Basal bone phenotype and increased anabolic responses to intermittent parathyroid hormone in healthy male COX-2 knockout mice.

Cyclooxygenase-2 (COX-2) knockout (KO) mice in inbred strains can have renal dysfunction with secondary hyperparathyroidism (HPTH), making direct effects of COX-2 KO on bone difficult to assess. COX-2 KO mice in an outbred CD-1 background did not have renal dysfunction but still had two-fold elevated PTH compared to wild type (WT) mice. Compared to WT mice, KO mice had increased serum markers of bone turnover, decreased femoral bone mineral density (BMD) and cortical bone thickness, but no differences in trabecular bone volume by microCT or dynamic histomorphometry. Because PTH is a potent inducer of COX-2 and prostaglandin (PG) production, we examined the effects of COX-2 KO on bone responses after 3 weeks of intermittent PTH. Intermittent PTH increased femoral BMD and cortical bone area more in KO mice than in WT mice and increased trabecular bone volume in the distal femur in both WT and KO mice. Although not statistically significant, PTH-stimulated increases in trabecular bone tended to be greater in KO mice than in WT mice. PTH increased serum markers of bone formation and resorption more in KO than in WT mice but increased the ratio of osteoblastic surface-to-osteoclastic surface only in KO mice. PTH also increased femoral mineral apposition rates and bone formation rates in KO mice more than in WT mice. Acute mRNA responses to PTH of genes that might mediate some anabolic and catabolic effects of PTH tended to be greater in KO than WT mice. We conclude that (1) the basal bone phenotype in male COX-2 KO mice might reflect HPTH, COX-2 deficiency or both, and (2) increased responses to intermittent PTH in COX-2 KO mice, despite the presence of chronic HPTH, suggest that absence of COX-2 increased sensitivity to PTH. It is possible that manipulation of endogenous PGs could have important clinical implications for anabolic therapy with PTH.

Serum sclerostin levels negatively correlate with parathyroid hormone levels and free estrogen index in postmenopausal women.

CONTEXT: Sclerostin is a negative regulator of bone formation. OBJECTIVE: The aim of the study was to compare serum sclerostin levels in premenopausal and postmenopausal women and evaluate its relationship to estrogen, TH, bone turnover, and bone mass. DESIGN, SETTING, AND PARTICIPANTS: We conducted a cross-sectional observational study of healthy community-dwelling pre- and postmenopausal women. INTERVENTION(S): There were no interventions. MAIN OUTCOME MEASURE(S): We compared serum sclerostin levels in pre- and postmenopausal women and correlated sclerostin levels with female sex hormones, calciotropic hormones, bone turnover markers, and bone mineral density. RESULTS: Premenopausal women were 26.8 yr old, and postmenopausal women were 56.8 yr old. Postmenopausal women had lower values for estradiol (30 +/- 23 vs. 10 +/- 4 pg/ml; P < 0.001), estrone (61 +/- 24 vs. 29 +/- 10 pg/ml; P <0.001), and free estrogen index (FEI) (6 +/- 4 vs. 3 +/- 2 pmol/nmol; P = 0.008) and significantly lower bone mineral density at all sites compared to premenopausal women, with no significant differences in levels of PTH, 25-hydroxy or 1,25-dihydroxy vitamin D levels. Postmenopausal women had significantly higher serum sclerostin levels (1.16 +/- 0.38 ng/ml vs. 0.48 +/- 0.15 ng/ml; P < 0.001). Because most of the premenopausal women were on oral contraceptives, subsequent analyses were limited to postmenopausal women. There were significant negative correlations between sclerostin and FEI and sclerostin and PTH in this group. Using multiple regression analysis, both FEI (beta = -0.629; P = 0.002) and PTH (beta = -0.554; P = 0.004) were found to be independent predictors of sclerostin levels in postmenopausal women. CONCLUSIONS: Our findings suggest that serum sclerostin levels are regulated by both estrogens and PTH in postmenopausal women. These findings need to be explored further in larger prospective studies.

Prostaglandins in bone: bad cop, good cop?

