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.

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.

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.

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.

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.

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.

Extracellular calcium is a potent inducer of cyclo-oxygenase-2 in murine osteoblasts through an ERK signaling pathway.

UNLABELLED: [Ca2+]e may be important in bone turnover. We found [Ca2+]e induces COX-2 transcription and PGE2 production in primary calvarial osteoblasts through an ERK signaling pathway. Inhibition of PGE2 production inhibited the [Ca2+]e stimulation of osteoblastic differentiation but not the increase in cell number. Hence, some effects of [Ca2+]e on bone may be mediated by COX-2. INTRODUCTION: Local changes in extracellular calcium ([Ca2+]e) may play an important role in bone turnover. We examined the possibility that prostaglandins produced by cyclo-oxygenase-2 (COX-2) could mediate some of the effects of [Ca2+]e on osteoblasts. METHODS: We examined the [Ca2+]e induction of COX-2 expression and prostaglandin E2 (PGE2) production in primary osteoblasts (POBs) obtained by sequential enzymatic digestion of mouse calvariae. We measured mRNA and protein levels by Northern and Western analyses and PGE2 production in culture medium by radioimmunoassay (RIA). COX-2 promoter activity was measured as luciferase activity in calvarial osteoblasts derived from mice transgenic for 371 bp of the COX-2 promoter fused to a luciferase reporter gene. RESULTS AND CONCLUSIONS: COX-2 mRNA and protein expression were induced by 3-40 mM of [Ca2+]e. [Ca2+]e (5 mM) induced COX-2 mRNA within 30 minutes; levels peaked at 6-9 h and remained elevated at 24 h. Cumulative medium PGE2 was increased at 3 h, with levels rising to 30 nM at 24 h. PGE2 production in POBs from mice with only COX-1 gene expression was 1/40th of that in POBs from mice with both COX-1 and COX-2 gene expression. [Ca2+]e increased alkaline phosphatase activity and osteocalcin mRNA, and this increase was blocked by inhibiting PGE2 production. [Ca2+]e stimulation of COX-2 promoter activity correlated with the induction of COX-2 mRNA expression. [Ca2+]e induced rapid and transient phosphorylation of extracellular signal-regulated kinase (ERK) in POBs, which peaked at 5-10 minutes. Inhibition of ERK phosphorylation with the specific inhibitors, PD-98059 and U-0126, decreased the [Ca2+]e induction of both COX-2 mRNA and luciferase activity by 70-80%. Although less effective than [Ca2+]e, strontium [Sr2+]e also induced COX-2 mRNA and promoter activity in POBs through an ERK signaling pathway. We conclude that [Ca2+]e is a potent transcriptional inducer of COX-2 expression and PGE2 production in osteoblasts through an ERK signaling pathway.

Fluid flow induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular signal-regulated kinase signaling pathway.

Mechanical loading of bone may be transmitted to osteocytes and osteoblasts via shear stresses at cell surfaces generated by the flow of interstitial fluid. The stimulated production of prostaglandins, which mediates some effects of mechanical loading on bone, is dependent on inducible cyclo-oxygenase 2 (COX-2) in bone cells. We examined the fluid shear stress (FSS) induction of COX-2 gene expression in immortalized MC3T3-E1 osteoblastic cells stably transfected with -371/+70 base pairs (bp) of the COX-2 5'-flanking DNA (Pluc371) and in primary osteoblasts (POBs) from calvaria of mice transgenic for Pluc371. Cells were plated on collagen-coated glass slides and subjected to steady laminar FSS in a parallel plate flow chamber. FSS, from 0.14 to 10 dynes/cm2, induced COX-2 messenger RNA (mRNA) and protein. FSS (10 dynes/cm2) induced COX-2 mRNA within 30 minutes, with peak effects at 4 h in MC3T3-E1 cells and at > or = 8 h in POBs. An inhibitor of new protein synthesis puromycin blocked the peak induction of COX-2 mRNA by FSS. COX-2 promoter activity, measured as luciferase activity, correlated with COX-2 mRNA expression in both MC3T3-E1 and POB cells. FSS induced phosphorylation of extracellular signal-regulated kinase (ERK) in MC3T3-E1 cells, with peak effects at 5 minutes. Inhibiting ERK phosphorylation with the specific inhibitor PD98059 inhibited FSS induction of COX-2 mRNA by 55-70% and FSS stimulation of luciferase activity by > or = 80% in both MC3T3-E1 and POB cells. We conclude that FSS transcriptionally induces COX-2 gene expression in osteoblasts, that the maximum induction requires new protein synthesis, and that induction occurs largely via an ERK signaling pathway.

