Characterization of prostaglandin E₂ receptors and their role in 24,25- (OH)₂D₃-mediated effects on resting zone chondrocytes

Resting zone chondrocyte differentiation is modulated by the vitamin D metabolite, 24,25-(OH)₂D₃, via activation of protein kinase C (PKC). In previous studies, inhibition of prostaglandin production with indomethacin caused an increase in PKC activity, suggesting that changes in prostaglandin levels may mediate the 24,25-(OH)₂D₃-dependent response and act as autocrine or paracrine regulators of chondrocyte metabolism. Supporting this hypothesis is the fact that resting zone cells respond directly to prostaglandin E₂ (PGE₂). The aim of the present study was to identify which PGE₂ receptor subtypes (EP) mediate the effects of PGE₂ on resting 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. A variant form of the EP1 cDNA, EPIv, was also amplified in an RT-dependent manner. In parallel experiments, we used EP subtype-specific agonists to examine the role of EP receptors in 24,25-(OH)₂D₃-mediated cell proliferation and differentiation. 17-phenyl-trinor-PGE₂ (PTPGE₂), an EP1 agonist, increased [³H]-thymidine incorporation in a dose-dependent manner and reversed the 24,25-(OH)₂D₂-induced inhibition of [³H]-thymidine incorporation. SC-19220, an EP1 antagonist, caused a further dose-dependent decrease in 24,25-(OH)₂D₃-induced inhibition of [³H]-thymidine incorporation. PTPGE₂ also caused a biphasic increase in [³⁵S]-sulfate incorporation and increased alkaline phosphatase enzyme activity at high concentrations (10^-8 M). 24,25-(OH)₂D₃-induced alkaline phosphatase activity was synergistically stimulated in a dose-dependent manner by PTPGE₂. In contrast, 24,25-(OH)₂D₃-induced PKC activity was inhibited in a dose-dependent manner by PTPGE₂ and SC-19220, the EP1 antagonist, elevated PKC activity at high concentrations (10^-8 M). The EP2 agonist, misoprostol, only affected [³⁵S]-sulfate incorporation, but in a dose-dependent manner. The EP3 and EP4 agonists had no effect on cell response. These results suggest that the EP1 receptor subtype mediates some of the PGE₂-induced cellular responses in resting zone cells that lead to both increased proliferation and differentiation. Because 24,25-(OH)₂D₃ inhibits PGE₂ synthesis in these cells, EP1-mediated induction of proliferation is blocked, encouraging cellular maturation and activation of PKC activity.