Prostaglandins (PGs) are multifunctional regulators of bone metabolism that stimulate both bone resorption and formation. PGs have been implicated in bone resorption associated with inflammation and metastatic bone disease, and also in bone formation associated with fracture healing and heterotopic ossification. Recent studies have identified roles for inducible cyclooxygenase (COX)-2 and PGE(2) receptors in these processes. Although the effects of PGs have been most often associated with cAMP production and protein kinase A activation, PGs can engage an extensive G-protein signaling network. Further analysis of COX-2 and PG receptors and their downstream G-protein signaling in bone could provide important clues to the regulation of skeletal cell growth in both health and disease.

Bone morphogenetic protein 2 enhances PGE(2)-stimulated osteoclast formation in murine bone marrow cultures.

Bone morphogenetic protein 2 (BMP-2) is used clinically to stimulate bone formation and accelerate fracture repair. Adding prostaglandin (PG) E(2) or PGE(2) receptor agonists to BMP-2 has been proposed to improve BMP-2 efficacy. However, this may enhance bone resorption, since PGE(2) can increase receptor activator of NF-kappaB ligand (RANKL) expression and decrease osteoprotegerin (OPG) expression in osteoblasts, and the RANKL:OPG ratio is critical for osteoclast formation. We used bone marrow (BM) cultures and BM macrophage (BMM) cultures from outbred CD1 mice to examine effects on osteoclast formation of BMP-2 and PGE(2). In BM cultures, which contain both osteoblastic and osteoclastic lineage cells, BMP-2 (100 ng/ml) alone did not increase osteoclast formation but enhanced the peak response to PGE(2) by 1.6-9.6-fold. In BMM cultures, which must be treated with RANKL because they do not contain osteoblastic cells, BMP-2 did not increase osteoclast formation, with or without PGE(2). Our results suggest that BMP-2 can increase osteoclast formation in response to PGE(2) by increasing the RANKL:OPG ratio in osteoblasts, which may have therapeutic implications for the use of BMP-2.

Effects of prostaglandin E2 on bone in mice in vivo.

We have examined the effects of varying doses, schedules and routes of administration of prostaglandin E(2) (PGE(2)) on bone in mice. Male C57BL/6 mice treated with a high dose of PGE(2) (6 mg/kg/d) showed decreased trabecular bone volume (BV/TV) by 14 d, indicating increased bone resorption. However, there was also stimulation of bone formation at 14 d after 3 d treatment with PGE(2,) since mineral apposition rate (MAR) and bone formation rate (BFR/BS) were increased. In CD-1 male and female mice, PGE(2) (3mg/kg, 2/wk for 4 wk) increased MAR by 50% and BFR/BS by 100%, but there was no significant change in BV/TV. Tibial mRNA showed an increase in BMP-2 and RUNX-2 expression with PGE(2). Additional experiments using a higher dose or longer exposure did not increase bone mass. We conclude that exposure to high doses of PGE(2) in mice may be anabolic but is balanced by catabolic effects. Studies of PGE(2) in combination with an inhibitor of resorption could lead to development of a true anabolic model and permit assessment of the roles of specific PGE(2) receptors and signal transduction pathways.

Effects of global or targeted deletion of the EP4 receptor on the response of osteoblasts to prostaglandin in vitro and on bone histomorphometry in aged mice.

Because global deletion of the prostaglandin EP4 receptor results in neonatal lethality, we generated a mouse with targeted EP4 receptor deletion using Cre-LoxP methodology and a 2.3 kb collagen I a1 promoter driving Cre recombinase that is selective for osteoblastic cells. We compared wild type (WT), global heterozygote (G-HET), targeted heterozygote (T-HET) and knockout (KO) mice. KO mice had one targeted and one global deletion of the EP4 receptor. All mice were in a mixed background of C57BL/6 and CD-1. Although there were one third fewer G-HET or KO mice at weaning compared to WT and T-HET mice, G-HET and KO mice appeared healthy. In cultures of calvarial osteoblasts, prostaglandin E(2) (PGE(2)) increased alkaline phosphatase (ALP) activity in cells from WT mice, and this effect was significantly decreased in cells from either G-HET or T-HET mice and further decreased in cells from KO mice. A selective agonist for EP4 receptor increased ALP activity and osteocalcin mRNA levels in cells from WT but not KO mice. A selective COX-2 inhibitor, NS-398, decreased osteoblast differentiation in WT but not KO cells. At 15 to 18 months of age there were no differences in serum creatinine, calcium, PTH, body weight or bone mineral density among the different genotypes. Static and dynamic histomorphometry showed no consistent changes in bone volume or bone formation. We conclude that expression of the EP4 receptor in osteoblasts is critical for anabolic responses to PGE(2) in cell culture but may not be essential for maintenance of bone remodeling in vivo.

