Characterization of PGE2 receptors (EP) and their role as mediators of 1α,25-(OH)2D3 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 previous studies with cells derived from prehypertrophic and upper hypertrophic zones of rat costochondral cartilage (growth zone cells), inhibition of prostaglandin production with indomethacin caused a decrease in the stimulation of PKC activity, suggesting that changes in prostaglandin levels mediate the 1 alpha ,25-(OH)(2)D-3-dependent response in these cells. Growth zone cells also respond to PGE(2) directly, indicating that prostaglandins act as autocrine or paracrine regulators of chondrocyte metabolism in the growth plate. The aim of the present study was to identify which PGE, receptor subtypes (EP) mediate the effects of PGE2 on growth zone cells. Using primers specific for EP1-EP4, reverse transcription-polymerase chain reaction (RT-PCR) amplified EP1 and EP2 cDNA in a RT-dependent manner. In parallel experiments, we used EP subtype-specific agonists to examine the role of EP receptors in 1 alpha ,25-(OH)(2)D-3-mediated cell proliferation and differentiation. 17-Phenyl-trinor-PGE(2) (PTPGE(2)), an EP1 agonist, decreased [H-3]-thymidine incorporation in a dose-dependent manner and augmented the 1 alpha ,25-(OH)(2)D-2-induced inhibition of [H-3]-thymidine incorporation. PTPGE(2) also caused significant increases in proteoglycan production, as measured by [S-35]-sulfate incorporation, and alkaline phosphatase specific activity. 1 alpha ,25-(OH)(2)D-3-induced alkaline phosphatase activity was only slightly stimulated by PTPGE(2). In contrast, 1 alpha ,25-(OH)(2)D-3-induced PKC 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 responses measured. EP1 receptor-induced responses were blocked by the phospholipase C inhibitor U73122, and reduced by PKA inhibitors. EP1 receptor-induced PKC activity was insensitive to pertussis toxin or choleratoxin but blocked 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 decreased proliferation and enhanced differentiation, as well as the effect of la,25-(OH)2D3 on cellular maturation. (C) 2001 Elsevier Science Ltd. All rights reserved.