Sp1 and Smad proteins cooperate to mediate transforming growth factor-beta1-induced alpha 2(I) collagen expression in human glomerular mesangial cells

The mechanism(s) by which Smads mediate and modulate the transforming growt h factor (TGF)-beta signal transduction pathway in fibrogenesis are not wel l characterized. We previously showed that Smads promotes alpha2(I) collage n gene (COL1A2) activation in human glomerular mesangial cells, potentially contributing to glomerulosclerosis. Here, we report that Sp1 binding is ne cessary for TGF-beta1-induced type I collagen mRNA expression. Deletion of three Spl sites (GC box) between -376 and -268 or mutation of a CAGA box at -268/-260 inhibited TGF-beta1-induced alpha2(I) collagen promoter activity . TGF-beta1 inducibility was also blocked by a SmadS dominant negative muta nt, Chemical inhibition of Spl binding with mithramycin A, or deletion of t he GC boxes, inhibited COLIA2 activation by SmadS, suggesting cooperation b etween SmadS and Spl in the TGF-beta1 response. Electrophoretic mobility sh ift assay showed that Spl and Smads form complexes with -283/-250 promoter sequences. Coimmunoprecipitation experiments demonstrate that endogenous Sp l, Smad3, and Smad4 form complexes in mesangial cells. In a Gal4-LUC report er assay system, Spl stimulated the TGF-beta1-induced transcriptional activ ity of Gal4-Smads, Gal4-Smad4 (266-552), or both. Using the transactivation domain B of Spl fused to the Gal4 DNA binding domain, we show that, in our system, the transcriptional activity of this Spl domain is not regulated b y TGF-beta1, but it becomes responsive to this factor when Smad8 is coexpre ssed. Finally, combined Spl and Smads overexpression induces marked ligand- independent and ligand-dependent promoter activity of COLIA2, Thus, Spl and Smad proteins form complexes and their synergy plays an important role in mediating TGF-beta1-induced alpha2(I) collagen expression in human mesangia l cells.