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.

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.

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.