UNLABELLED: Signaling intermediates for PTH and phorbol activation of PLD in UMR-106 cells were determined. Calcium was required, and the effects of PTH, phorbol, and calcium were dependent on p42/44 MAP kinase and small G proteins, specifically RhoA, acting through Rho kinase. INTRODUCTION: Phospholipase D (PLD) plays a key signaling role in numerous cellular processes. PLD-stimulated hydrolysis of phosphatidylcholine (PC) generates phosphatidic acid, a source of diacylglycerol (DAG). We previously reported that parathyroid hormone (PTH) stimulates PLD activity in UMR-106 osteoblastic cells by a protein kinase C (PKC)-independent mechanism. The current study investigated the roles of calcium, MAP kinase, and small G proteins in PTH- and phorbol-12,13-dibutyrate (PDBu)-stimulated transphosphatidylation of ethanol, a reaction catalyzed by PLD. METHODS: UMR-106 cells were labeled with 3H-palmitic and treated in the presence of ethanol. Phosphatidylethanol was separated by thin-layer chromatography and detected by autoradiography, and the bands were scraped and counted. Statistical significance of the responses from three to nine replicates was determined by ANOVA and Tukey's post-test. RESULTS AND CONCLUSIONS: PTH and PDBu effects were attenuated by EGTA, BAPTA, nifedipine, and dantrolene, whereas ionomycin or 2X calcium increased basal PLD activity. PTH activated p42/p44 MAP kinase, and the effects of PTH, PDBu, and ionomycin on PLD, but not on calcium influx, were prevented by the MEK inhibitors PD98059 and U0126. Small G proteins were shown to be involved in the effects of PTH, PDBu, and ionomycin on PLD. Inhibition of ARF by brefeldin prevented the PLD activation by all three agonists. A nonselective Rho/Rac/cdc-42 inhibitor, Clostridium difficile toxin B, also inhibited the effects of all three agonists on PLD. More selective inhibition of RhoA with a dominant negative RhoA construct or by inhibiting geranylgeranyltransferase I antagonized the effects of PTH, PDBu, and ionomycin, as did inhibiting the downstream kinase, Rho kinase. The current results reveal the importance of calcium, MAP kinase, and small G proteins in PTH and PDBu stimulation of PLD activity in UMR-106 cells.
UNLABELLED: Signaling intermediates for PTH and phorbol activation of PLD in UMR-106 cells were determined. Calcium was required, and the effects of PTH, phorbol, and calcium were dependent on p42/44 MAP kinase and small G proteins, specifically RhoA, acting through Rho kinase. INTRODUCTION: Phospholipase D (PLD) plays a key signaling role in numerous cellular processes. PLD-stimulated hydrolysis of phosphatidylcholine (PC) generates phosphatidic acid, a source of diacylglycerol (DAG). We previously reported that parathyroid hormone (PTH) stimulates PLD activity in UMR-106 osteoblastic cells by a protein kinase C (PKC)-independent mechanism. The current study investigated the roles of calcium, MAP kinase, and small G proteins in PTH- and phorbol-12,13-dibutyrate (PDBu)-stimulated transphosphatidylation of ethanol, a reaction catalyzed by PLD. METHODS: UMR-106 cells were labeled with 3H-palmitic and treated in the presence of ethanol. Phosphatidylethanol was separated by thin-layer chromatography and detected by autoradiography, and the bands were scraped and counted. Statistical significance of the responses from three to nine replicates was determined by ANOVA and Tukey's post-test. RESULTS AND CONCLUSIONS:PTH and PDBu effects were attenuated by EGTA, BAPTA, nifedipine, and dantrolene, whereas ionomycin or 2X calcium increased basal PLD activity. PTH activated p42/p44 MAP kinase, and the effects of PTH, PDBu, and ionomycin on PLD, but not on calcium influx, were prevented by the MEK inhibitors PD98059 and U0126. Small G proteins were shown to be involved in the effects of PTH, PDBu, and ionomycin on PLD. Inhibition of ARF by brefeldin prevented the PLD activation by all three agonists. A nonselective Rho/Rac/cdc-42 inhibitor, Clostridium difficile toxin B, also inhibited the effects of all three agonists on PLD. More selective inhibition of RhoA with a dominant negative RhoA construct or by inhibiting geranylgeranyltransferase I antagonized the effects of PTH, PDBu, and ionomycin, as did inhibiting the downstream kinase, Rho kinase. The current results reveal the importance of calcium, MAP kinase, and small G proteins in PTH and PDBu stimulation of PLD activity in UMR-106 cells.