24-25-dihydroxyvitamin-d-3 and chelerythrine

Growth plate cartilage is responsible for long bone growth in children and adolescents and is regulated by vitamin D metabolites in a cell zone-specific manner. Resting zone chondrocytes (RC cells) are regulated by 24,25-dihydroxyvitamin D3 via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH)2D3 stimulates rat costochondral RC chondrocytes to release lysophosphatidic acid (LPA) and, therefore sought to determine the role of LPA signaling in these cells. RC cells expressed the G-protein coupled receptors LPA1-5 and peroxisome proliferator-activated receptor gamma (PPAR-gamma). LPA and the LPA1/3 selective agonist OMPT increased proliferation and two maturation markers, alkaline phosphatase activity and [35S]-sulfate incorporation. LPA and 24R,25(OH)2D3's effects were inhibited by the LPA1/3 selective antagonist VPC32183(S). Furthermore, apoptosis induced by either inorganic phosphate or chelerythrine was attenuated by LPA, based on DNA fragmentation, TUNEL staining, caspase-3 activity, and Bcl-2:Bax protein ratio. LPA prevented apoptotic signaling by decreasing the abundance, nuclear localization, and transcriptional activity of the tumor-suppressor p53. LPA treatment also regulated the expression of the p53-target genes Bcl-2 and Bax to enhance cell survival. Collectively, these data suggest that LPA promotes differentiation and survival in RC chondrocytes, demonstrating a novel physiological function of LPA-signaling.

Topics: 24,25-Dihydroxyvitamin D 3; Adolescent; Animals; Antineoplastic Agents; Apoptosis; Autocrine Communication; bcl-2-Associated X Protein; Benzophenanthridines; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Child; Chondrocytes; Cyclin-Dependent Kinase Inhibitor p21; Growth Plate; Humans; Lysophospholipids; Male; Osteogenesis; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Receptors, Lysophosphatidic Acid; Signal Transduction; Tumor Suppressor Protein p53

Prior studies have shown that 1,25-(OH)2D3 stimulates alkaline phosphatase, phospholipase A2 (PLA2), and protein kinase C (PKC)-specific activities, and production of prostaglandin E2 (PGE2) in growth zone chondrocytes. In contrast, 24,25-(OH)2D3 stimulates alkaline phosphatase and PKC-specific activities but inhibits PLA2-specific activity and PGE2 production in resting zone cells. This indicates that different mechanisms are involved in the action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on their respective target cells. In this study, we examined the hypothesis that differential regulation of prostaglandin production modulates the activity of PKC and alkaline phosphatase. To do this, we examined the effect of the cyclooxygenase inhibitor indomethacin (Indo) on alkaline phosphatase, PLA2, and PKC-specific activities in growth plate chondrocytes treated with these two vitamin D metabolites. In addition, we examined whether inhibition of PKC altered PGE2 production. In growth zone cells, Indo inhibited basal alkaline phosphatase and blocked the 1,25-(OH)2D3-dependent increase in alkaline phosphatase. This effect was due to inhibition of both plasma membrane and matrix vesicle alkaline phosphatase. In resting zone cells, Indo increased basal alkaline phosphatase activity in a dose-dependent manner, but it did not further enhance the 24,25-(OH)2D3-dependent stimulation of this enzyme. The effect of Indo was found in both plasma membranes and matrix vesicles. These data indicate that 1,25-(OH)2D3-dependent increases in alkaline phosphatase-specific activity in growth zone cells are mediated through increased prostaglandin production, whereas 24,25-(OH)2D3-mediated changes in enzyme activity in resting zone cells are mediated through decreased prostaglandin production. Regulation of PLA2 by either 1,25-(OH)2D3 or 24,25-(OH)2D3 in their target cells was unaffected by Indo, indicating that the effect of the vitamin D metabolites on this enzyme is not dependent on changes in PGE2 production. The rapid increase in 1,25-(OH)2D3-dependent PKC-specific activity in growth zone cells was inhibited by Indo, whereas there was a potentiation of the effect of 24,25-(OH)2D3 on PKC activity in resting zone cells. In addition, inhibition of PKC blocked the 1,25-(OH)2D3-dependent increase in PGE2 production in growth zone cells and the 24,25-(OH)2D3-dependent decrease in PGE2 production by resting zone cells. These data indicate that prostaglandins are involved in mediating the rapi

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 24,25-Dihydroxyvitamin D 3; Alkaline Phosphatase; Alkaloids; Animals; Benzophenanthridines; Calcitriol; Cells, Cultured; Chondrocytes; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Enzyme Inhibitors; Growth Plate; Indomethacin; Male; Phenanthridines; Phospholipases A; Phospholipases A2; Protein Kinase C; Rats; Rats, Sprague-Dawley

We have previously shown that 24,25-(OH)2D3 plays a major role in resting zone (RC) chondrocyte differentiation and that this vitamin D metabolite regulates protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 24,25-(OH)2D3 to stimulate PKC activation. Confluent, fourth passage RC cells from rat costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of RC cultures with 24,25-(OH)2D3 for 90 min produced a dose-dependent increase in diacylglycerol (DAG). Addition of R59022, a diacylglycerol kinase inhibitor, significantly increased PKC activity in cultures treated with 24,25-(OH)2D3. Addition of dioctanoylglycerol (DOG) to plasma membranes isolated from RC increased PKC activity 447-fold. Addition of pertussis toxin or cholera toxin to control cultures elevated basal PKC activity. When added together with 10(-9) M 24,25-(OH)2D3, there was an additive effect on PKC activity but in cultures treated with 10(-8) M 24,25-(OH)2D3, only the hormone-dependent stimulation of PKC was observed. The phospholipase C inhibitor, U73-122, had no effect on PKC activity, indicating that the DAG produced in response to 24,25-(OH)2D3 is not derived from phosphatidylinositol. Addition of the tyrosine kinase inhibitor, genistein, also had no effect on 24,25-(OH)2D3-stimulated PKC, further supporting the hypothesis that phospholipase C is not involved in the mechanism and that phospholipase D is responsible for the increase in DAG production. Phospholipase A2 inhibitors, quinacrine and AACOCF3, and the cyclooxygenase inhibitor indomethacin increased PKC activity in the RC cultures. Exogenous PGE2, one of the downstream products of phospholipase A2 action, inhibited PKC activity. These results suggest that 24,25-(OH)2D3 regulates PKC activity by two distinct phospholipid-dependent mechanisms: production of DAG via phospholipase D and inhibition of the production of PGE2 via inhibition of phospholipase A2 and cyclooxygenase.

Topics: 24,25-Dihydroxyvitamin D 3; Alkaloids; Benzophenanthridines; Cyclooxygenase Inhibitors; Diacylglycerol Kinase; Diglycerides; Enzyme Inhibitors; GTP-Binding Proteins; Phenanthridines; Phospholipases A; Phospholipases A2; Phosphotransferases (Alcohol Group Acceptor); Prostaglandin-Endoperoxide Synthases; Prostaglandins; Protein Kinase C; Protein-Tyrosine Kinases; Signal Transduction; Type C Phospholipases