Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G/S phase arrest in rat basilar arterial smooth muscle cells.
OBJECTIVE
Previously, we have found that the ClC-3 chloride channel is involved in endothelin-1 (ET-1)-induced rat aortic smooth muscle cell proliferation. The present study was to investigate the role of ClC-3 in cell cycle progression/distribution and the underlying mechanisms of proliferation.


METHODS
Small interference RNA (siRNA) is used to silence ClC-3 expression. Cell proliferation, cell cycle distribution and protein expression were measured or detected with cell counting, bromodeoxyuridine (BrdU) incorporation, Western blot and flow cytometric assays respectively.


RESULTS
ET-1-induced rat basilar vascular smooth muscle cell (BASMC) proliferation was parallel to a significant increase in endogenous expression of ClC-3 protein. Silence of ClC-3 by siRNA inhibited expression of ClC-3 protein, prevented an increase in BrdU incorporation and cell number induced by ET-1. Silence of ClC-3 also caused cell cycle arrest in G(0)/G(1) phase and prevented the cells' progression from G(1) to S phase. Knockdown of ClC-3 potently inhibited cyclin D1 and cyclin E expression and increased cyclin-dependent kinase inhibitors (CDKIs) p27(KIP) and p21(CIP) expression. Furthermore, ClC-3 knockdown significantly attenuated phosphorylation of Akt and glycogen synthase kinase-3beta (GSK-3beta) induced by ET-1.


CONCLUSIONS
Silence of ClC-3 protein effectively suppressed phosphorylation of the Akt/GSK-3beta signal pathway, resulting in down-regulation of cyclin D1 and cyclin E, and up-regulation of p27(KIP) and p21(CIP). In these BASMCs, integrated effects lead to cell cycle G(1)/S arrest and inhibition of cell proliferation.
Tang
Y-B
YB
Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China.
Liu
Y-J
YJ
Zhou
J-G
JG
Wang
G-L
GL
Qiu
Q-Y
QY
Guan
Y-Y
YY
D000818:Animals; D001488:Basilar Artery / Q000166:cytology* / Q000201:enzymology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016196:S Phase / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects
D018118:Chloride Channels; C115376:ClC-3 channel; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D019332:Endothelin-1; D034741:RNA, Small Interfering; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3