Synergistic cooperation between Sp1 and Smad3/Smad4 mediates transforming growth factor beta1 stimulation of alpha 2(I)-collagen (COL1A2) transcription.
Transforming growth factor-beta1 (TGFbeta) is a strong activator of extracellular matrix accumulation. TGFbeta stimulates the gene coding for human alpha2(I)-collagen (COL1A2) by inducing binding of an Sp1-containing complex to an upstream promoter element (TGFbeta responsive element or TbRE) that contains a CAGA box. Here we report that the CAGA box of the TbRE is the binding site of the Smad3/Smad4 complex, and that the binding of the complex is required for TGFbeta-induced COL1A2 up-regulation. Recombinant Smad3 and Smad4 bind in vitro to the CAGA box of COL1A2; TGFbeta treatment of cultured fibroblasts induces Smad3/Smad4 binding to the TbRE; transient overexpression of Smad3 and Smad4 in fibroblasts transactivates TbRE-driven transcription; and COL1A2 gene up-regulation by TGFbeta is abolished in cells stably transfected with plasmids that express dominant negative forms of Smad3 or Smad4. In Sp1-deficient Drosophila Schneider cells, there was cooperative synergy between Smad3/Smad4 and Sp1 at the TbRE site. The analysis also emphasized the requirement of both Sp1- and Smad-binding sites for optimal promoter transactivation. In cells stably transfected with a plasmid expressing a dominant negative form of Sp1, the synergy was shown to be promoter-specific and dependent on the binding of Sp1 to the TbRE. Interestingly, overexpression of dominant negative Sp1 was found to block the antagonistic signal of tumor necrosis factor-alpha on COL1A2 transcription, as well. These results provide the first linkage between the Smad3 and Smad4 proteins and TGFbeta stimulation of type I collagen biosynthesis.
Zhang
W
W
Brookdale Center, Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York University, New York, New York 10029, USA.
Ou
J
J
Inagaki
Y
Y
Greenwel
P
P
Ramirez
F
F
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D003094:Collagen / Q000235:genetics / Q000378:metabolism*; D024042:Collagen Type I; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins; D020218:Response Elements; D015398:Signal Transduction; D051900:Smad3 Protein; D051901:Smad4 Protein; D016329:Sp1 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1; D015854:Up-Regulation
D024042:Collagen Type I; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; D016329:Sp1 Transcription Factor; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D003094:Collagen