Regulation of collagen I gene expression by ras.

Although transformation of rodent fibroblasts can lead to dramatic changes in expression of extracellular matrix genes, the molecular basis and physiological significance of these changes remain poorly understood. In this study, we have investigated the mechanism(s) by which ras affects expression of the genes encoding type I collagen. Levels of both alpha 1(I) and alpha 2(I) collagen mRNAs were markedly reduced in Rat 1 fibroblasts overexpressing either the N-rasLys-61 or the Ha-rasVal-12 oncogene. In fibroblasts conditionally transformed with N-rasLys-61, alpha 1(I) transcript levels began to decline within 8 h of ras induction and reached 1 to 5% of control levels after 96 h. In contrast, overexpression of normal ras p21 had no effect on alpha 1(I) or alpha 2(I) mRNA levels. Nuclear run-on experiments demonstrated that the transcription rates of both the alpha 1(I) and alpha 2(I) genes were significantly reduced in ras-transformed cells compared with those in parental cells. In addition, the alpha 1(I) transcript was less stable in transformed cells. Chimeric plasmids containing up to 3.6 kb of alpha 1(I) 5'-flanking DNA and up to 2.3 kb of the 3'-flanking region were expressed at equivalent levels in both normal and ras-transformed fibroblasts. However, a cosmid clone containing the entire mouse alpha 1(I) gene, including 3.7 kb of 5'- and 4 kb of 3'-flanking DNA, was expressed at reduced levels in fibroblasts overexpressing oncogenic ras. We conclude that oncogenic ras regulates the type I collagen genes at both transcriptional and posttranscriptional levels and that this effect, at least for the alpha 1(I) gene, may be mediated by sequences located either within the body of the gene itself or in the distal 3'-flanking region.

Elements in the first intron of the alpha 1(I) collagen gene interact with Sp1 to regulate gene expression.

Sequences within the first intron of the alpha 1(I) collagen gene act both positively and negatively to regulate expression of the gene. We have further characterized a 274 bp intronic sequence that contains an orientation-specific inhibitory activity and represents a constitutive DNase I-hypersensitive site in the gene. We show that this sequence contains two tandem, unique binding elements for the transcription factor Sp1. In addition, an Sp1-like site, capable of competing for protein binding to the intronic elements, resides in the distal promoter of the collagen gene. The results of experiments with site-directed mutations that abolish binding to the intronic elements indicate that these protein-DNA interactions have an inhibitory effect on the transcriptional efficiency of alpha 1(I) collagen-reporter gene constructs in transient transfection analysis. These data support our conclusion that the first intron plays a complex role, involving multiple protein-DNA binding interactions, in the regulation of expression of the alpha 1(I) collagen gene.