Induction of MMP-1 (collagenase-1) by relaxin in fibrocartilaginous cells requires both the AP-1 and PEA-3 promoter sites.
OBJECTIVES - Relaxin induces the matrix metalloproteinase MMP-1 (collagenase-1) in TMJ fibrocartilaginous cells, and this response is potentiated by beta-estradiol. We identified the MMP-1 promoter sites and transcription factors that are induced by relaxin with or without beta-estradiol in fibrocartilaginous cells. MATERIAL AND METHODS - Early passage cells were transiently transfected with the pBLCAT2 plasmid containing specific segments of the human MMP-1 promoter regulating the chloramphenicol acyl transferase (CAT) gene and co-transfected with a plasmid containing the beta-galactosidase gene. The cells were cultured in serum-free medium alone or medium containing 0.1 ng/ml relaxin, or 20 ng/ml beta-estradiol or both hormones, and lysates assayed for CAT and beta-galactosidase activity. RESULTS - Cells transfected with the -1200/-42 or -139/-42 bp MMP-1 promoter-reporter constructs showed 1.5-fold and 3-fold induction of CAT by relaxin in the absence or presence of beta-estradiol, respectively. Relaxin failed to induce CAT in the absence of the -137/-69 region of the MMP-1 promoter, which contains the AP-1-and PEA3-binding sites. Using wild type or mutated minimal AP-1 and PEA-3 promoters we found that both these promoter sites are essential for the induction of MMP-1 by relaxin. The mRNAs for transcription factors c-fos and c-jun, which together form the AP-1 heterodimer, and Ets-1 that modulates the PEA-3 site, were upregulated by relaxin or beta-estradiol plus relaxin. CONCLUSION - These studies show that both the AP-1 and PEA-3 promoter sites are necessary for the induction of MMP-1 by relaxin in fibrocartilaginous cells.
Kapila
S
S
Department of Orthodontics and Pediatric Dentistry, The University of Michigan School of Dentistry, Ann Arbor, MI 48109-1078, USA. skapila@umich.edu
Xie
Y
Y
Wang
W
W
D000818:Animals; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000187:drug effects; D019902:Chondrocytes / Q000201:enzymology; D016895:Culture Media, Serum-Free; D004790:Enzyme Induction / Q000187:drug effects / Q000235:genetics; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D051445:Fibrocartilage / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000096:biosynthesis* / Q000187:drug effects; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D050784:Proto-Oncogene Protein c-ets-1 / Q000187:drug effects; D016760:Proto-Oncogene Proteins c-fos / Q000187:drug effects; D016755:Proto-Oncogene Proteins c-jun / Q000187:drug effects; D011817:Rabbits; D012065:Relaxin / Q000494:pharmacology*; D013704:Temporomandibular Joint / Q000166:cytology; D018808:Transcription Factor AP-1 / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects; D001616:beta-Galactosidase / Q000032:analysis / Q000187:drug effects
D016895:Culture Media, Serum-Free; C494041:ETS1 protein, human; D050784:Proto-Oncogene Protein c-ets-1; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C065788:transcription factor PEA3; D004958:Estradiol; D012065:Relaxin; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1