Parathyroid hormone induces rat interstitial collagenase mRNA through Ets-1 facilitated by cyclic AMP response element-binding protein and Ca2+/calmodulin-dependent protein kinase II in osteoblastic cells

Parathyroid hormone (PTH), a powerful bone-resorbing agent, is capable of s timulating interstitial collagenase (MMP-13) mRNA production in osteoblasti c cells. In this study, a PEA3 consensus binding sequence (-80; AGGAAGT) in addition to a 'TRE-like' sequence (-89; CGACTCA) in the 5' upstream regula tory 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 oligonuc leotide containing the PEA3 region (-95 to -71). This increase in binding w as 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 t ransferase (CAT) construct containing 5' upstream regulatory sequence of th e rat MMP-13 gene significantly decreased activation by PTH. PTH-mediated b inding 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 Ca2+/calmodulin-dep endent 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 prote in (CBP) and CREB were capable of supershifting proteins binding to the oli gonucleotide containing the PEA3 region. These data suggest a possible co-o perative interaction of factors binding to the PEA3 and TRE-like sequences and provide the first indication of a role for a calcium-mediated pathway i n the PTH induction of MMP-13 mRNA in osteoblastic cells.