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.

The Prostanoid EP4 Receptor and Its Signaling Pathway

“…Osteoclasts have also been shown to express EP4 abundantly (Mano et al, 2000). Although there are convincing in vitro and in vivo collective data indicating that EP2 receptors may have a role in PGE 2 -mediated RANKL expression and anabolic effects on bone, to date no functional EP2 receptors have been identified on human osteoblasts or osteoclasts (Akhter et al, 2001;Woodward et al, 2011).…”

“…The major effect of PGE 2 on resorption is generally considered to occur indirectly via upregulation of RANKL expression and by inhibition of osteoprotegrin expression in osteoblastic cells (Blackwell et al, 2010). It has been suggested that PGE 2 enhances osteoclast formation through EP4 receptor activation on osteoblasts (Mano et al, 2000;Sakuma et al, 2000). Osteoclast formation was enhanced by the presence of an EP4 agonist in coculture of mouse primary osteoblastic cells and bone marrow cells, but not in cocultures of primary osteoblastic cells from EP4 KO mice (Sakuma et al, 2000).…”

12-O-tetradecanoylphorbol-13-acetate (TPA) Inhibits Osteoclastogenesis by Suppressing RANKL-Induced NF-κB Activation

“…(2) 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, decreased osteoclastic bone-resorbing activity. (3) A synthetic peptide fragment, PKC(530-558), which activates PKC, significantly inhibits osteoclastic bone resorption, and this effect is abolished in the presence of a synthetic peptide fragment PKC (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36), which inhibits PKC, suggesting an important physiological role for the PKC pathway in osteoclast function. (4) In addition, synthetic diacylglycerols, analogs of the physiological activators of PKC, also inhibited bone resorption, providing further evidence for an important role of the PKC pathway in the regulation of osteoclast activity.…”

Prostaglandin E2 Induces Interaction Between hSlo Potassium Channel and Syk Tyrosine Kinase in Osteosarcoma Cells

“…(10) Moreover, PGE 2 can inhibit bone resorption by a direct action on mature osteoclasts. (11) In osteoblasts, PGE 2 , PTH, and mechanical loading stimulate an increase in intracellular calcium, likely via the production of inositol triphosphate, diacylglycerol, and cyclic adenosine monophosphate (cAMP). (12)(13)(14) This increase in intracellular Ca 2ϩ has been shown to trigger the opening of Ca 2 -dependent K ϩ channels of high conductance (BK).…”