Background and Aims Liver injury serves as an excellent model of wound healing, characterized by increased synthesis of various cytokines and peptides, including the vasoactive peptide endothelin-1. In the liver, wound healing is mediated by effector cells such as hepatic stellate cells, which cause tissue contraction. Endothelin-1 has autocrine effects on stellate cells and induces their contractile activities. We explored the role of various extracellular matrix components, particularly of fibronectin, in regulating endothelin-1 production during liver injury. Methods Hepatic stellate cells were isolated from normal and injured rats. Real-time PCR, immunoblot and ELISA analyses were used to measure specific variables, including endothelin-1 production. Preproendothelin-1 promoter activity was determined by a luciferase assay. Stellate cell contraction was measured by a gel contraction assay. Results Fibronectin stimulated transcription of preproendothelin-1 mRNA and expression of endothelin through an integrin-dependent pathway in activated hepatic stellate cells. In these cells, fibronectin induced phosphorylation/activation of extracellular signal-regulated kinase (ERK) through a Shc- and Src-dependent mechanism; ERK activation was required for fibronectin-induced endothelin-1 expression. Fibronectin stimulation by stellate cells induced expression of smooth muscle α-actin and endothelin-1–mediated autocrine stellate cell contraction. Stellate cells isolated from injured livers of rats exhibited increased basal phosphorylation levels of Src, Shc and ERK, as well as increased endothelin-1 synthesis. Conclusions Fibronectin stimulates activated stellate cells to produce endothelin-1 and contract, via an ERK-dependent signaling pathway. The resulting autocrine functional effects of endothelin-1 are likely to be important in the wound-healing process in injured liver.

Endothelin-1 (ET-1), a potent vasoconstrictor peptide up-regulated during wound healing and fibrosis, induces myofibroblasts to contract tissue. Here we have used a liver injury model to test the hypothesis that TNFα may be an important stimulator of ET-1 production in hepatic wound healing. We examined primary rat hepatic stellate cells, isolated from either normal or injured livers and used standard methodology to measure preproET-1 mRNA and mature ET-1 peptide, specific kinases, and preproET-1 promoter activity. Chromatin immunoprecipitation analysis was used to determine basal binding of transcription factors to the preproET-1 promoter. TNFα induced preproET-1 expression in activated hepatic stellate cells in a c-Jun N-terminal kinase (JNK)/AP-1-dependent fashion. TNFα activated JNK through an IκB kinase (IKK) pathway, which activated the transcriptional factor, c-Jun, leading to preproET-1 promoter mediated ET-1 transcription. The TNFα mediated induction of ET-1 synthesis also had functional effects, specifically mediating autocrine induced stellate cell contraction. TNFα stimulated activated stellate cells to produce ET-1 via a novel IKK-JNK-dependent signaling pathway. The resulting autocrine functional effects of ET-1 are likely to be important in the wound-healing process.