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.
|Original language||English (US)|
|Number of pages||13|
|Journal||Journal of Cellular Physiology|
|Publication status||Published - Jan 13 2000|
ASJC Scopus subject areas
- Clinical Biochemistry
- Cell Biology