Bone morphogenetic protein 2 induces cyclo-oxygenase 2 in osteoblasts via a Cbfal binding site: role in effects of bone morphogenetic protein 2 in vitro and in vivo.

We tested the hypothesis that induction of cyclo-oxygenase (COX) 2 mediates some effects of bone morphogenetic protein (BMP) 2 on bone. BMP-2 induced COX-2 mRNA and prostaglandin (PG) production in cultured osteoblasts. BMP-2 increased luciferase activity in calvarial osteoblasts from mice transgenic for a COX-2 promoter-luciferase reporter construct (Pluc) and in MC3T3-E1 cells transfected with Pluc. Deletion analysis identified the -300/-213-bp region of the COX-2 promoter as necessary for BMP-2 stimulation of luciferase activity. Mutation of core-binding factor activity 1 (muCbfal) consensus sequence (5'-AACCACA3') at -267/-261 bp decreased BMP-2 stimulation of luciferase activity by 82%. Binding of nuclear proteins to an oligonucleotide spanning the Cbfal site was inhibited or supershifted by specific antibodies to Cbfal. In cultured osteoblasts from calvariae of COX-2 knockout (-/-) and wild-type (+/+) mice, the absence of COX-2 expression reduced the BMP-2 stimulation of both ALP activity and osteocalcin mRNA expression. In cultured marrow cells flushed from long bones, BMP-2 induced osteoclast formation in cells from COX-2(+/+) mice but not in cells from COX-2(-/-) mice. In vivo, BMP-2 (10 microg/pellet) induced mineralization in pellets of lyophilized collagen implanted in the flanks of mice. Mineralization of pellets, measured by microcomputed tomography (microCT), was decreased by 78% in COX-2(-/-) mice compared with COX-2(+/+) mice. We conclude that BMP-2 transcriptionally induces COX-2 in osteoblasts via a Cbfal binding site and that the BMP-2 induction of COX-2 can contribute to effects of BMP-2 on osteoblastic differentiation and osteoclast formation in vitro and to the BMP-2 stimulation of ectopic bone formation in vivo.

The effect of micronized estradiol on bone turnover and calciotropic hormones in older men receiving hormonal suppression therapy for prostate cancer.

To examine the effect of estradiol (E(2)) without the confounding effect of hypothalamic-pituitary feedback, we studied men with prostate cancer in whom gonadotropin secretion was suppressed by LH-releasing hormone agonists (LHRH-A). Fourteen men over 65 yr of age and receiving established LHRH-A treatment (EST group) without bony metastases and 12 men who received LHRH-A as neoadjuvant therapy for locally advanced prostate cancer (NEO group) were randomized (double blind) to receive either 1 mg/d micronized E(2) (n = 12) or placebo (PL; n = 13) for 9 wk. E(2), estrone, testosterone, SHBG, PTH, and 25-hydroxy- and 1,25-dihydroxyvitamin D levels as well as markers of bone resorption [N- and C-telopeptide cross-links (NTX and CTX) and deoxypyridinoline] and bone formation (bone-specific alkaline phosphatase, osteocalcin, and N-terminal type I collagen) were measured before LHRH-A in the NEO group, before [baseline (BL)] and after 9 wk of E(2) or PL in all patients, and 6 wk after E(2) treatment in the EST group. In the NEO group, hormone levels fell 3 wk after the initial LHRH-A injection, and deoxypyridinoline increased significantly (P = 0.006). At BL, the EST group had higher bone turnover due to the longer duration of LHRH-A treatment. With E(2) treatment, E(2) levels rose into the normal male range, and two resorption markers decreased significantly from BL by 33% for NTX (P < 0.001) and 28% for CTX (P = 0.009). Bone formation markers did not change. PTH increased by 43% from BL (P < 0.01) in the E(2) group and decreased 16% from BL in the PL group (P < 0.01). Ionized calcium did not change in the E(2) group, but increased in the PL group by 2.3% (P < 0.01). NTX and CTX increased 6 wk after E(2) withdrawal in the EST group. We conclude that E(2) inhibits bone resorption in hypogonadal men through a direct skeletal effect that is independent of PTH. Low dose estrogen may be an option for the prevention and/or treatment of bone loss in this population.

The effect of deletion of cyclooxygenase-2, prostaglandin receptor EP2, or EP4 in bone marrow cells on osteoclasts induced by mouse mammary cancer cell lines.

