Anabolic effects of PTH in cyclooxygenase-2 knockout osteoblasts in vitro.
PTH is a potent bone anabolic agent in vivo but anabolic effects on osteoblast differentiation in vitro are difficult to demonstrate. This study examined the role of cyclooxygenase (COX)-2 and prostaglandin (PG) production in the effects of PTH on osteoblast differentiation in vitro using marrow stromal cell (MSC) and calvarial osteoblast (COB) cultures from COX-2 knockout (KO) and wild type (WT) mice. Cells were treated with PTH (10 nM) or vehicle throughout culture. Alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA levels were measured at days 14 and 21, respectively, and mineralization at day 21. cAMP concentrations were measured in the presence of a phosphodiesterase inhibitor. PTH did not stimulate differentiation in cultures from WT mice but significantly increased ALP and OCN mRNA expression 6- to 7-fold in KO MSC cultures and 2- to 4-fold in KO COB cultures. PTH also increased mineralization in both KO MSC and COB cultures. Effects in KO cells were mimicked in WT MSC cultures treated with NS-398, an inhibitor of COX-2 activity. PTH increased cAMP concentrations similarly in WT and KO COBs. Differential gene responses to PTH in COX-2 KO COBs relative to WT COBs included greater fold-increases in the cAMP-mediated early response genes, c-fos and Nr4a2; increased IGF-1 mRNA expression; and decreased mRNA expression of MAP kinase phosphatase-1. PTH inhibited SOST mRNA expression 91% in COX-2 KO MSC cultures compared to 67% in WT cultures. We conclude that endogenous PGs inhibit the anabolic responses to PTH in vitro, possibly by desensitizing cAMP pathways.
Choudhary
Shilpa
S
Musculoskeletal Institute, Department of Medicine, Division of Endocrinology, University of Connecticut Health Center, 263 Farmington Avenue, MC5456, Farmington, CT 06030, USA.
Huang
Hechang
H
Raisz
Lawrence
L
Pilbeam
Carol
C
D048868:Adaptor Proteins, Signal Transducing; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D002113:Calcification, Physiologic / Q000187:drug effects / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D000242:Cyclic AMP / Q000096:biosynthesis; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D005819:Genetic Markers / Q000235:genetics; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D018345:Mice, Knockout; D010006:Osteoblasts / Q000166:cytology* / Q000201:enzymology; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics; D010281:Parathyroid Hormone / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D012886:Skull / Q000166:cytology
D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D005819:Genetic Markers; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D010281:Parathyroid Hormone; D012333:RNA, Messenger; C493716:Runx2 protein, mouse; C486030:Sost protein, mouse; D015675:Osteocalcin; D000242:Cyclic AMP; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D000469:Alkaline Phosphatase; D015232:Dinoprostone