title: Characterization of PGE(2) receptors (EP) and their role as mediators of 1alpha,25-(OH)(2)D(3) effects on growth zone chondrocytes.
abstract: Growth plate chondrocyte function is modulated by the vitamin D metabolite 1alpha,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 1alpha,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(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 1alpha,25-(OH)(2)D(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 1alpha,25-(OH)(2)D(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. 1alpha,25-(OH)(2)D(3)-induced alkaline phosphatase activity was only slightly stimulated by PTPGE(2). In contrast, 1alpha,25-(OH)(2)D(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 GDPbetaS, 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 1alpha,25-(OH)(2)D(3) on cellular maturation.
authors.affiliation: Department of Orthopaedics, The University of Texas Health Science Center at San Antonio, Mail Code 7774, 7703 Floyd Curl Drive, 78229-3900, USA.
authors.forename: V L
authors.initials: VL
authors.lastname: Sylvia
authors.forename: F
authors.initials: F
authors.lastname: Del Toro
authors.forename: R R
authors.initials: RR
authors.lastname: Hardin
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: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D015232:Dinoprostone / Q000378:metabolism / Q000494:pharmacology; D006132:Growth Plate / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018078:Receptors, Prostaglandin E / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D058306:Receptors, Prostaglandin E, EP1 Subtype; D058307:Receptors, Prostaglandin E, EP2 Subtype; D013936:Thymidine / Q000378:metabolism
chemical_list: D017931:DNA Primers; D011509:Proteoglycans; C545637:Ptger1 protein, rat; C545646:Ptger2 protein, rat; D012333:RNA, Messenger; D018078:Receptors, Prostaglandin E; D058306:Receptors, Prostaglandin E, EP1 Subtype; D058307:Receptors, Prostaglandin E, EP2 Subtype; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D002117:Calcitriol; D015232:Dinoprostone; D013936:Thymidine