The inducible prostaglandin (PG) synthesis enzyme, cyclooxygenase-2 (COX-2), is involved in osteoclast (OC) formation in cocultures of mouse mammary cancer cell lines (MMT060562 or BALB/c-MC) and bone marrow cells through production of PGE(2). There are four PGE(2) receptors but only the EP2 and EP4 receptors are reported to be important for OC formation. We have investigated the role of COX-2, EP2 receptor, and EP4 receptor in marrow cells for osteoclastogenesis in cocultures of cancer cells and bone marrow cells. We cocultured cancer cell lines with bone marrow cells from COX-2 knockout (-/-), EP2 -/- or EP4 -/- mice compared to wild-type mice. In addition, an EP4 receptor antagonist (EP4 RA) was added in some cocultures. Disruption of COX-2 gene in bone marrow cells had no effect on PGE(2) production and OC formation in cocultures with MMT060562, while it abrogated PGE(2) production and OC formation in cocultures with BALB/c-MC. Disruption of the EP2 gene in bone marrow cells had no effect on OC formation in the cocultures, while disruption of the EP4 gene in bone marrow cells abrogated OC formation in the cocultures. Furthermore, EP4 RA suppressed OC formation and prevented the increase in receptor activator of nuclear factor kappaB ligand (RANKL) mRNA levels in the cocultures. We conclude that COX-2 in cancer cells is responsible for PGE(2) and OC production in cocultures with MMT060562, while COX-2 in bone marrow cells, not cancer cells, is responsible for PGE(2) and OC production in cocultures with BALB/c-MC, and EP4 receptors are essential for OC formation in both cocultures.

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.

Pathogenesis of osteoporosis.

There are many pathways that might lead to decreased bone mass, skeletal fragility, and increased fracture risk in osteoporosis. Some of these have been clearly identified, such as estrogen deficiency. Others that were conceived on the basis of experimental findings and recent scientific discoveries such as abnormalities of cytokines, bone growth factors, and osteoblast transcription factors remain interesting but speculative. The recent revolution in genomics and proteomics opens new avenues for pursuing in great depth the pathways leading to osteoporosis. Animal models developed largely in rodents can suggest specific factors that can be further studied in primate models and in osteoporotic patients. Identification of specific pathogenetic mechanisms should lead to new approaches to the diagnosis and management of this disorder.

Effects of selective prostaglandin EP2 and EP4 receptor agonists on bone resorption and formation in fetal rat organ cultures.

Prostaglandin E2 (PGE2) can stimulate bone resorption and formation through receptors which activate adenylyl cyclase. We examined the effects of selective EP2 and EP4 agonists (EP2A and EP4A) on the release of previously incorporated 45Ca from fetal rat long bones and the incorporation of [3H]-proline or [3H]-thymidine (TdR) in fetal rat calvaria to assess the relative effects of these selective agonists on bone formation and resorption. Only EP4A was effective in increasing 45Ca release. Both agonists increased [3H]-TdR incorporation and [3H]-proline incorporation into calvariae, particularly in the presence of cortisol.

Prostaglandin receptor EP2 mediates PGE2 stimulated hypercalcemia in mice in vivo.

Prostaglandin E2 (PGE2) can stimulate bone resorption by a cyclic AMP-dependent pathway. Two PGE2 receptors, EP2 and EP4 have been shown to play a role in PGE2 stimulation of osteoclast formation. In primary osteoblastic cell cultures from EP2 wild type (EP2 +/+) mice, PGE2 (0.1 microM) increased cyclic AMP production 3.5-fold, but PGE2 had no effect on cells from mice in which the EP2 receptor had been deleted (EP2 -/-). To examine the role of the EP2 receptor in the resorption response in vivo we injected PGE2 in EP2 -/- mice, and compared them with EP2 +/+ mice. Injection of PGE2 (3 mg/kg, four times daily for three days) in 9- to 12-month-old male mice on a 129 SvEv background increased serum calcium from 9.8 +/- 0.5 to 10.7 +/- 0.3 mg/dl (P < 0.01) in EP2 +/+ mice but not in EP2 -/- mice (10.1 +/- 0.3 vs. 10.2 +/- 0.3 mg/dl). PGE2 injection (6 mg/kg twice a day for three days) in 3-4 month old male mice on a C57 BL/6 X 129 SvEv background increased calcium from 8.2 +/- 0.1 to 9.0 +/- 0.3 mg/dl (P < 0.05) in EP2 +/+ mice but had no effect in EP2-/- mice (8.4 +/- 0.1 vs. 8.3 +/- 0.2 mg/dl). Injection of PGE2 over the calvariae of EP2 +/+ and EP2-/- mice increased the expression of receptor activator of nuclear factor kappaB ligand (RANKL) both locally and in the tibia, but RANKL responses were lower in EP2 -/- mice. We conclude that EP2 receptor plays a role in the hypercalcemic response to PGE2. This impaired response in EP2 -/- mice may be due to decreased ability to stimulate cyclic AMP and in part, to a smaller increase in the expression of RANKL mRNA.

