Abstract
The mechanism(s) by which Smads mediate and modulate the transforming growth factor (TGF)-beta signal transduction pathway in fibrogenesis are not well characterized. We previously showed that Smad3 promotes alpha2(I) collagen gene (COL1A2) activation in human glomerular mesangial cells, potentially contributing to glomerulosclerosis. Here, we report that Sp1 binding is necessary for TGF-beta1-induced type I collagen mRNA expression. Deletion of three Sp1 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 Smad3 dominant negative mutant. Chemical inhibition of Sp1 binding with mithramycin A, or deletion of the GC boxes, inhibited COL1A2 activation by Smad3, suggesting cooperation between Smad3 and Sp1 in the TGF-beta1 response. Electrophoretic mobility shift assay showed that Sp1 and Smads form complexes with -283/-250 promoter sequences. Coimmunoprecipitation experiments demonstrate that endogenous Sp1, Smad3, and Smad4 form complexes in mesangial cells. In a Gal4-LUC reporter assay system, Sp1 stimulated the TGF-beta1-induced transcriptional activity of Gal4-Smad3, Gal4-Smad4 (266), or both. Using the transactivation domain B of Sp1 fused to the Gal4 DNA binding domain, we show that, in our system, the transcriptional activity of this Sp1 domain is not regulated by TGF-beta1, but it becomes responsive to this factor when Smad3 is coexpressed. Finally, combined Sp1 and Smad3 overexpression induces marked ligand-independent and ligand-dependent promoter activity of COL1A2. Thus, Sp1 and Smad proteins form complexes and their synergy plays an important role in mediating TGF-beta1-induced alpha2(I) collagen expression in human mesangial cells.
Highlights
The Smads are a series of proteins that function downstream from the serine/threonine kinase receptors of the transforming growth factor (TGF)- family to transduce signal to the nucleus [2,3,4]
We previously showed that a construct containing the sequence from Ϫ376 to ϩ58 of the human ␣2(I) collagen gene (COL1A2) promoter is responsive to TGF-1 in human mesangial cells and that overexpression of Smad3 stimulates COL1A2 promoter activity [29]
The inhibitory effect of mithramycin A was specific since transcription of other genes such as tissue inhibitor of metalloproteinase (TIMP)-1 was not blocked by this chemical antagonist (Fig. 1B)
Summary
Materials—Reagents were purchased from the following vendors: active, human recombinant TGF-1 from R&D Systems; goat polyclonal anti-Smad2/3 IgG (N-19), mouse monoclonal anti-Smad1/2/3 IgG (H-2), mouse monoclonal anti-Smad IgG (B-8), rabbit anti-Sp1 IgG (PEP-2), normal goat IgG, normal mouse IgG, normal rabbit IgG, anti-goat IgG-horseradish peroxidase, anti-mouse IgG-horseradish peroxidase antibody, Sp1 consensus binding site, AP-1 consensus binding site, Smad3/4 binding site (CAGA), and mutated CAGA oligonucleotide from Santa Cruz Biotechnology; rabbit anti-phospho-Smad IgG from Upstate Biotechnology Inc.; recombinant protein G-Sepharose and rabbit polyclonal anti-phosphoserine antibody from Zymed Laboratories Inc.; anti-rabbit IgG-horseradish peroxidase, luciferase, and -galactosidase assay systems from Promega; Pwo DNA polymerase and rapid DNA ligation kit from Roche Molecular Biochemicals; T4 polynucleotide kinase from Life Technologies, Inc.; mithramycin A and curcumin from Sigma. RNA Isolation and Northern Blot—Cells were plated in 100-mm culture dishes (3–5 ϫ 105 cells/dish) The day, they were preincubated with mithramycin A (100 mM) for 17 h or curcumin (20 M) for 30 min before addition of 1 ng/ml TGF-1 or control vehicle for 24 h. Plasmid Constructs—The 376COL1A2-LUC construct containing 376 bp of the ␣2(I) collagen (COL1A2) promoter and 58 bp of the transcribed sequence fused to the luciferase (LUC) reporter gene was described previously [29]. Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSA)—Nuclear extracts were prepared from control and TGF-1-treated cells as described by Schreiber et al [45]. For competition and supershift assays, nuclear extracts were preincubated with a 100-fold molar excess of cold oligonucleotide or 1 g of antibody for 30 min or 1 h before addition of the labeled probe.
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