Suppression of collagenase gene expression by all‐trans and 9‐cis retinoic acid is ligand dependent and requires both RARs and RXRs

Retinoic acids (RA) are active metabolites of vitamin A which affect the expression of many genes involved in embryonic development, cell differentiation, and homeostasis. One important target gene for RA is matrix metalloproteinase (MMP-1, collagenase), the only enzyme active at neutral pH that can degrade interstitial collagen, a major component of extracellular matrix. Using a cell line of normal rabbit synovial fibroblasts, HIG82 cells, as a model, we report that both all-trans- and 9-cis-RA inhibit collagenase synthesis. This inhibition occurs at a transcriptional level and is ligand-dependent. Constitutive levels of retinoic acid receptor (RAR) mRNA levels are low, but are increased by all-trans and by 9-cis RA. In contrast, constitutive levels of retinoid X receptor (RXR) mRNA are higher and are not affected by RA. To measure DNA/protein interactions, we used a gel mobility shift assay with oligonucleotides containing either an AP-1 site or a 40 bp region between -182/-141, nuclear extracts from RT-treated cells, and antibodies to RARs and RXRs. We found that both RARs and RXRs interact with these regions of the collagenase promoter, perhaps as part of a complex with other proteins. Our results suggest that heterodimers between RARs and RXRs mediate suppression of the collagenase gene by RA, and that RAR is a limiting factor in this negative regulation.