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Regulation of Parathyroid Hormone‐Stimulated Phospholipase D in UMR‐106 Cells by Calcium, MAP Kinase, and Small G Proteins
Author(s) -
Singh Amareshwar TK,
Bhattacharyya Rumi S,
Radeff Julie M,
Stern Paula H
Publication year - 2003
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.2003.18.8.1453
Subject(s) - ionomycin , phospholipase d , protein kinase c , phosphatidic acid , diacylglycerol kinase , endocrinology , parathyroid hormone , phosphatidylethanol , medicine , phorbol , calcium , chemistry , phospholipase c , egta , bapta , biology , signal transduction , biochemistry , phospholipid , membrane
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 3 H‐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 2× 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.