Basic fibroblast growth factor (bFGF) is a pleiotropic factor that is implicated in tissue remodeling. The growth factor is capable of up-regulating the expression of the interstitial collagenase (matrix metalloproteinase-1 or MMP-1) gene. In this study, the full-length human MMP-1 promoter, spanning 4.3 kb, was sequenced and the regulatory control of its activity by bFGF was examined in NIH3T3 fibroblasts. Several regulatory sequences, including five activator protein-1 (AP-l), five activator protein-2 (AP-2), five glucocorticoid-response elements and multiple ets/polyoma enhancer-binding 3 elements, were identified. Deletion constructs were prepared and transiently transfected into fibroblast cultures incubated with and without bFGF. The results showed that bFGF enhanced the activity of the deletion promoter fragments and the full-length MMP-1 promoter by sixfold or more in the cell cultures. Stimulation of the MMP-1 promoter activity by bFGF was reflected in substantial increase of the collagenase mRNA levels. A bFGF-responsive element appeared to be the AP-1 consensus sequence. Mutation of the first AP-1 site resulted in major reduction of the basal level of the MMP-1 promoter activity, supporting the notion that the AP-1 consensus sequence is essential for the constitutive expression of the MMP-1 gene. Furthermore, bFGF induction of the activity of the promoter constructs containing a mutant AP-1 site was essentially absent, suggesting that the regulatory element is necessary for the induction of the promoter activity by the growth factor. Thus, bFGF up-regulates MMP-1 gene expression in NIH3T3 fibroblasts via induction of its promoter activity that is dependent on an AP-1 consensus sequence.Keywords: basic fibroblast-growth factor; interstitial collagenase; promoter activity ; activator protein-1.Degradation of collagen is an integral aspect of tissue remodeling which occurs in pathologic and physiologic conditions, such as wound repair, angiogenesis, and embryonic development. At the cellular level, dissolution of collagen in the extracellular matrix is thought to facilitate cellular migration [l]. Various cytokines, growth factors, inflammatory mediators, and phorbol esters have been shown to stimulate enzymatic degradation of collagen [2, 31.The major matrix metalloproteinase that is involved in the degradation of collagens, specifically types I, 11, 111, V, VII, and X collagens, is the interstitial collagenase, known as MMP-1 [2, 31. Many cell types, such as keratinocytes, fibroblasts, monocytes/macrophages, chondrocytes, as well as vascular endothelial and smooth muscle cells secrete MMP-1. The activity of MMP-1 is stringently regulated, either at the promoter level, or The induction of the MMP-1 gene expression via stimulation of its promoter activity involves transcription factors. Activator protein, known more commonly as AP-1, is a well-characterized dimeric complex composed of products encoded by the fos and jun proto-oncogenes. AP-1 regulates expression of the stromelysin and collagenase genes [...

Glucocorticoids exert their action on gene expression through activation of cytoplasmic glucocorticoid receptors (GRs) that bind to glucocorticoid response elements (GREs). The consensus GRE consists of two half sites (underlined), AGAACANNNTGTTCT. We have recently cloned the entire human elastin gene. Nucleotide sequencing of the promoter region disclosed the presence of three putative GREs with the downstream half-site sequence TGTTCC that has homology with the consensus GRE, although the upstream half site showed no homology. To examine the functionality of these putative GREs in binding to the GRs, we performed gel mobility shift and supershift assays with synthetic oligomers containing the putative GREs and a recombinant GR protein, expressed in a baculovirus system. All three GREs identified in the elastin promoter bound the receptor. A chimeric oligonucleotide containing the upstream consensus GRE half site and the downstream elastin promoter GRE half site was capable of binding the receptor, and this binding could be competed with the elastin promoter GRE. Nonconservative substitution of single nucleotides (positions 1-6) in the elastin GRE indicated that mutations in the positions 1-3 and 6 had relatively little effect, but substitutions in positions 4 and 5 rendered the oligomer less effective in competing for the binding. These observations suggest that the downstream half site of GREs in the human elastin promoter is sufficient for receptor binding and certain nucleotides are critical for the efficient binding. The results also imply that the three GREs within the human elastin promoter are active and mediate the glucocorticoid-induced up-regulation of human elastin promoter activity.

Platelet-derived growth factor (PDGF) is a mitogen associated with tissue repair, a process involving collagen synthesis and remodelling by interstitial collagenase. This study examines and compares the regulation of interstitial collagenase and collagen gene expression by PDGF-AA and -BB in human fibroblasts. Time-course analysis showed that neither PDGF-AA or -BB had a consistent effect on the expression of pro-alpha 1(I) or pro-alpha 2(I) type-I collagen genes. In contrast, interstitial collagenase gene expression was found to be consistently up-regulated severalfold by PDGF-BB. Enhanced expression of the collagenase gene was not apparently due to up-regulation of its promoter activity in human dermal fibroblasts, as indicated by transient and stable transfection experiments. Unlike PDGF-BB, PDGF-AA did not alter collagenase mRNA levels under low-serum culture conditions. Thus, the biological activities of the PDGF homodimers are different, with PDGF-BB being clearly more potent than PDGF-AA in its regulation of collagenase gene expression.

Basic fibroblast growth factor (bFGF) is a mitogenic factor that is implicated in smooth muscle cell growth in atherosclerosis and vascular restenosis. In this study, we examined the effect of bFGF on the expression of the interstitial collagenase gene in human vascular smooth muscle cells. Results from Northern transfer analysis showed that bFGF increased collagenase mRNA levels greater than threefold as early as 24 h. Collagenase pre-mRNA levels were elevated approximately threefold by bFGF, according to RT-PCR analysis. Transient transfections of the smooth muscle cells with a 4.4-kb human collagenase promoter-CAT reporter gene, however, failed to show upregulation of the promoter activity by bFGF. Interestingly, transfections with deleted fragments containing promoter sequences from -1047 to -2271 resulted in modest stimulation of the collagenase-CAT promoter activity by bFGF, bFGF did not alter the stability of the collagenase mRNA, as demonstrated by degradation studies. The enhanced collagenase mRNA levels elicited by bFGF were reflected in increased amounts of collagenase protein that were detected by Western blot analysis. In summary, bFGF upregulates the interstitial collagenase expression, resulting in turnover of the extracellular matrix, an event that could facilitate smooth muscle cell migration and proliferation during the early stages of atherosclerosis and restenosis.