Human SP1 but not human AP1 binding to the TGF-beta element in the 5 ' flanking region of the rat PRO alpha 1(I) collagen gene

The consensus TGF-beta element (TGCCCACGGCCAG) located at approximately -16 1Obp from the start site of transcription of the rat pro alpha1(I) collagen gene has recently been shown to be required for the basal promoter activit y of this gene (Meisler et al., J. Cell Biochem. 75: 196, 1999). Site direc ted mutation of this TGF-beta element resulted in almost complete abolishme nt of the basal promoter activity of the fibroblasts transfected with the 3 .6 ColCat plasmid which contains a 3.6 kb portion of the 5' flanking region of the rat pro alpha1(I) collagen gene linked to the reporter gene, chlora mphenicol acetyltransferase (CAT). Southwestern analysis of the nuclear pro tein binding to the TGF-beta element revealed a 34 000 Da complex while aft er UV-crosslinking, studies revealed a TGF-beta element nuclear protein com plex of 82 000 Da (Ritzenthaler et al., J. Biol. Chem. 268: 13625, 1993). T hus, a multiple protein TGF-beta DNA element complex may exist which may pr omote the transcription of the rat pro alpha1(I) collagen gene. Since liter ature findings indicate that a nuclear factor interacts with an SP1-like bi nding site of the human pro alpha1(I) collagen promoter and an AP-1 binding sequence has been shown to be involved in the regulation of the human pro alpha2(I) collagen gene and both these binding sequences are TGF-beta1 resp onsive, we determined whether the TGF-beta element located in the 5' flanki ng region of the rat pro alpha1(I) collagen gene formed complexes with eith er of these nuclear factors or both.