title: Characterization of prostaglandin E(2) receptors and their role in 24,25-(OH)(2)D(3)-mediated effects on resting zone chondrocytes.
abstract: Resting zone chondrocyte differentiation is modulated by the vitamin D metabolite, 24,25-(OH)(2)D(3), 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)(2)D(3)-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 E(2) (PGE(2)). The aim of the present study was to identify which PGE(2) receptor subtypes (EP) mediate the effects of PGE(2) 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, EPlv, 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)(2)D(3)-mediated cell proliferation and differentiation. 17-phenyl-trinor-PGE(2) (PTPGE(2)), an EP1 agonist, increased [(3)H]-thymidine incorporation in a dose-dependent manner and reversed the 24, 25-(OH)(2)D(2)-induced inhibition of [(3)H]-thymidine incorporation. SC-19220, an EP1 antagonist, caused a further dose-dependent decrease in 24,25-(OH)(2)D(3)-induced inhibition of [(3)H]-thymidine incorporation. PTPGE(2) also caused a biphasic increase in [(35)S]-sulfate incorporation and increased alkaline phosphatase enzyme activity at high concentrations (10(-8) M). 24, 25-(OH)(2)D(3)-induced alkaline phosphatase activity was synergistically stimulated in a dose-dependent manner by PTPGE(2). In contrast, 24,25-(OH)(2)D(3)-induced PKC activity was inhibited in a dose-dependent manner by PTPGE(2) and SC-19220, the EP1 antagonist, elevated PKC activity at high concentrations (10(-8) M). The EP2 agonist, misoprostol, only affected [(35)S]-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 PGE(2)-induced cellular responses in resting zone cells that lead to both increased proliferation and differentiation. Because 24,25-(OH)(2)D(3) inhibits PGE(2) synthesis in these cells, EP1-mediated induction of proliferation is blocked, encouraging cellular maturation and activation of PKC activity.
authors.affiliation: Department of Orthodontics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas.
authors.forename: F
authors.initials: F
authors.lastname: Del Toro
authors.forename: V L
authors.initials: VL
authors.lastname: Sylvia
authors.forename: S R
authors.initials: SR
authors.lastname: Schubkegel
authors.forename: R
authors.initials: R
authors.lastname: Campos
authors.forename: D D
authors.initials: DD
authors.lastname: Dean
authors.forename: B D
authors.initials: BD
authors.lastname: Boyan
authors.forename: Z
authors.initials: Z
authors.lastname: Schwartz
mesh_terms: D015650:24,25-Dihydroxyvitamin D 3 / Q000494:pharmacology*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D020033:Protein Isoforms / Q000819:agonists / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018078:Receptors, Prostaglandin E / Q000819:agonists / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013431:Sulfates / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D011509:Proteoglycans; D012333:RNA, Messenger; D018078:Receptors, Prostaglandin E; D013431:Sulfates; D015650:24,25-Dihydroxyvitamin D 3; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D013936:Thymidine