Effects of Misoprostol on Bone Resorption and Formation in Organ Culture.

Prostaglandins are potent stimulators of bone resorption and formation. Because misoprostol is an analog of prostaglandin E(1) (PGE(1)), we have examined its effects on resorption and formation in organ culture. The results were compared with PGE(2) which can stimulate resorption and both stimulate and inhibit bone formation. Resorption, measured as the release of previously incorporated (45)Ca from 5-day cultures of 19-day fetal-rat long bones, was increased by misoprostol 1.5-fold at 10(minus sign6) M and twofold at 10(minus sign5) M. The effect at 10(minus sign6) M was abolished by addition of indomethacin (10(minus sign6) M). PGE(2) was approximately 100 times more potent and was not affected by indomethacin in this system. In 21-day fetal-rat calvariae, cultured for 24 h in the presence of cortisol (10(minus sign7) M), misoprostol produced a dose-related increase in TdR incorporation between 10(minus sign5) and 10(minus sign7) M. PGE(2) appeared to be only 10-fold more potent in this response. The effects of misoprostol on incorporation of [(3)H]proline into collagenase digestible protein (CDP) and noncollagen protein (NCP) were measured in 72-h cultures, either with continuous treatment or 24-h treatment followed by 48 h in control medium. With continuous treatment at 10(minus sign6) M, misoprostol increased labeling of CDP twofold. A similar effect was observed with 24 h of treatment at 10(minus sign5) M, followed by 48 h in control medium. Again, PGE(2) was approximately 10-fold more potent than misoprostol. When calvariae were treated with insulin-like growth factor I, which increases CDP labeling by 2.5-fold, the effects of misoprostol and PGE(2) were inhibitory. We conclude that misoprostol resembles PGE(2) in its effects on bone but is less potent. Moreover, misoprostol may be relatively less effective in stimulating resorption than in stimulating formation. Therefore, an increase in bone turnover, possibly with a net anabolic effect, might occur in vivo with long-term misoprostol treatment. Misoprostol effects on bone turnover in humans could be evaluated using the sensitive biochemical markers for bone resorption and formation which are currently available.

Prevention and treatment of osteoporosis.

Osteoporosis develops in older adults when the normal processes of bone formation and resorption become uncoupled or unbalanced, resulting in bone loss. Fractures are the result of decreased bone mass and strength and, in the case of wrist and hip fractures, usually involve a fall. Osteoporosis prevention and treatment programs should then focus on strategies that minimize bone resorption and maximize bone formation as well as on strategies that reduce falls. Optimal treatment and prevention of osteoporosis require modification of risk factors, particularly smoking cessation, adequate physical activity, and attention to diet, in addition to pharmacologic intervention. A number of pharmacologic options are now available to health care providers. This article focuses on US Food and Drug Administration-approved medications for osteoporosis and emphasizes the importance of using these agents as part of a comprehensive program that includes nonpharmacologic measures, complete diagnostic evaluation, and adequate follow-up with bone mineral density measurement.

Mechanisms of bone remodeling: implications for clinical practice.

The adult skeleton undergoes continuous remodeling. The remodeling cycle involves the interaction of cells of osteoblastic and osteoclastic lineage and is regulated by both systemic hormones and local factors. In addition to the systemic calcium-regulating hormones, parathyroid hormone, 1,25-dihydroxy vitamin D and calcitonin, sex hormones play an important role. Estrogen has been identified as the major inhibitor of bone resorption in both men and women. Androgen is important not only as a source of estrogen, through the action of aromatase, but also for its direct effect in stimulating bone formation. The effects of sex hormones may be mediated by their ability to alter the secretion of local cytokines, prostaglandins and growth factors. Sex hormone action is also modulated by the level of sex hormone-binding globulin in the circulation. A more precise analysis of these effects has been made possible by the development of new methods of measuring not only bone mineral density, but also relative rates of bone formation and resorption using biochemical markers. These new approaches have allowed us to define more precisely the specific roles of androgens, estrogens and other regulatory hormones in human skeletal physiology and pathophysiology.