Regulation of COX-2 mediates acid-induced bone calcium efflux in vitro.
Chronic metabolic acidosis induces net Ca efflux from bone; this osteoclastic bone resorption is mediated by increased osteoblastic prostaglandin synthesis. Cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis, is present in both constitutive (COX-1) and inducible (COX-2) forms. We report here that acidosis increases both osteoblastic RNA and protein levels for COX-2 and that genetic deficiency or pharmacologic inhibition of COX-2 significantly reduces acid-induced Ca efflux from bone.


BACKGROUND
Incubation of neonatal mouse calvariae in medium simulating physiologic metabolic acidosis induces an increase in osteoblastic prostaglandin E2 (PGE2) release and net calcium (Ca) efflux from bone. Increased PGE2 is necessary for acid-induced bone resorption, because inhibition of cyclooxygenase activity with indomethacin significantly decreases not only PGE2 production but also Ca release. Cyclooxygenase is present in both constitutive (COX-1) and inducible (COX-2) forms. Because COX-2 activity has been implicated in several forms of pathological bone resorption, we tested the hypothesis that COX-2 is critical for acid-induced, cell-mediated bone Ca efflux.


METHODS
To determine the effect of metabolic acidosis on COX-2 RNA and protein, primary cells isolated from neonatal CD-1 mouse calvariae were cultured in neutral (Ntl) or physiologically acidic medium (Met). RNA levels for COX-2 and COX-1 were measured by quantitative real-time PCR. Levels of COX-2 and COX-1 protein were measured by immunoblot analysis. To determine the effect of acidosis on bone Ca efflux in genetically deficient COX-2 mice, mice heterozygous for the COX-2 knockout (strain B6;129S7-Ptgs2(tm1Jed)/J) were used as breeders, and neonatal calvariae were cultured in Ntl or Met. To determine the effects of the specific COX-2 inhibitor, NS398, on acid-induced bone resorption, CD-1 calvariae were incubated in Ntl or Met with or without NS398 (1 microM). Medium PGE2 was assayed by ELISA.


RESULTS
Incubation of mouse calvarial cells in Met significantly increased COX-2 RNA and protein levels without a change in COX-1. Increased COX-2 protein levels in response to Met were also observed in cultured calvariae. Acid-induced, cell-mediated Ca efflux from B6;129S7-Ptgs2(tm1Jed)/J calvariae was dependent on genotype. From 0 to 24 h, when physicochemical Ca efflux predominates, Met significantly increased net Ca efflux in all genotypes. After 24 h, when cell-mediated Ca efflux predominates, Met induced greater Ca efflux from (+/+) than from (+/-), and there was no increase from (-/-). In calvariae from CD-1 mice, NS398 significantly inhibited both the acid-induced increase in PGE2 and Ca release.


CONCLUSIONS
The specific acid-induced increase in COX-2 RNA and protein levels and the dependency of the increased Ca efflux on COX-2 activity, as determined by both genetic deficiency and pharmacologic inhibition, show that COX-2 is critical for acid-induced, cell-mediated bone resorption.
Krieger
Nancy S
NS
Department of Medicine, Division of Nephrology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Frick
Kevin K
KK
LaPlante Strutz
Kelly
K
Michalenka
Anne
A
Bushinsky
David A
DA
D000138:Acidosis / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001862:Bone Resorption / Q000378:metabolism; D001842:Bone and Bones / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D051545:Cyclooxygenase 1 / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005838:Genotype; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D009578:Nitrobenzenes / Q000494:pharmacology; D009924:Organ Culture Techniques; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012886:Skull / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors
D003470:Culture Media; D052246:Cyclooxygenase 2 Inhibitors; D008565:Membrane Proteins; D009578:Nitrobenzenes; D013449:Sulfonamides; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D012313:RNA; C077623:Ptgs2 protein, mouse; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496512:Ptgs1 protein, mouse; D015232:Dinoprostone; D002118:Calcium