SP1 IS REQUIRED FOR THE EARLY RESPONSE OF ALPHA-2(I) COLLAGEN TO TRANSFORMING GROWTH-FACTOR-BETA-1

It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor beta 1 (TGF-beta) stimulation of the human alpha 2(I) collagen (COL1A2) gene by binding to an upstream promoter element (TbRE). The present study was designed to resolve this controv ersy by correlating expression of COL1A2, AP1, and Sp1 in the same cel l line and under different experimental conditions. The results strong ly indicate that Sp1 is required for the immediate early response of C OL1A2 to TGF-beta and AP1 is not. The Sp1 inhibitor mithramycin blocke d stimulation of alpha 2(I) collagen mRNA accumulation by TGF-beta, wh ereas the AP1 inhibitor curcumin had no effect. Furthermore, antibodie s against Jun-B and c-Jun failed to identify immunologically related i n the TbRE-bound complex, irrespective of whether they were purified f rom untreated or TGF-beta-treated cells. AP1 did bind to the TbRE prob e in vitro, but only in the absence of the upstream Sp1 recognition se quence. Based on this finding and DNA transfection results, we conclud e that the AP1 sequence of the TbRE represents a cryptic site used und er experimental conditions that either eliminate the more favorable Sp 1 binding site or force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays excluded t hat COL1A2 transactivation involves the retinoblastoma gene product (p Rb), an activator of Sp1, the pRb-related protein p107, an inhibitor o f Sp1, or the Sp1-related repressor, Sp3.