Characterization of PGE(2) receptors (EP) and their role as mediators of 1alpha,25-(OH)(2)D-3 effects on growth zone chondrocytes

Growth plate chondrocyte function is modulated by the vitamin D metabolite 1 alpha ,25-(OH)(2)D-3 via activation of protein kinase C (PKC). In previou s studies with cells derived from prehypertrophic and upper hypertrophic zo nes of rat costochondral cartilage (growth zone cells), inhibition of prost aglandin production with indomethacin caused a decrease in the stimulation of PKC activity, suggesting that changes in prostaglandin levels mediate th e 1 alpha ,25-(OH)(2)D-3-dependent response in these cells. Growth zone cel ls also respond to PGE(2) directly, indicating that prostaglandins act as a utocrine or paracrine regulators of chondrocyte metabolism in the growth pl ate. The aim of the present study was to identify which PGE, receptor subty pes (EP) mediate the effects of PGE2 on growth zone cells. Using primers sp ecific for EP1-EP4, reverse transcription-polymerase chain reaction (RT-PCR ) amplified EP1 and EP2 cDNA in a RT-dependent manner. In parallel experime nts, we used EP subtype-specific agonists to examine the role of EP recepto rs in 1 alpha ,25-(OH)(2)D-3-mediated cell proliferation and differentiatio n. 17-Phenyl-trinor-PGE(2) (PTPGE(2)), an EP1 agonist, decreased [H-3]-thym idine incorporation in a dose-dependent manner and augmented the 1 alpha ,2 5-(OH)(2)D-2-induced inhibition of [H-3]-thymidine incorporation. PTPGE(2) also caused significant increases in proteoglycan production, as measured b y [S-35]-sulfate incorporation, and alkaline phosphatase specific activity. 1 alpha ,25-(OH)(2)D-3-induced alkaline phosphatase activity was only slig htly stimulated by PTPGE(2). In contrast, 1 alpha ,25-(OH)(2)D-3-induced PK C activity was synergistically increased by PTPGE21 whereas EP1 antagonists SC-19220 and AH6809 inhibited PKC activity in a dose-dependent manner. The EP2, EP3 and EP4 agonists had no effect on the various cell-induced respon ses measured. EP1 receptor-induced responses were blocked by the phospholip ase C inhibitor U73122, and reduced by PKA inhibitors. EP1 receptor-induced PKC activity was insensitive to pertussis toxin or choleratoxin but blocke d by the G-protein inhibitor GDP betaS, suggesting the involvement of G(q). These results suggest that the EP1 receptor subtype mediates various PGE(2 )-induced cellular responses in growth zone chondrocytes leading to decreas ed proliferation and enhanced differentiation, as well as the effect of la, 25-(OH)2D3 on cellular maturation. (C) 2001 Elsevier Science Ltd. All right s reserved.