Parathyroid hormone induces rat interstitial collagenase mRNA through Ets-1 facilitated by cyclic AMP response element-binding protein and Ca(2+)/calmodulin-dependent protein kinase II in osteoblastic cells.
Parathyroid hormone (PTH), a powerful bone-resorbing agent, is capable of stimulating interstitial collagenase (MMP-13) mRNA production in osteoblastic cells. In this study, a PEA3 consensus binding sequence (-80; AGGAAGT) in addition to a 'TRE-like' sequence (-89; CGACTCA) in the 5' upstream regulatory region of the rat MMP-13 gene were examined. In response to PTH, there was a time-dependent increase in binding of nuclear factors to an oligonucleotide containing the PEA3 region (-95 to -71). This increase in binding was first observed at 0.5 h, peaked at 4 h (7. 6-fold) then returned to basal levels by 24 h. Mutagenesis of the PEA3 site in a chloramphenicol acetyl transferase (CAT) construct containing 5' upstream regulatory sequence of the rat MMP-13 gene significantly decreased activation by PTH. PTH-mediated binding of nuclear factors to an oligonucleotide containing the mutant PEA3 sequence was decreased as compared with the wild type. Mutation or deletion of the TRE-like sequence affected basal as well as PTH-mediated induction of corresponding CAT constructs. Treatment with KN93, a Ca(2+)/calmodulin-dependent protein kinase II specific inhibitor, greatly reduced the amount of protein binding to the PEA3 region in response to PTH which correlated to a notable decrease in the amount of MMP-13 mRNA produced in response to PTH. Antibodies against Ets-1, cyclic AMP response element (CREB)-binding protein (CBP) and CREB were capable of supershifting proteins binding to the oligonucleotide containing the PEA3 region. These data suggest a possible co-operative interaction of factors binding to the PEA3 and TRE-like sequences and provide the first indication of a role for a calcium-mediated pathway in the PTH induction of MMP-13 mRNA in osteoblastic cells.
Department of Pediatrics, Pediatric Research Institute, St Louis University Health Sciences Center, St Louis, Missouri 63104, USA. Quinnco@slu.edu
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Quinn
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Rajakumar
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Agapova
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017364:Collagenases / Q000235:genetics*; D016384:Consensus Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D005809:Genes, Regulator; D053509:Matrix Metalloproteinase 13; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics*; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism
D017362:Cyclic AMP Response Element-Binding Protein; C494043:Ets1 protein, rat; D010281:Parathyroid Hormone; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D012333:RNA, Messenger; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017364:Collagenases; D053509:Matrix Metalloproteinase 13; C507083:Mmp13 protein, rat