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.
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Department of Medicine, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
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Brinckerhoff
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D017364:Collagenases / Q000096:biosynthesis / Q000235:genetics*; D005347:Fibroblasts; D005786:Gene Expression Regulation / Q000187:drug effects; D008024:Ligands; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011817:Rabbits; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D047488:Retinoid X Receptors; D013237:Stereoisomerism; D013489:Suppression, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D014212:Tretinoin / Q000378:metabolism* / Q000494:pharmacology*
D008024:Ligands; D009687:Nuclear Proteins; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014212:Tretinoin; D017364:Collagenases