Prostaglandin E2 directly inhibits bone-resorbing activity of isolated mature osteoclasts mainly through the EP4 receptor

Prostaglandins (PGs) are well known to be important local factors in regulating bone formation and resorption. PGE(2) is a potent stimulator of bone resorption because of enhancing osteoclast formation by its indirect action through stromal cells. However, the direct action of PGE(2) on functionally mature osteoclasts is still controversial. In this study using highly purified rabbit mature osteoclasts, we examined the direct effect of PGE(2) on osteoclastic bone-resorbing activity and its mechanism. PGE(2) inhibited resorption pit formation on a dentine slice by the purified osteoclasts in a dose- and time-dependent manner. The inhibitory effect appeared as early as 4 hours after the PGE(2) addition. Forskolin and 12-0-tetradecanoyl phorbol-13-acetate (TPA), respective activators of adenylate cyclase and protein kinase C, also decreased the osteoclastic bone-resorbing activity. PGE(2) increased the content of intracellular cAMP in a dose range effective for the inhibition of bone resorption, whereas the prostanoid did not alter the intracellular level of inositol triphosphate. The inhibition of osteoclastic bone resorption by PGE(2) was amplified and diminished by a cAMP phosphodiesterase inhibitor (isobutyl methylxanthine) and a protein kinase A inhibitor (Rp-cAMP), respectively. Of four different subtypes of PGE(2) receptors (EPs), EP4 mRNA was predominantly expressed in isolated osteoclasts, whereas the other types of EP mRNA were detected in only small amounts. These results suggest that the PGE(2) inhibitory effect was mediated by an adenylate cyclase system coupled with EP4, This possible association of PGE(2) with EP4 in mature osteoclasts was supported by the finding that a specific agonist of EP4 (AE-604) inhibited the bone-resorbing activity and elevated the intracellular cAMP content. However, butaprost, a selective EP2 agonist, also mimicked the PGE(2) effects on isolated osteoclasts although EP2 mRNA expression was minimal. In conclusion, PGE(2) directly inhibits bone-resorbing activity of functionally mature osteoclasts by activation of the adenylate cyclase system, perhaps mainly through EP4.