Bisphosphonate-associated osteonecrosis of the jaw: report of a task force of the American Society for Bone and Mineral Research.

UNLABELLED: ONJ has been increasingly suspected to be a potential complication of bisphosphonate therapy in recent years. Thus, the ASBMR leadership appointed a multidisciplinary task force to address key questions related to case definition, epidemiology, risk factors, diagnostic imaging, clinical management, and future areas for research related to the disorder. This report summarizes the findings and recommendations of the task force. INTRODUCTION: The increasing recognition that use of bisphosphonates may be associated with osteonecrosis of the jaw (ONJ) led the leadership of the American Society for Bone and Mineral Research (ASBMR) to appoint a task force to address a number of key questions related to this disorder. MATERIALS AND METHODS: A multidisciplinary expert group reviewed all pertinent published data on bisphosphonate-associated ONJ. Food and Drug Administration drug adverse event reports were also reviewed. RESULTS AND CONCLUSIONS: A case definition was developed so that subsequent studies could report on the same condition. The task force defined ONJ as the presence of exposed bone in the maxillofacial region that did not heal within 8 wk after identification by a health care provider. Based on review of both published and unpublished data, the risk of ONJ associated with oral bisphosphonate therapy for osteoporosis seems to be low, estimated between 1 in 10,000 and <1 in 100,000 patient-treatment years. However, the task force recognized that information on incidence of ONJ is rapidly evolving and that the true incidence may be higher. The risk of ONJ in patients with cancer treated with high doses of intravenous bisphosphonates is clearly higher, in the range of 1-10 per 100 patients (depending on duration of therapy). In the future, improved diagnostic imaging modalities, such as optical coherence tomography or MRI combined with contrast agents and the manipulation of image planes, may identify patients at preclinical or early stages of the disease. Management is largely supportive. A research agenda aimed at filling the considerable gaps in knowledge regarding this disorder was also outlined.

Effects of selective prostaglandins E2 receptor agonists on cultured calvarial murine osteoblastic cells.

We compared the direct effects of selective EP4 and EP2 receptor agonists (EP4A and EP2A) with prostaglandin E(2) (PGE(2)) on the differentiation of cultured murine calvarial osteoblastic cells. EP4A increased alkaline phosphatase activity and osteocalcin mRNA levels in these cultures similar to PGE(2). This effect was seen with both "direct plating" immediately after isolating the cells, or "indirect plating" in which the cells were grown to confluence and replated. EP2A had a smaller effect, significant only in "indirect plating" experiments. All three agents decreased the DNA and protein content in indirect plating experiments, but not in direct plating experiments. We conclude that the anabolic effect of PGE(2) in calvarial osteoblastic cell cultures is largely mediated by activation of the EP4 receptor, while activation of the EP2 receptor is less effective.

Pathogenesis of osteoporosis: concepts, conflicts, and prospects.

Osteoporosis is a disorder in which loss of bone strength leads to fragility fractures. This review examines the fundamental pathogenetic mechanisms underlying this disorder, which include: (a) failure to achieve a skeleton of optimal strength during growth and development; (b) excessive bone resorption resulting in loss of bone mass and disruption of architecture; and (c) failure to replace lost bone due to defects in bone formation. Estrogen deficiency is known to play a critical role in the development of osteoporosis, while calcium and vitamin D deficiencies and secondary hyperparathyroidism also contribute. There are multiple mechanisms underlying the regulation of bone remodeling, and these involve not only the osteoblastic and osteoclastic cell lineages but also other marrow cells, in addition to the interaction of systemic hormones, local cytokines, growth factors, and transcription factors. Polymorphisms of a large number of genes have been associated with differences in bone mass and fragility. It is now possible to diagnose osteoporosis, assess fracture risk, and reduce that risk with antiresorptive or other available therapies. However, new and more effective approaches are likely to emerge from a better understanding of the regulators of bone cell function.

