Prostaglandin E₂ Directly Inhibits Bone-Resorbing Activity of Isolated Mature Osteoclasts Mainly Through the EP4 Receptor

Abstract.

Prostaglandins (PGs) are well known to be important local factors in regulating bone formation and resorption. PGE₂ is a potent stimulator of bone resorption because of enhancing osteoclast formation by its indirect action through stromal cells. However, the direct action of PGE₂ on functionally mature osteoclasts is still controversial. In this study using highly purified rabbit mature osteoclasts, we examined the direct effect of PGE₂ on osteoclastic bone-resorbing activity and its mechanism. PGE₂ 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 PGE₂ 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. PGE₂ 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 PGE₂ 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 PGE₂ 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 PGE₂ inhibitory effect was mediated by an adenylate cyclase system coupled with EP4. This possible association of PGE₂ 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 PGE₂ effects on isolated osteoclasts although EP2 mRNA expression was minimal. In conclusion, PGE₂ directly inhibits bone-resorbing activity of functionally mature osteoclasts by activation of the adenylate cyclase system, perhaps mainly through EP4.