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

Abstract

Objectives: 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.

Materials and 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.

Conclusion: 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.

Figure 1

Effects of ClC‐3 siRNA transfection on endogenous ClC‐3 protein expression in BASMCs. (a) Representative image of fluorescence in BASMCs (200×). Stealth siRNA labelled with FITC was transfected to BASMCs, and fluorescence was detected in these cells after 6 h incubation (transfection), while there was no fluorescence detectable in untransfected (control) cells, indicating where siRNA had been successfully transfected. (b) Western blot results show that 40 nmol/L ClC‐3 siRNA decreased significantly endogenous ClC‐3 expression, but transfection of Lipofectamine^TM RNAiMAX (lipo) and negative siRNA control (negative siRNA) did not significantly change ClC‐3 expression (n = 5; ‡P < 0.01 versus control).

Figure 2

Effects of ClC‐3 knockdown on ET‐1‐dependent ClC‐3 protein expression and cell proliferation. (a) Densitometric analysis shows stealth siRNA inhibits ClC‐3 protein expression induced by 10 nmol/L ET‐1, while transfections of Lipofectamine^TM RNAiMAX and negative siRNA control had no significant effect on ClC‐3 protein expression (n = 5; *P < 0.01 versus control; **P < 0.01 versus ET‐1). Representative Western blots for ClC‐3 protein expression are shown in the bottom. (b) Increase in cell growth induced by 10 nmol/L ET‐1 was determined by counting the number of cells, and was significantly inhibited by incubation of ClC‐3 siRNA for 48 h, but not by transfection of Lipofectamine^TM RNAiMAX and negative siRNA. (c) The effects of ClC‐3 siRNA on cell proliferation induced by ET‐1 were further determinated by assaying BrdU incorporation. Incubation of ClC‐3 siRNA for 48 h also significantly decreased BrdU incorporation (n = 5; ‡P < 0.01 versus control; *P < 0.01 versus ET‐1). These results show that the inhibitory effect of ClC‐3 siRNA on ET‐1 induced ClC‐3 protein expression matches that of cell proliferation.

Figure 3

Flow cytometric analysis shows ET‐1 accelerated entrance into G₁ and arrested entrance from G₁ to S phase. (a) Representative images of cell cycle analysis. (b) Statistical analysis shows the percent distribution of cells in G₀/G₁, S, G₂/M stages of the cell cycle (n = 5; ‡P < 0.01 versus control; *P < 0.01 versus ET‐1).

Figure 4

Effects of ClC‐3 knockdown by incubation of 40 nm ClC‐3 siRNA for 48 h, on protein expression of cyclin E, cyclin D1 and cyclin‐dependent kinase inhibitors (CDKIs) p21 and p27. (a) Representive Western blot images of cyclins and CDKIs. (b and c) Densitometric analysis of the effects of ClC‐3 knockdown on expression of cyclin D1 and cyclin E (b), and p21 and p27 (c), respectively. Expression of CDK2 was not changed by ClC‐3 knockdown. (a) control; (b) ET‐1; (c) ET‐1 + Lipofectamine^TM RNAiMAX; (d) ET‐1 + negative siRNA; (e) ET‐1 + ClC‐3 siRNA (n = 5; †P < 0.05, ‡P < 0.01 versus control; *P < 0.01 versus ET‐1).

Figure 5

Effects of ClC‐3 knockdown by incubation of 40 nm ClC‐3 siRNA for 48 h, on phosphorylation of Akt and GSK‐3β. (a) Representative images of Western blots show that loss of ClC‐3 channel affects the time course response to ET‐1 after ClC‐3 was knocked own by siRNA, and ET‐1 was added to observe phosphorylation of Akt (Ser473) at 10‐min, 1‐h and 12‐h time point (n = 5). (b) Representative images of Western blots to show that ET‐1 increased phosphorylationsof Thr308 and its downstream target, GSK‐3β, at Ser9, but failed to increase this phosphorylation when ClC‐3 was knocked down. However, deficiency of ClC‐3 did not significantly alter protein levels of Akt and GSK‐3β. (c) Densitometric analysis of the effects of ClC‐3 knockdown on inactivation of Akt/GSK‐3β signal transduction pathway. (a) control; (b) ET‐1; (c) ET‐1 + Lipofectamine^TM RNAiMAX; (d) ET‐1 + negative siRNA; (e) ET‐1 + ClC‐3 siRNA; (n = 6; ‡P < 0.01 versus control; *P < 0.01 versus ET‐1).