Effect of deletion of the prostaglandin EP4 receptor on stimulation of calcium release from cultured mouse calvariae: impaired responsiveness in heterozygotes.

The ability of prostaglandin E2 (PGE2), selective receptor agonists for EP2 and EP4 receptors (EP2A and EP4A) and parathyroid hormone (PTH) to stimulate calcium release from cultured fetal mouse calvariae was compared in wild type (WT) mice and in mice heterozygous (HET) or homozygous (KO) for deletion of the EP4 receptor. Calvariae from 19 day fetal mice were used in order to avoid the problem of high neonatal mortality. Calcium release was increased by PGE2, EP4A or PTH in WT mice, but EP2A had no significant effect. There was a significant decrease in calcium release in response to PGE2, EP4A and PTH in calvariae from HET mice compared to WT mice. The response to PGE2 and EP4A was abrogated and the response to PTH was further diminished in EP4 receptor KO mice. These results suggest that the EP4 receptor may be rate limiting not only for PGE2 stimulated resorption but also for resorption stimulated by other agonists, like PTH that induce PGE2 production.

Prostaglandins differently regulate FGF-2 and FGF receptor expression and induce nuclear translocation in osteoblasts via MAPK kinase.

We have previously reported that prostaglandin F(2alpha) (PGF(2alpha)) and its selective agonist fluprostenol increase basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends our previous studies by showing that Py1a cells express FGF receptor-2 (FGFR2) and that treatment with PGF(2alpha) or fluprostenol decreases FGFR2 mRNA. We have used confocal and electron microscopy to show that, under PGF(2alpha) stimulation, FGF-2 and FGFR2 proteins accumulate near the nuclear envelope and colocalize in the nucleus of Py1a cells. Pre-treatment with cycloheximide blocks nuclear labelling for FGF-2 in response to PGF(2alpha). Treatment with SU5402 does not block prostaglandin-mediated nuclear internalization of FGF-2 or FGFR2. Various effectors have been used to investigate the signal transduction pathway. In particular, pre-treatment with phorbol 12-myristate 13-acetate (PMA) prevents the nuclear accumulation of FGF-2 and FGFR2 in response to PGF(2alpha). Similar results are obtained by pre-treatment with the protein kinase C (PKC) inhibitor H-7. In addition, cells treated with PGF(2alpha) exhibit increased nuclear labelling for the mitogen-activated protein kinase (MAPK), p44/ERK2. Pre-treatment with PMA blocks prostaglandin-induced ERK2 nuclear labelling, as confirmed by Western blot analysis. We conclude that PGF(2alpha) stimulates nuclear translocation of FGF-2 and FGFR2 by a PKC-dependent pathway; we also suggest an involvement of MAPK/ERK2 in this process.

Biphasic effect of prostaglandin E2 on osteoclast formation in spleen cell cultures: role of the EP2 receptor.

UNLABELLED: We examined the effect of PGE2 on OC formation from spleen cells treated with M-CSF and RANKL. PGE2 decreased OC number at 5-6 days of culture and increased OC number, size, and resorptive activity at 7-8 days. A selective EP2 receptor agonist mimicked these effects. Deletion of the EP2 receptor or depletion of T-cells abrogated the increase in OC number. INTRODUCTION: Prostaglandin E2 (PGE2) has been reported to increase osteoclast (OC) number in spleen cells cultured with RANKL and macrophage-colony-stimulating factor (M-CSF). In this study, we examined the time course of PGE2 effects on spleen cells cultured with RANKL and M-CSF. We then investigated which PGE receptors and cell types were involved in these effects. MATERIALS AND METHODS: Spleen cells were cultured from wildtype C57BL/6 mice and EP2 or EP4 receptor-deficient (-/-) and wildtype (+/+) mice on a mixed genetic background. Spleen cells were cultured with M-CSF and RANKL for 5-9 days with or without PGE2 or selective agonists for the four PGE2 receptors (EP1A, EP2A, EP3A, or EP4A). Some cultures were performed using T-cell-depleted spleen cells. OC number and size were quantitated. OC apoptosis and pit formation were measured at 7 or 8 days. RESULTS: PGE2 decreased the number of OCs formed in the presence of RANKL and M-CSF at 5-6 days of culture and increased OC number at 8-9 days compared with cultures without PGE2. PGE2 also increased OC size at 7 and 8 days, decreased apoptosis of OC at 7 days, and increased pit formation at 8 days. EP1A or EP4A had no effect on OC. EP3A decreased OC number. EP2A mimicked effect of PGE2. EP2(-/-) spleen cells showed no increase in OC number in response to PGE2, whereas deletion of EP4 had no effect. Depletion of T-cells abrogated the late increase of OC number. CONCLUSIONS: We conclude that PGE2 has an initial inhibitory effect on OC formation in spleen cell cultures, possibly mediated by both EP2 and EP3 receptors, and a later stimulatory effect, mediated by the EP2 receptor, possibly acting on T-cells.

