Role of Smad4 on TGF-β–induced extracellular matrix stimulation in mesangial cells

Role of Smad4 on TGF-β–induced extracellular matrix stimulation in mesangial cells. The best characterized signaling pathway employed by transforming growth factor-β (TGF-β) is the Smad pathway; however, its role in matrix production in mesangial cells is unclear. We focused on Smad4, as Smad4 is essential for the activation of Smad-dependent target genes. To investigate the function of Smad4 in extracellular matrix (ECM) production, we generated several stably transfected mesangial cell lines (MMC) that have a deletion in the linker region (Smad4ΔM4: Δ275-322) or have a deletion in MH1 of Smad4 (Smad4N4: Δ1-136). The ECM genes, α1 type I collagen (COL1A1), plasminogen activator inhibitor-1 (PAI-1) and fibronectin (FN) were assessed in wild-type mesangial cells and stably transfected Smad4-DN cell lines in the absence and presence of TGF-β. As compared to wild-type MMC that had a 10.8-fold stimulation of TGF-β–induced p3TP-Lux activity, MMC stably transfected with Smad4ΔM4 and Smad4N4 had only a 2.0-fold and 1.3-fold stimulation, respectively, indicating that they had dominant-negative effects on TGF-β signaling. Basal and TGF-β–induced COL1A1 expression in Smad4 dominant-negative cells were dramatically reduced to very low levels. The early (2 hours) TGF-β–induced PAI-1 mRNA expression was inhibited; however, the sustained (24 to 48 hours) TGF-β–induced expression was not affected in Smad4 dominant-negative cells. For FN, TGF-β–induced expression was maintained in Smad4-dominant negative cells. These results indicate that Smad4 is essential for basal and TGF-β–induced COL1A1 expression, and contributes to the early, but not sustained TGF-β–induced PAI-1 expression in mesangial cells. However, TGF-β–induced FN expression is independent of Smad4. In conclusion, Smad4 has a discriminate effect in mediating specific ECM molecules stimulated by TGF-β in mesangial cells.