Smads 2 and 3 Are Differentially Activated by Transforming Growth Factor-β (TGF-β) in Quiescent and Activated Hepatic Stellate Cells

Abstract

Hepatic stellate cells are the primary cell type responsible for matrix deposition in liver fibrosis, undergoing a process of transdifferentiation into fibrogenic myofibroblasts. These cells, which undergo a similar transdifferentiation process when cultured in vitro, are a major target of the profibrogenic agent transforming growth factor-β (TGF-β). We have studied activation of the TGF-β down-stream signaling molecules Smads 2, 3, and 4 in hepatic stellate cells (HSC) cultured in vitro for 1, 4, and 7 days, with quiescent, intermediate, and fully transdifferentiated phenotypes, respectively. Total levels of Smad4, common to multiple TGF-β superfamily signaling pathways, do not change as HSC transdifferentiate, and the protein is found in both nucleus and cytoplasm, independent of treatment with TGF-β or the nuclear export inhibitor leptomycin B. TGF-β mediates activation of Smad2 primarily in early cultured cells and that of Smad3 primarily in transdifferentiated cells. The linker protein SARA, which is required for Smad2 signaling, disappears with transdifferentiation. Additionally, day 7 cells demonstrate constitutive phosphorylation and nuclear localization of Smad 2, which is not affected by pretreatment with TGF-β-neutralizing antibodies, a type I TGF-β receptor kinase inhibitor, or activin-neutralizing antibodies. These results demonstrate essential differences between TGF-β-mediated signaling pathways in quiescent and in vitro transdifferentiated hepatic stellate cells.