Sp1 and Smad Proteins Cooperate to Mediate Transforming Growth Factor-β1-induced α2(I) Collagen Expression in Human Glomerular Mesangial Cells

Abstract

The mechanism(s) by which Smads mediate and modulate the transforming growth factor (TGF)-␤ signal transduction pathway in fibrogenesis are not well characterized. We previously showed that Smad3 promotes ␣2(I) collagen gene (COL1A2) activation in human glomerular mesangial cells, potentially contributing to glomerulosclerosis. Here, we report that Sp1 binding is necessary for TGF-␤1-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-␤1-induced ␣2(I) collagen promoter activity. TGF-␤1 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-␤1 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-␤1-induced transcriptional activity of Gal4-Smad3, Gal4-Smad4 (266 -552), 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-␤1, but it becomes responsive to this factor when Smad3 is coexpressed. Finally, combined Sp1 and Smad3 overexpression induces marked ligand-independent and liganddependent promoter activity of COL1A2. Thus, Sp1 and Smad proteins form complexes and their synergy plays an important role in mediating TGF-␤1-induced ␣2(I) collagen expression in human mesangial cells. Glomerulosclerosis is a scarring process involving extracellular matrix (ECM)1 accumulation and obliteration of glomerular capillaries. It is considered to be the final pathway leading to the progressive loss of renal function in several kidney diseases. Mechanical factors such as hyperfiltration and intraglomerular hypertension, as well as a variety of mediators including cytokines, growth factors, and eicosanoids derived from circulating or glomerular cells, have been implicated in initiating or maintaining sclerosis (1). However, little information is available regarding the cellular mechanisms by which these factors affect matrix turnover. Our laboratory has been studying the mechanisms by which transforming growth factor (TGF)-␤ stimulates ECM accumulation. Previously, we determined that the Smad pathway plays a role in activating type I collagen gene expression in human glomerular mesangial cells.

Expression and regulation of intracellular SMAD signaling in scleroderma skin fibroblasts

“…Therefore, the present results demonstrating signal-independent activation of SMAD3 in scleroderma fibroblasts may provide a mechanistic explanation to account for the reduced collagenase expression previously described in scleroderma (59). Recent reports indicate that optimal induction of COL1A2 transcription by TGF␤ involves an interaction between activated SMAD3 and the ubiquitous DNA binding transcription factor Sp1 (32,33). In this regard, it is of interest that Sp1 has been shown to be constitutively phosphorylated and activated in scleroderma fibroblasts (60).…”

“…The SMAD pathway plays a fundamental role in regulation of collagen synthesis, and SMADs are necessary to mediate TGF␤-dependent stimulation (29)(30)(31)(32)(33). In light of the singular importance of TGF␤ in both initiating and sustaining fibroblast activation, and given the pivotal role of SMADs as major intracellular effectors of TGF␤-induced profibrotic responses, we undertook extensive characterization of SMAD signaling in a large panel of scleroderma fibroblasts.…”

Transcriptional control of human T‐BET expression: The role of Sp1

“…Since Sp1 interacts with many other proteins [29], it is reasonable to speculate that its specificity may be brought about by the recruitment of other nuclear factors onto the T-BET promoter. For example, it is well-documented that both in vivo and in vitro, SMAD proteins associate with Sp1 [33][34][35][36]. Specifically, Sp1 physically interacts with SMAD2 and SMAD4 , and indirectly with SMAD3 through SMAD3 0 s association with SMAD2 and/or SMAD4 [33].…”

Transcription Factor Hnf–6/Oc–1 Inhibits the Stimulation of the Hnf–3 /Foxa1 Gene by Tgf–B in Mouse Liver

“…The sequence has similarity with the P15 promoter, which is known to be stimulated by both Smad and Erk signal transducers 36,45 and in which Sp1 binding is critical for its response to Smad proteins. 46 It is known that Sp1 and other transcription factors can physically interact with Smad proteins and are required for the expression of TGF-␤ target genes 47 ; whether this is the case for Foxa1 remains to be determined.…”