Calphostin-C induction of vascular smooth muscle cell apoptosis proceeds through phospholipase D and microtubule inhibition.
Calphostin-C, a protein kinase C inhibitor, induces apoptosis of cultured vascular smooth muscle cells. However, the mechanisms are not completely defined. Because apoptosis of vascular smooth muscle cells is critical in several proliferating vascular diseases such as atherosclerosis and restenosis after angioplasty, we decided to investigate the mechanisms underlying the calphostin-C-induced apoptotic pathway. We show here that apoptosis is inhibited by the addition of exogenous phosphatidic acid, a metabolite of phospholipase D (PLD), and that calphostin-C inhibits completely the activities of both isoforms of PLD, PLD1 and PLD2. Overexpression of either PLD1 or PLD2 prevented the vascular smooth muscle cell apoptosis induced by serum withdrawal but not the calphostin-C-elicited apoptosis. These data suggest that PLDs have anti-apoptotic effects and that complete inhibition of PLD activity by calphostin-C induces smooth muscle cell apoptosis. We also report that calphostin-C induced microtubule disruption and that the addition of exogenous phosphatidic acid inhibits calphostin-C effects on microtubules, suggesting a role for PLD in stabilizing the microtubule network. Overexpressing PLD2 in Chinese hamster ovary cells phenocopies this result, providing strong support for the hypothesis. Finally, taxol, a microtubule stabilizer, not only inhibited the calphostin-C-induced microtubule disruption but also inhibited apoptosis. We therefore conclude that calphostin-C induces apoptosis of cultured vascular smooth muscle cells through inhibiting PLD activity and subsequent microtubule polymerization.
Zheng
Xi-Long
XL
Smooth Muscle Research Group, Department of Biochemistry & Molecular Biology, The University of Calgary, Calgary, Alberta T2N 4N1,Canada. xlzheng@ucalgary.ca
Gui
Yu
Y
Du
Guangwei
G
Frohman
Michael A
MA
Peng
Dao-Quan
DQ
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000473:pathology*; D009281:Naphthalenes / Q000494:pharmacology*; D017239:Paclitaxel / Q000494:pharmacology; D010712:Phosphatidic Acids / Q000737:chemistry; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011233:Precipitin Tests; D020033:Protein Isoforms; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D009281:Naphthalenes; D010712:Phosphatidic Acids; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010739:Phospholipase D; C058819:calphostin C; D017239:Paclitaxel