Singh Amareshwar T K, Bhattacharyya Rumi S, Radeff Julie M, Stern Paula H
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611-3008, USA.
J Bone Miner Res. 2003 Aug;18(8):1453-60. doi: 10.1359/jbmr.2003.18.8.1453.
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.
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.
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.
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.
确定了甲状旁腺激素（PTH）和佛波醇激活UMR - 106细胞中磷脂酶D（PLD）的信号中间体。钙是必需的，并且PTH、佛波醇和钙的作用依赖于p42/44丝裂原活化蛋白激酶（MAP激酶）和小G蛋白，特别是通过Rho激酶起作用的RhoA。
磷脂酶D（PLD）在众多细胞过程中发挥关键的信号传导作用。PLD刺激的磷脂酰胆碱（PC）水解产生磷脂酸，这是二酰基甘油（DAG）的一个来源。我们之前报道过甲状旁腺激素（PTH）通过一种不依赖蛋白激酶C（PKC）的机制刺激UMR - 106成骨细胞中的PLD活性。本研究调查了钙、MAP激酶和小G蛋白在PTH和佛波醇 - 12,13 - 二丁酸酯（PDBu）刺激的乙醇转磷脂酰化反应（由PLD催化的反应）中的作用。
用3H - 棕榈酸标记UMR - 106细胞，并在乙醇存在的情况下进行处理。通过薄层层析分离磷脂酰乙醇，并通过放射自显影检测，然后刮下条带并计数。通过方差分析（ANOVA）和Tukey事后检验确定三到九个重复样本反应的统计学显著性。
EGTA、BAPTA、硝苯地平和丹曲林可减弱PTH和PDBu的作用，而离子霉素或2倍钙可增加基础PLD活性。PTH激活p42/p44 MAP激酶，MEK抑制剂PD98059和U0126可阻止PTH、PDBu和离子霉素对PLD的作用，但不影响钙内流。小G蛋白参与了PTH、PDBu和离子霉素对PLD的作用。布雷菲德菌素对ARF的抑制可阻止所有三种激动剂对PLD的激活。一种非选择性的Rho/Rac/cdc - 42抑制剂，艰难梭菌毒素B，也抑制了所有三种激动剂对PLD的作用。用显性负性RhoA构建体或通过抑制香叶基香叶基转移酶I对RhoA进行更具选择性的抑制，以及抑制下游激酶Rho激酶，均拮抗了PTH、PDBu和离子霉素的作用。目前的结果揭示了钙、MAP激酶和小G蛋白在PTH和PDBu刺激UMR - 106细胞中PLD活性方面的重要性。