Extracellular calcium induces COX-2 in osteoblasts via a PKA pathway.

We have shown that extracellular calcium [Ca(+2)](e) induces cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production via an ERK signaling pathway in osteoblasts. In this study, we examined the roles of protein kinase C (PKC) and A (PKA) signaling pathways in the [Ca(+2)](e) induction of COX-2 in primary calvarial osteoblasts from mice transgenic for -371 bp of the COX-2 promoter fused to a luciferase reporter. Neither PKC specific inhibitors nor downregulation of the PKC pathway by phorbol myristate acetate (PMA) affected the [Ca(+2)](e) stimulation of COX-2 mRNA or promoter activity. In contrast, PKA inhibitors, used at doses that inhibited forskolin-stimulated luciferase activity by 90%, reduced [Ca(+2)](e)-stimulated COX-2 mRNA expression and promoter activity by 80-90%. [Ca(+2)](e) also stimulated a 2- to 3-fold increase in cAMP production. Hence, the [Ca(+2)](e) induction of COX-2 mRNA expression and promoter activity was independent of the PKC pathway and dependent on the PKA signaling pathway.

Stimulation of cAMP production and cyclooxygenase-2 by prostaglandin E(2) and selective prostaglandin receptor agonists in murine osteoblastic cells.

Prostaglandins (PGs), particularly PGE(2), can stimulate bone resorption and formation and auto-amplify their effects by inducing cyclooxygenase (COX)-2. We examined the role of different PG receptors in stimulating cAMP production and COX-2 expression in murine calvarial osteoblasts. Cells were obtained from PGE(2) receptor (EP2R and EP4R) wild-type and knockout (KO) mice and from mice transgenic for the COX-2 promoter fused to a luciferase reporter. We analyzed effects of selective agonists, EP2A and EP4A, for EP2R and EP4R, which mediate the increase in cAMP in response to PGE(2). We also tested agonists for other PGE(2) receptors (EP1A and EP3A) and for prostacyclin (IPA), prostaglandin D(2) (DPA), thromboxane (TPA), and prostaglandin F(2alpha) (FPA) receptors. PGE(2) and EP2A were the most effective stimulators of cAMP production. EP4A, IPA, and DPA produced smaller responses, and EP1A, EP3A, FPA, and TPA were ineffective. In EP2R KO cells, cAMP responses to PGE(2) were reduced by 80%, and responses to EP2A were abrogated. In EP4R KO cells, cAMP responses to PGE(2) and EP2A showed a small reduction, while the response to EP4A was abrogated. Pretreatment with PGE(2), EP2A, or EP4A down-regulated the subsequent response to the respective ligands. COX-2 induction was measured by increased luciferase activity and mRNA expression. PGE(2) was the most effective agonist; EP2A and another selective EP2R agonist, butaprost, showed similar efficacy, and EP4A was less effective. EP2A and EP4A effects on luciferase activity were additive, and effects of the combination were similar to PGE(2) itself. IPA, TPA, and DPA produced 2- to 6-fold increases in COX-2 expression. FPA was a weak agonist, while EP1A and EP3A were inactive. Treatment with specific inhibitors indicated that PGE(2), EP2A, and EP4A induced COX-2 expression largely through protein kinase A (PKA). We conclude that the PG induction of COX-2 in this system generally paralleled effects on cAMP production and was mediated predominantly via the PKA pathway.