Sp1 is required for the early response of alpha2(I) collagen to transforming growth factor-beta1.
It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor beta1 (TGF-beta) stimulation of the human alpha2(I) collagen (COL1A2) gene by binding to an upstream promoter element (TbRE). The present study was designed to resolve this controversy by correlating expression of COL1A2, AP1, and Sp1 in the same cell line and under different experimental conditions. The results strongly indicate that Sp1 is required for the immediate early response of COL1A2 to TGF-beta and AP1 is not. The Sp1 inhibitor mithramycin blocked stimulation of alpha2(I) collagen mRNA accumulation by TGF-beta, whereas the AP1 inhibitor curcumin had no effect. Furthermore, antibodies against Jun-B and c-Jun failed to identify immunologically related proteins in the TbRE-bound complex, irrespective of whether they were purified from untreated or TGF-beta-treated cells. AP1 did bind to the TbRE probe in vitro, but only in the absence of the upstream Sp1 recognition sequence. Based on this finding and DNA transfection results, we conclude that the AP1 sequence of the TbRE represents a cryptic site used under experimental conditions that either eliminate the more favorable Sp1 binding site or force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays excluded that COL1A2 transactivation involves the retinoblastoma gene product (pRb), an activator of Sp1, the pRb-related protein p107, an inhibitor of Sp1, or the Sp1-related repressor, Sp3.
Greenwel
P
P
Brookdale Center for Developmental and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Inagaki
Y
Y
Hu
W
W
Walsh
M
M
Ramirez
F
F
D003094:Collagen / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D016755:Proto-Oncogene Proteins c-jun; D016160:Retinoblastoma Protein; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D051705:Sp3 Transcription Factor; D003094:Collagen