These studies indicate that t-RA downregulates the promoter activity of the rat pro alpha1(I) collagen gene by decreasing the binding of nuclear protein to the RARE sequence in the 5' flanking region of the gene. The same assays performed with the mutated oligonucleotide resulted in only slight binding. Gel mobility shift assays using nuclear protein extracts prepared from t-RA-treated FRS fibroblasts showed that binding to the oligonucleotide containing the RARE was decreased from control values. Mutation of the idealized half-site eliminated the binding of receptor proteins to the oligonucleotide. ACID Music Studio 5 Specifications: Microsoft Windows 2000 or XP 500 MHz processor 128MB RAM Windows-compatible sound card CD-ROM drive (for installation. Two nuclear retinoid receptors that were expressed in bacteria, retinoic acid receptor-gamma and retinoid X receptor-alpha, were found to bind specifically to a double-stranded oligonucleotide containing the RARE in gel mobility shift assays. Computer sequence analysis of the 3.6-kb DNA fragment that contains the promoter for the rat pro alpha1(I) collagen gene identified a direct repeat RARE sequence composed of one diverse (5'-AGTAGA-3') and one idealized (5'-GGGTCA-3') half site located at positions -1345 and -1335, respectively. Transforming growth factor-beta1 did not block the inhibitory effect of t-RA on CAT activity. Maximal inhibition of CAT activity by t-RA occurred at 10(-8) M after 48 h of treatment. The effect of t-RA on CAT activity was determined as a function of concentration and incubation time. FRS fibroblasts were stably transfected with the ColCat3.6 plasmid, which contains a portion of the 5' flanking region of the rat pro alpha1(I) collagen gene linked to a reporter gene, chloramphenicol acetyltransferase. The current study was undertaken to determine the mechanism by which the retinoid all-trans-retinoic acid regulates pro alpha1(I) collagen gene expression in fetal rat skin fibroblasts.

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