Transforming Growth Factor-β and Platelet-Derived Growth Factor Signal via c-Jun N-Terminal Kinase-Dependent Smad2/3 Phosphorylation in Rat Hepatic Stellate Cells after Acute Liver Injury

Abstract

After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro.

Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor β signaling, promoting cirrhosis and hepatocellular carcinoma

“…Immunohistochemical analyses were performed as described previously. 17 Primary antibodies used in this study included mouse monoclonal anti-␣SMA Ab (1.7 g/ml; DAKO, Glostrup, Denmark), mouse monoclonal anti-PAI-1 Ab (0.4 g/ml; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-p21 WAF1 Ab (0.5 g/ml; DAKO), in addition to rabbit polyclonal anti-pSmad3L (2 g/ml), and rabbit polyclonal antipSmad3C (0.5 g/ml) described above.…”

“…13 We therefore have focused on Smad3 signaling, 14,15 and have recently reported different roles of Smad3 phosphoisoform-mediated signaling in epithelial cells and mesenchymal cells. 16,17 Thus, TGF-␤ activates not only TGF-␤ type I receptor (T␤RI) but also c-Jun N-terminal kinase (JNK), converting Smad3 into two distinctive phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). 16 The T␤RI/pSmad3C pathway inhibits growth of epithelial cells including hepatocytes, whereas JNK/pSmad3L-mediated signaling promotes ECM deposition by activated mesenchymal cells such as hepatic stellate cells (HSCs).…”

“…16 The T␤RI/pSmad3C pathway inhibits growth of epithelial cells including hepatocytes, whereas JNK/pSmad3L-mediated signaling promotes ECM deposition by activated mesenchymal cells such as hepatic stellate cells (HSCs). 17 According to these phosphorylation-defined activities, we sought the molecular mechanisms by which Smad3 phosphoisoforms govern progression from chronic hepatitis C through cirrhosis to HCC.…”

TGFβ pathobiology in the eye

“…[30][31][32] Although Smad2/3 signaling is relatively specific to the binding of ligands of the TGFb/activin family to cell surface receptors, investigators are discovering that MAPKs (p42/p44 Erk1/2, JNK, or p38MAPK) phosphorylate specific sites in the middle linker region of Smad2/3, sites that are distinct from the C-terminus that is phosphorylated by the TGFb II receptor (Figure 2). [33][34][35][36] Thus, ligands capable of activating MAPKs potentially modulate Smad signaling induced by TGFb. It has recently been demonstrated that p38MAPK activate phosphorylation of Smad3 in the middle linker region, which enhances Smad3/4 complex formation and nuclear translocation, 37 consistent with our finding of diminished Smad3/4 reporter gene activity in the presence of a p38MAPK inhibitor.…”

A zebrafish model of intrahepatic cholangiocarcinoma by dual expression of hepatitis B virus X and hepatitis C virus core protein in liver

“…TGF-b-dependent JNK/pSmad3L oncogenic pathway activation has been observed in rat hepatic stellate cells and myofibroblasts after liver injury. 24,25 The status of the TGF-b-dependent JNK/pSmad3L oncogenic pathway can be used to evaluate the risk for developing hepatocellular carcinoma (HCC) in HBVinfected patients. 26 Based on the results of the gene expression analysis, TGF-b-dependent epithelial-mesenchymal transition (EMT) and cell proliferation occurred among the top 10 rankings (Fig.…”