The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells

Background. Transforming growth factor-beta (TGF-beta) signals through a un ique set of intracellular proteins, called SMADs, that have been characteri zed mainly in transient overexpression systems. Because several models of g lomerulosclerosis suggest a role for TGF-beta in the extracellular matrix a ccumulation, we sought to characterize the role of SMAD proteins in mediati ng TGF-beta 1 responses in a more physiological system using nontransformed human mesangial cells. Methods. Endogenous SMAD expression and its modulation by TGF-beta 1 were e valuated by Western and Northern blot analyses. Phosphorylation of Smad2 an d Smad3 was determined by both phospholabeling and immunoblot. SMAD functio n and its role in type I collagen transcription were investigated in cotran sfection experiments using promoter-luciferase reporter gene constructs. Results. Cultured human mesangial cells express Smad2, SmadS, and Smad4 pro teins. TGF-beta 1 down-regulated Smad3 mRNA and protein expression, respect ively, after 4 and 24 hours of treatment, whereas Smad2 and Smad4 were less affected. Both Smad2 and Smad3 were phosphorylated in response to TGF-beta 1 beginning at 5 minutes, with maximal phosphorylation at 15 minutes, and decreasing phosphorylation by 2 hours. Smad2/3 and Smad4 coimmunoprecipitat e only after TGF-beta 1 treatment. The activity of a transiently transfecte d, TGF-beta-responsive construct, p3TP-Lux, was stimulated 3.6-fold by TGF- beta 1. Overexpressed wild-type Smad3 increased basal luciferase activity, which was further stimulated by TGF-beta 1. A dominant negative mutant form of Smad3 lacking the C-terminal serine phosphoacceptor sites (Smad3A) inhi bited TGF-beta 1-induced luciferase activity. TGF-beta 1 also increased the activation of an alpha 2(I) collagen promoter-luciferase reporter construc t transfected into mesangial cells. This activation was inhibited by cotran sfection with the Smad3A mutant. Conclusions. Smad2, Smad3, and Smad4 are present and activated by TGF-beta 1 in human mesangial cells. The SMAD pathway is functional in these cells a nd appears to be involved in TGF-beta 1-induced type I collagen gene transc ription. These findings raise the possibility that SMAD signaling plays a r ole in glomerular matrix accumulation.