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 I collagen in fibrotic liver, and transforming growth factor-beta (TGF-beta) plays critical roles in stimulating collagen gene expression in the cells mainly at the level of transcription. We have previously identified an upstream sequence of alpha2(I) collagen gene (COL1A2) that is essential for its basal and TGF-beta-stimulated transcription in skin fibroblasts and HSC. We designated this region the TGF-beta-responsive element (TbRE). Recently Smad3, an intracellular mediator of TGF-beta signal transduction, has been shown to bind to the TbRE and stimulate COL1A2 transcription when overexpressed in skin fibroblasts. In the present study, we demonstrate increased transcription of COL1A2 and plasminogen activator inhibitor-1 (PAI-1) genes and low response to TGF-beta in an activated HSC clone derived from a cirrhotic liver. Western blot analyses indicated constitutive phosphorylation of Smad3 in the cells. Immunofluorescence studies revealed that, in contrast to Smad2 that translocated 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 abnormality in TGF-beta/Smad signaling with pathologically accelerated collagen gene transcription in activated HSC.
Inagaki
Y
Y
Department of Internal Medicine and Division of Clinical Research, National Kanazawa Hospital, 1-1 Shimoishibiki-machi, Kanazawa 920-8650, Japan. inagaki@kinbyou.hosp.go.jp
Mamura
M
M
Kanamaru
Y
Y
Greenwel
P
P
Nemoto
T
T
Takehara
K
K
Ten Dijke
P
P
Nakao
A
A
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D008103:Liver Cirrhosis / Q000378:metabolism*; D010766:Phosphorylation; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics; D020033:Protein Isoforms / Q000378:metabolism; D051381:Rats; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D004268:DNA-Binding Proteins; D017395:Plasminogen Activator Inhibitor 1; D020033:Protein Isoforms; D051900:Smad3 Protein; C497787:Smad3 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D003094:Collagen