Involvement of chloride channels in IGF-I-induced proliferation of porcine arterial smooth muscle cells

Abstract

Objective: The existence of Cl- channels in vascular smooth muscle cells (VSMCs) has been increasingly investigated, but the biological functions are not yet clear. Insulin-like growth factor (IGF)-I affects proliferation and migration of VSMCs, and dysregulation of this axis may be involved in atherogenesis and intimal hyperplasia. We examined the effects of Cl- channel blockers on IGF-I-induced proliferation in porcine VSMCs. The siRNA approach was used to support the role of ClC-2, a member of the volume-regulated Cl- channel family, in cell proliferation of VSMCs.

Methods and results: The IGF-I-induced VSMC proliferation was significantly suppressed by the Cl- channel blockers NPPB and IAA94 but not by DIDS. IGF-I-induced cell proliferation parallels a significant increase in the endogenous expression of ClC-2 mRNA and protein. Inhibitors of PI3-kinase, LY294002 and wortmannin, significantly attenuated the IGF-I-upregulated ClC-2 expression and cell proliferation. We observed ClC-2-like Cl- current, and this current was augmented by IGF-I. SiRNA specifically targeted to ClC-2 abolished IGF-I-induced cell proliferation.

Conclusion: Our data demonstrate that ClC-2 plays a role in IGF-1-induced regulation of VSMC proliferation in cardiovascular diseases.

Figure 1

Concentration-dependent stimulation of VSMC proliferation induced by IGF-I. A: VSMCs were exposed to serum free media in the presence of absence of chosen concentrations of IGF-I. B: Effect of Cl^- channel blockers on VSMC proliferation. Both NPPB and IAA94 inhibited IGF-I induced VSMCs proliferation, whereas DIDS had no effect. #, p < 0.05 compared with corresponding control group. *,p < 0.05 compared with corresponding IGF-I group. Data represent mean ± SEM (n = 5).

Figure 2

Measurement of porcine VSMC DNA synthesis using bromo-deoxyuridine (BrdU). VSMCs were stimulated with IGF-I (100 ng/ml) in the presence and absence of Cl^- channel blockers for 48 h, and BrdU incorporation was analyzed by photometric immunoassay. Data represent mean ± SEM (n = 5). #p < 0.05 vs control, *P < 0.05 vs. IGF-I stimulation.

Figure 3

Dose response effect of Cl-channel blockers on IGF-I induced DNA synthesis. Both NPPB and IAA94 dose-dependently inhibited coronary VSMC proliferation induced by 100 ng/ml IGF-I. (*P < 0.05 vs. IGF-I stimulation alone). Data represent mean ±SEM of n = 5.

Figure 4

RT-PCR and Western blot analyses of ClC expression in porcine vascular smooth muscle cells. A: RT-PCR of CIC-2 and ClC-3 mRNA from unstimulated porcine coronary artery SMCs (top). qRT-PCR analysis of CIC-2 mRNA expression after 24h and 48h stimulation by IGF-I reported in relative quantitative expression (bottom panel). Total protein lysates from porcine coronary VSMCs were prepared for immunoblotting. Membranes were probed with anti-ClC-2 antibody and reprobed with anti-GAPDH antibody. Densitometric analysis shows dose- and time-dependent response for IGF-I induced ClC-2 upregulation (B and C). Data represent mean ± SEM (n = 5).

Figure 5

LY294002 and wortmannin inhibit IGF-I-induced ClC-2 protein expression in cultured VSMCs. Serum-starved cells were pretreated with either LY294002 (10 μM), wortmannin (20 μM), or PD98059 (40 μM) for 2 h prior to 24h stimulation by IGF-I (100 ng/ml). Western blotting was performed on the cell lysates utilizing antibody specific for ClC-2. Bar graphs correspond to representative immunoblots. Data represent mean ± SEM (n= 5). #P < 0.05 vs control, *P < 0.05 vs. IGF-I stimulation.

Figure 6

Effect of PI3 kinase and MAP kinase inhibitors on IGF-I-induced proliferation in cultured VSMCs. Serum-starved cells were pretreated with either LY294002 (10 μM), wortmannin (20 μM), or PD98059 (40 μM) for 2 h prior to 24h stimulation by IGF-I (100 ng/ml). BrdU incorporation was analyzed by photometric immunoassay. Data represent mean ± SEM (n = 5). *P < 0.05 vs. IGF-I stimulation.

Figure 7

Effect of IGF-1 on whole-cell Cl^- currents in porcine VSMCs. Whole-cell Cl^- currents were recorded at different voltage before in control (A) and after (B) perfusion of IGF-I (100ng/ml) in coronary VSMCs. (C) Whole-cell Cl^- currents were also recorded from ClC-2 siRNA transfected coronary SMCs. The I-V curve (D) showed relationship between control (A) and after (B) perfusion of IGF-I (100ng/ml) in pig coronary artery smooth muscle cells. Whole-cell Cl^- currents in control cells (A) showed an inward rectification. Representative recording of Cl-currents from individual experimental groups are shown (n = 7). The bathing medium and the pipette solution contained CsCl saline. Holding potential was 0 mV. Voltage pulses, 500 ms in duration, were applied at 20 mV increments from -100 to +100 mV.

Figure 8

Effect of wortmannin on IGF-I induced whole-cell Cl^- current in porcine VSMCs. Whole-cell Cl^- currents were recorded after perfusion of IGF-I (100ng/ml) in VSMCs (A) and pre-incubated with 20 μM wortmannin (B). Representative recording of Cl-currents from individual experimental groups are shown (n = 7).

Figure 9

Effect of ClC-2 siRNA on ClC-2 expression in coronary artery SMCs. (A) mRNA expression and protein abundance were assessed by RT-PCR (top) and Western blotting (bottom), respectively, in VSMC transfected with ClC-2 siRNA or nonsilencing (NS) siRNA. (B) Essential role of ClC-2 in IGF-I stimulated VSMC DNA synthesis was examined. #p < 0.05 vs control, *P < 0.05 vs. IGF-I stimulation