Parathyroid hormone induction of cyclooxygenase-2 in murine osteoblasts: role of the calcium-calcineurin-NFAT pathway.
Murine MC3T3-E1 and MC-4 cells were stably transfected with -371/+70 bp of the murine cyclooxygenase-2 (COX-2) promoter fused to a luciferase reporter (Pluc371) or with Pluc371 carrying site-directed mutations. Mutations were made in (1) the cAMP response element (CRE) at -57/-52 bp, (2) the activating protein-1 (AP-1)-binding site at -69/-63 bp, (3) the nuclear factor of activated T-cells (NFAT)-binding site at -77/-73 bp, and (4) both the AP-1 and NFAT sites, which comprise a composite consensus sequence for NFAT/AP-1. Single mutation of CRE, AP-1, or NFAT sites decreased parathyroid hormone (PTH)-stimulated COX-2 promoter activity 40% to 60%, whereas joint mutation of NFAT and AP-1 abrogated the induction. On electrophoretic mobility shift analysis, PTH stimulated binding of phosphorylated CREB to an oligonucleotide spanning the CRE and binding of NFATc1, c-Fos, and c-Jun to an oligonucleotide spanning the NFAT/AP-1 composite site. Mutation of the NFAT site was less effective than mutation of the AP-1 site in competing binding to the composite element, suggesting that cooperative interactions of NFATc1 and AP-1 are more dependent on NFAT than on AP-1. Both PTH and forskolin, an activator of adenylyl cyclase, stimulated NFATc1 nuclear translocation. PTH- and forskolin-stimulated COX-2 promoter activity was inhibited 56% to 80% by calcium chelation or calcineurin inhibitors and 60% to 98% by protein kinase A (PKA) inhibitors. These results indicate an important role for the calcium-calcineurin-NFAT signaling pathway in the PTH induction of COX-2 and suggest that cross-talk between the cAMP/PKA pathway and the calcium-calcineurin-NFAT pathway may play a role in other functions of PTH in osteoblasts.
Huang
Hechang
H
Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA.
Chikazu
Daichi
D
Voznesensky
Olga S
OS
Herschman
Harvey R
HR
Kream
Barbara E
BE
Drissi
Hicham
H
Pilbeam
Carol C
CC
D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002614:Chelating Agents / Q000378:metabolism; D005576:Colforsin / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics*; D016572:Cyclosporine / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D051379:Mice; D009154:Mutation; D050778:NFATC Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology*; D010281:Parathyroid Hormone / Q000494:pharmacology / Q000502:physiology*; D011401:Promoter Regions, Genetic; D016559:Tacrolimus / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D065095:Calcineurin Inhibitors; D002614:Chelating Agents; D017362:Cyclic AMP Response Element-Binding Protein; D050778:NFATC Transcription Factors; D010281:Parathyroid Hormone; D018808:Transcription Factor AP-1; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D005576:Colforsin; D004533:Egtazic Acid; D016572:Cyclosporine; D051546:Cyclooxygenase 2; D017868:Cyclic AMP-Dependent Protein Kinases; D019703:Calcineurin; D002118:Calcium; D016559:Tacrolimus