Integrin β3 mediates cerebrovascular remodelling through Src/ClC‐3 volume‐regulated Cl⁻ channel signalling pathway

Abstract

Background and PurposeCerebrovascular remodelling is one of the 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 remodelling and related Cl− signalling pathway.Experimental ApproachCl− currents were recorded using a 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.Key ResultsIntegrin β3 expression 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 the basilar artery of 2‐kidney 2‐clip hypertensive rats. Knockdown of integrin β3 inhibited the proliferation of rat basilar vascular smooth muscle cells 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 pathway was shown to be involved in the activation of volume‐regulated chloride channels induced by both hypo‐osmotic stress and angiotensin II. 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 remodelling.Conclusions and ImplicationsIntegrin β3 mediates cerebrovascular remodelling during hypertension via Src/ClC‐3 signalling pathway.

Endophilin A2 Influences Volume-Regulated Chloride Current by Mediating ClC-3 Trafficking in Vascular Smooth Muscle Cells

“…17 Our recent work has demonstrated that tyrosine 284 phosphorylation targeted by Src is associated with ClC-3 channel activation upon hypotonic stress in VSMCs. 25, 40 However, the tyrosine phosphorylation signaling appears not to be involved in the endophilin A2-mediated ClC-3 trafficking mechanism, which may be due to their different influences on the subcellular localization of ClC-3. In the basal state, exogenous endophilin A2 transfection could regulate ClC-3 subcellular localization, but it is not the case for tyrosine 284 phosphorylation.…”

Threonine532 phosphorylation in ClC‐3 channels is required for angiotensin II‐induced Cl− current and migration in cultured vascular smooth muscle cells

“…We have already shown that phosphorylation of Tyr 284 in ClC‐3 channels by Src kinase is an important mechanism for the activation of these channels by AngII (Wang et al ., 2013 ; Zeng et al ., 2014). Therefore, we examined the combined effects of ROCK2 and Src on ClC‐3 channel activation and found that either the ROCK2 inhibitor Y27632 or the Src kinase inhibitor SU6656 inhibited but did not abolish the Cl − current carried by ClC‐3 channels.…”

“…The tyrosine phosphorylation in ClC‐3 channels was upregulated, as vascular cells switched from contractile to proliferative phenotype (Kang et al ., 2012). Another study also demonstrated that the phosphorylation of Tyr 284 in ClC‐3 channels by Src kinase, was important for activation of these channels (Wang et al ., 2013; Zeng et al ., 2014). These results suggested that many phosphorylation sites in the ClC‐3 protein were involved in the channel activity.…”

“…Mutation of Ser 109 within the N ‐terminal of ClC‐3 channels abolished the CaMKII‐dependent Cl − current (Robinson et al ., 2004). We have also demonstrated that the phosphorylation of Tyr 284 in ClC‐3 channels, by Src kinase was an important mechanism for activation of these channels (Wang et al ., 2013; Zeng et al ., 2014). Moreover, the Rho/Rho‐kinase (ROCK) pathway was involved in the regulation of ClC‐3 channel activity (Liu et al ., 2010).…”

Interactions of the Mechanosensitive Channels with Extracellular Matrix, Integrins, and Cytoskeletal Network in Osmosensation

“…Silencing integrin β1 expression reduces RVD of the adherent cells (Sørensen et al, 2015). Interrupting an integrin β3/Src/ClC-3 signaling pathway influences the hyposmotic activation of volume-regulated chloride channels (Zeng et al, 2014). Deletion of the integrin alpha1 subunit inhibited the intracellular Ca 2+ transients of chondrocytes to hyposmotic stress ex vivo and in vitro (Jablonski et al, 2014).…”