title: Parathyroid hormone induction of rat interstitial collagenase mRNA in osteosarcoma cells is mediated through an AP-1-binding site.
abstract: Interstitial collagenase is secreted by the osteoblast in response to bone-resorbing agents. Previously, we cloned the rat interstitial collagenase cDNA from UMR 106-01 rat osteoblastic osteosarcoma cells. We demonstrated that induction of collagenase by PTH, a powerful resorbing agent, in UMR 106-01 cells is in part transcriptional. In the present study we isolate and characterize the rat interstitial collagenase gene. The gene consists of 10 exons and spans approximately 12 kbp. The major transcriptional start site, determined by primer extension analysis and confirmed by RNase protection assay, is 25 nucleotides upstream of the translational start site. The previously isolated cDNA was missing the 5'-untranslated sequence in addition to 17 nucleotides of the signal sequence of the preproenzyme; therefore, we also present these data. Chloramphenicol acetyl transferase (CAT) analyses were performed on the 5'-upstream region of the gene. These data indicate that PTH appears to mediate its effect through an AP-1 consensus-binding sequence (-51). Footprint analysis demonstrates protein binding to this site. Site-specific mutagenesis markedly decreased protein binding, which correlated directly with a decrease in CAT activation by PTH. Supershift data indicate that cAMP response element binding protein (CREB) is binding to this AP-1 consensus sequence. In addition we demonstrate that PTH induces phosphorylation of CREB.
authors.affiliation: Cardinal Glennon Children's Hospital, Department of Pediatrics, St. Louis University Health Sciences Center, Missouri 63104, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Rajakumar
authors.forename: C O
authors.initials: CO
authors.lastname: Quinn
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D017364:Collagenases / Q000235:genetics* / Q000378:metabolism*; D017124:Conserved Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D018983:DNA Footprinting; D017931:DNA Primers; D003850:Deoxyribonuclease I / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D004789:Enzyme Activation; D016762:Genes, fos; D016758:Genes, jun; D020781:Matrix Metalloproteinase 1; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D010281:Parathyroid Hormone / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012260:Ribonucleases / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D010281:Parathyroid Hormone; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D015500:Chloramphenicol O-Acetyltransferase; D012260:Ribonucleases; D003850:Deoxyribonuclease I; D017364:Collagenases; D020781:Matrix Metalloproteinase 1