The B-type natriuretic peptide receptor (NPR-B) is a specific receptor for the C-type natriuretic peptide (CNP) and the binding of the peptide to NPR-B stimulates the bone formation by osteoblasts. However, the mechanism behind the regulation of NPR-B expression in osteoblasts remains unknown. In this study, we examined the role of prostaglandin E 2 (PGE 2 ) through the PGE 2 receptor subtypes, EP1, EP2, EP3 and EP4, in the regulation of NPR-B expression using calvarial osteoblasts from rats of various ages. Reverse Transcription-PCR (RT-PCR) and Western blotting analyses revealed that PGE 2 or 17-phenyl-ω-trinor PGE 2 , an EP1 agonist, increased the expression of NPR-B of calvarial osteoblasts from 25-week-old rats in a time-and dose-dependent manner. The PGE 2 -and EP1 agonist-induced increase in NPR-B expression was blocked by treating with SC19220, an EP1 antagonist. By contrast, agonists for EP2, EP3, and EP4 failed to affect the NPR-B expression. The basal mRNA level of NPR-B and EP1 continuously decreased with the age of cell donors between 10 to 60 weeks and remained constant over 60 weeks. The degree of EP1 agonist-induced increase in NPR-B mRNA level gradually decreased with age of cell donors between 10 to 60 weeks, and no significant effect of EP1 agonist on the NPR-B mRNA level was observed over 60 weeks. From these results, we concluded that PGE 2 acts as a regulator of NPR-B expression through the EP1 receptor in osteoblasts and age-related decrease in EP1 expression causes a decrease in NPR-B expression.

The C-type natriuretic peptide stimulates osteoblastic functions through the B-type natriuretic receptor (NPR-B). In this study, we examined the signaling pathway behind the regulation of NPR-B expression through the prostaglandin E 2 (PGE 2 ) receptor, EP1 subtype using rat calvarial osteoblasts. A23187 as a Ca 2+ ionophore increased NPR-B expression dose-dependently. PGE 2 or 17-phenyl-ω-trinor PGE 2 (EP1A), an EP1 agonist, increased NPR-B expression, and the potentiating effects were blocked by treating with BAPTA-AM as an intracellular Ca 2+ chelator. Activators of protein kinase C (PKC), 1-oleoyl-2-acetyl-sn-glycerol, a membrane-permeable diacylglycerol, and 12-o-tetradecanoyl-phorbol-13-acetate, also increased NPR-B expression, and the potentiating effects were blocked by treating with BAPTA-AM. The treatment of cells with GF109203X, a PKC inhibitor, blocked the PGE 2 -and EP1A-induced increase in NPR-B expression. From these results, we concluded that EP1-mediated increase in the expression of NPR-B requires not only Ca 2+ mobilization but also PKC activation through the activation of phosphatidylinositol-specific phospholipase C.