TGF-β1 Up-Regulates the Expression of PDGF-β Receptor mRNA and Induces a Delayed PI3K-, AKT-, and p70S6K-Dependent Proliferative Response in Activated Hepatic Stellate Cells
- RS and KR-G contributed equally to this manuscript.
- Marcos Rojkind: Deceased.
- The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
Transforming growth factor beta 1 (TGF-β1) is a pleiotropic cytokine that activates hepatic stellate cell (HSC) proliferation, but inhibits parenchymal cell proliferation. Therefore, we hypothesize that TGF-β1 regulates HSC proliferation and elucidated its molecular action.
In order to elucidate the molecular mechanism whereby TGF-β1 up-regulates platelet derived growth factor beta (PDGF-β) receptor mRNA and induces a delayed proliferation of HSC, we used proliferation and apoptosis assays as well as RT-PCR, Western blot analysis, immunostaining, and flow cytometry in mouse and rat HSC.
We show that TGF-β1 markedly induces the proliferation of mouse HSC in culture with concomitant 2.1-fold (p < 0.001) stimulation in [3H]-thymidine incorporation into cellular DNA. This induction is maximal between 24 and 36 hours postcytokine exposure that is triggered by 7.6-fold (p < 0.001) up-regulation of PDGF-β receptor mRNA and associated increase in PDGF-β receptor protein after 48 hours. TGF-β1-dependent HSC proliferation is mimicked by H2O2 that is inhibited by catalase, implying that TGF-β1 action is mediated via reactive oxygen species. HSC proliferation is blunted by PDGF-β receptor–neutralizing antibody as well as by specific inhibitors of PI3 kinase (PI3K), AKT, and p70S6K, indicating that the action of TGF-β1 involves the activation of PDGF-β receptor via the PI3K/AKT/p70S6K signaling pathway. TGF-β1 also induces a reorganization of actin and myosin filaments and cell morphology leading to the formation of palisades although their myosin and actin contents remained constant. These findings suggest that TGF-β1-mediated oxidative stress causes the transdifferentiation of HSC and primes them for extracellular matrix (ECM) deposition and scar contraction.
We conclude that liver injury up-regulates TGF-β1 that inhibits parenchymal cell proliferation, but stimulates HSC proliferation leading to the production of ECM and type I collagen resulting in fibrosis.