Constitutive phosphorylation and nuclear localization of Smad3 are correlated with increased collagen gene transcription in activated hepatic stellate cells

Hepatic stellate cells (HSC) are the main producers of type collagen in fib rotic liver, and transforming growth factor-beta (TGF-beta) plays critical roles in stimulating collagen gene expression in the cells mainly at the le vel of transcription. We have previously identified an upstream sequence of alpha2(I) collagen gene (COL1A2) that is essential for its basal and TGF-b eta -stimulated transcription in skin fibroblasts and HSC. We designated th is region the TGF-beta -responsive element (TbRE). Recently Smad3, an intra cellular mediator of TGF-beta signal transduction, has been shown to bind t o the TbRE and stimulate COL1A2 transcription when overexpressed in skin fi broblasts. In the present study, we demonstrate increased transcription of COL1A2 and plasminogen activator inhibitor-1 (PAI-1) genes and low response to TCF-beta in an activated HSC clone derived from a cirrhotic liver. West ern blot analyses indicated constitutive phosphorylation of Smad3 in the ce lls. Immunofluorescence studies revealed that, in contrast to Smad2 that tr anslocated from the cytoplasm to the nucleus upon TGF-beta treatment, Smad3 and Smad4 were present in the nucleus irrespective of ligand stimulation, increased COL1A2 and PAI-1 gene transcription in the cells was not affected by overexpression of inhibitory Smad7. Altogether. the results correlate a bnormality in TCF-beta /Smad signaling with pathologically accelerated coll agen gene transcription in activated HSC. (C) 2001 Wiley-Liss, Inc.