Functional Regulation of ClC-3 in the Migration of Vascular Smooth Muscle Cells

Inositol Signaling Group, Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC.
Hypertension (Impact Factor: 6.48). 11/2012; 61(1). DOI: 10.1161/HYPERTENSIONAHA.112.194209
Source: PubMed


Migration of vascular smooth muscle cells (VSMCs) into neointima contributes to atherosclerosis and restenosis. This migration requires coordinated plasmalemmal fluxes of water and ions. Here, we show that aortic VSMC migration depends on the regulation of transmembrane Cl(-) flux by ClC-3, a Cl(-) channel/transporter. The contribution of ClC-3 to plasmalemmal Cl(-) current was studied in VSMCs by electrophysiological recordings. Cl(-) current was negligible in cells perfused with 0 [Ca(2+)]. Raising intracellular [Ca(2+)] to 0.5 μM activated a Cl(-) current (I(Cl.Ca)), approximately half of which was eliminated on inhibition by KN-93 of calmodulin-dependent protein kinase II. I(Cl.Ca) was also halved by inositol-3,4,5,6-tetrakisphosphate, a cellular signal with the biological function of specifically preventing calmodulin-dependent protein kinase II from activating I(Cl.Ca). Gene disruption of ClC-3 reduced I(Cl.Ca) by 50%. Moreover, I(Cl.Ca) in the ClC-3 null VSMCs was not affected by either KN-93 or inositol-3,4,5,6-tetrakisphosphate. We conclude that I(Cl.Ca) is composed of 2 components, one is ClC-3 independent whereas the other is ClC-3 dependent, activated by calmodulin-dependent protein kinase II and inhibited by inositol-3,4,5,6-tetrakisphosphate. We also assayed VSMC migration in transwell assays. Migration was halved in ClC-3 null cells versus wild-type cells. In addition, inhibition of ClC-3 by niflumic acid, KN-93, or inositol-3,4,5,6-tetrakisphosphate each reduced cell migration in wild-type cells but not in ClC-3 null cells. These cell-signaling roles of ClC-3 in VSMC migration suggest new therapeutic approaches to vascular remodeling diseases.

3 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: In smooth muscle cells (SMCs), the intracellular chloride ion (Cl(-)) concentration is high due to accumulation by Cl(-)/HCO3 (-) exchange and Na(+)-K(+)-Cl(-) cotransportation. The equilibrium potential for Cl(-) (E Cl) is more positive than physiological membrane potentials (E m), with Cl(-) efflux inducing membrane depolarization. Early studies used electrophysiology and nonspecific antagonists to study the physiological relevance of Cl(-) channels in SMCs. More recent reports have incorporated molecular biological approaches to identify and determine the functional significance of several different Cl(-) channels. Both "classic" and cGMP-dependent calcium (Ca(2+))-activated (ClCa) channels and volume-sensitive Cl(-) channels are present, with TMEM16A/ANO1, bestrophins, and ClC-3, respectively, proposed as molecular candidates for these channels. The cystic fibrosis transmembrane conductance regulator (CFTR) has also been described in SMCs. This review will focus on discussing recent progress made in identifying each of these Cl(-) channels in SMCs, their physiological functions, and contribution to diseases that modify contraction, apoptosis, and cell proliferation.
    Pflügers Archiv - European Journal of Physiology 09/2013; 466(5). DOI:10.1007/s00424-013-1357-2 · 4.10 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cerebrovascular remodeling is one of important risk factors of stroke. The underlying mechanisms are unclear. Integrin β3 and volume regulated ClC-3 Cl(-) channels have recently been implicated as important contributors to vascular cell proliferation. Therefore, we investigated the role of integrin β3 in cerebrovascular remodeling and related Cl(-) signalling pathway. Cl(-) currents were recorded using patch clamp technique. The expression of integrin β3 in hypertensive animals was examined by western blot and immunohistochemisty. Immunoprecipitation, cDNA and siRNA transfection were employed to investigate the integrin β3/Src/ClC-3 signalling. The expression of integrin β3 was up-regulated in stroke-prone spontaneously hypertensive rats, 2-kidney 2-clip hypertensive rats and angiotensin II-infused hypertensive mice. Integrin β3 expression was positively correlated with medial cross-sectional area and ClC-3 expression in basilar artery of 2-kidney 2-clip hypertensive rats. Knockdown of integrin β3 inhibited rat basilar vascular smooth muscle cell proliferation induced by angiotensin II. Co-immunoprecipitation and immunofluorescence experiments revealed a physical interaction between integrin β3, Src and ClC-3 protein. The integrin β3/Src/ClC-3 signalling were involved both in hypoosmotic stress and angiotensin II- induced volume regulated chloride channel activation. Tyrosine 284 within a concensus Src-phosphorylation site was the key point for ClC-3 channel activation. ClC-3 knockout significantly attenuated angiotensin II-induced cerebrovascular remodeling. Integrin β3 mediated cerebrovascular remodeling during hypertension via Src/ClC-3 signalling pathway.
    British Journal of Pharmacology 02/2014; 171(13). DOI:10.1111/bph.12654 · 4.84 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.
    Oncotarget 12/2014; 6(4). DOI:10.18632/oncotarget.2966 · 6.36 Impact Factor


3 Reads
Available from