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

Abstract

Growth plate chondrocyte function is modulated by the vitamin D metabolite 1α,25-(OH) 2D 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 1α,25-(OH) 2D 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 2 receptor subtypes (EP) mediate the effects of PGE 2 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 1α,25-(OH) 2D 3-mediated cell proliferation and differentiation. 17-Phenyl-trinor-PGE 2 (PTPGE 2), an EP1 agonist, decreased [ 3H]-thymidine incorporation in a dose-dependent manner and augmented the 1α,25-(OH) 2D 2-induced inhibition of [ 3H]-thymidine incorporation. PTPGE 2 also caused significant increases in proteoglycan production, as measured by [ 35S]-sulfate incorporation, and alkaline phosphatase specific activity. 1α,25-(OH) 2D 3-induced alkaline phosphatase activity was only slightly stimulated by PTPGE 2. In contrast, 1α,25-(OH) 2D 3-induced PKC activity was synergistically increased by PTPGE 2, 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 GDPβS, 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 1α,25-(OH) 2D 3 on cellular maturation.