed increased expression of collagen in CLDH. (B) After 24 hours of incubation of both cell types with or without TGFβ, immunofluorescence images of collagen (green) and Hoechst 33342 (blue) were collected by confocal microscope. (C) After 24 hours of incubation with or without TGFβ, the expression of albumin, a hepatocyte marker, was assessed by immunofluorescence staining. Note that CLDH were positively stained with albumin despite the morphology representing EMT.

Fig. 7

The effect of MEK/ERK inhibition on TGFβ-induced apoptosis. (A) Inhibition of MAPK pathway followed by TGFβ treatment induced significant cell death in CLDH. (B) Quantitative analysis of apoptosis from each group. CLDH demonstrated significantly increased apoptosis with MAPK inhibition and TGFβ treatment (P < 0.001).