Functional cooperation between IKCa and TRPC1 channels regulates serum‐induced vascular smooth muscle cell proliferation via mediating Ca2+ influx and ERK1/2 activation

Abstract The increased proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of vascular diseases. The intermediate conductance calcium‐activated potassium (IKCa) channel plays a critical role in VSMC proliferation by raising the intracellular calcium concentration ([Ca2+]i), but the underlying mechanism is still not unclear. Here we investigated the cooperation between IKCa and transient receptor potential canonical 1 (TRPC1) channels in mediating extracellular Ca2+ entry, which in turn activates downstream Ca2+ signalling in the regulation of VSMC proliferation using serum‐induced cell proliferation model. Serum‐induced cell proliferation was accompanied with up‐regulation of IKCa expression and an increase in [Ca2+]i. Serum‐induced cell proliferation and increase in [Ca2+]i were suppressed by IKCa inhibition with TRAM‐34 or IKCa knockdown. Serum‐induced cell proliferation was strongly reduced by the removal of extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA‐AM and, additionally, by TRPC1 knockdown. Moreover, the increase in [Ca2+]i induced by serum or by IKCa activation with 1‐EBIO was attenuated by TRPC1 knockdown. Finally, serum induced ERK1/2 activation, which was attenuated by treatment with TRAM‐34 or BAPTA‐AM, as well as TRPC1 knockdown. Consistently, serum‐induced cell proliferation was suppressed by ERK1/2 inhibition with PD98059. Taken together, these results suggest that the IKCa and TRPC1 channels cooperate in mediating Ca2+ influx that activates the ERK1/2 pathway to promote cell proliferation, thus providing new mechanistic insights into VSMC proliferation.

unclear. Here we investigated the cooperation between IK Ca and transient receptor potential canonical 1 (TRPC1) channels in mediating extracellular Ca 2+ entry, which in turn activates downstream Ca 2+ signalling in the regulation of VSMC proliferation using serum-induced cell proliferation model. Serum-induced cell proliferation was accompanied with up-regulation of IK Ca expression and an increase in [Ca 2+ ] i . Serum-induced cell proliferation and increase in [Ca 2+ ] i were suppressed by IK Ca inhibition with TRAM-34 or IK Ca knockdown. Serum-induced cell proliferation was strongly reduced by the removal of extracellular Ca 2+ with EGTA or intracellular Ca 2+ with BAPTA-AM and, additionally, by TRPC1 knockdown. Moreover, the increase in [Ca 2+ ] i induced by serum or by IK Ca activation with 1-EBIO was attenuated by TRPC1 knockdown. Finally, serum induced ERK1/2 activation, which was attenuated by treatment with TRAM-34 or BAPTA-AM, as well as TRPC1 knockdown. Consistently, serum-induced cell proliferation was suppressed by ERK1/2 inhibition with PD98059. Taken together, these results suggest that the IK Ca and TRPC1 channels cooperate in mediating Ca 2+ influx that activates the ERK1/2 pathway to promote cell proliferation, thus providing new mechanistic insights into VSMC proliferation. Xiaoling Jia and Xinlan Chen have contributed equally to this work.

| INTRODUCTION
Vascular smooth muscle cells (VSMCs) constitute to form the media of the blood vessel wall and play a pivotal role in maintaining vasculature structure and function. 1 Unlike other mature cell types in adult body, VSMCs exhibit remarkable plasticity in their function. In normal arteries, VSMCs adopt a contractile phenotype to regulate vascular tone and blood pressure. [1][2][3] In response to environmental changes, VSMCs switch to a synthetic phenotype, and proliferate excessively and migrate into blood vessel lumen. It has been well documented that the proliferative functionality of VSMCs contributes to the pathogenesis of multiple vascular diseases, such as hypertension, atherosclerosis and restenosis. [4][5][6][7][8][9] Ion channels are strongly implicated in the regulation of VSMC function and pathogenesis of related vascular diseases. [10][11][12][13] The intermediate conductance Ca 2+ -activated K + (IK Ca ) channel, also known as K Ca 3.1 or IKI encoded by the KCNN4 gene, is highly expressed in synthetic VSMCs, and is critically engaged in VSMC proliferation. [14][15][16][17][18] Compelling evidence exists to show that VSMC proliferation induced by chemical or physical stimuli is accompanied with increased expression and function of the IK Ca channel and that its pharmacological and/or genetic interventions effectively decrease VSMC proliferation and alleviate the progression of diseases, such as atherosclerosis and restenosis. 19 It has been reported that the expression of Ca 2+ -permeable transient receptor potential canonical (TRPC) channels is increased in synthetic VSMCs and vascular diseases. 12,[22][23][24] In particular, the TRPC1 expression was up-regulated in balloon-induced human internal mammary arteries 25 and also in human vein samples after angioplasty. 26 Importantly, treatment with inhibitory anti-TRPC1 antibodies prevented neointima progression in rodents. 26 Furthermore, there is evidence that TRPC1 interacts with Orai1 and Homer to mediate store-operated Ca 2+ entry (SOCE) and promotes VSMC proliferation and migration, contributing to neointima formation and stenosis in balloon-injured carotid arteries in rats. 27 Evidence also exists that the TRPC1 channel mediates extracellular Ca 2+ entry induced by activation of the IK Ca channel in breast cancer cells. 28 However, the interplay between the IK Ca and TRPC1 channels in modulating the [Ca 2+ ] i in VSMCs and its role in cell proliferation are not established. Ca 2+ is a universal second messenger and controls a wide range of cellular processes including cell proliferation 11,29-31 via diverse Ca 2+ -dependent signalling pathways, including ERK, PKA and Akt/PI 3 K, [32][33][34][35] and ERK is one of the most common mechanisms in the regulation of VSMC proliferation. 33,[36][37][38][39] In this study, we investigated the interplay between the IK Ca and TRPC1 channels in raising the [ Figure S1).

| MTT assay
Cell proliferation was assessed using the MTT assay.

| Calcium imaging
The change in [Ca 2+ ] i was monitored by single cell calcium imaging as previously described. 41

| Data presentation and statistical analysis
All data are presented as means ± SD, where appropriate. Western blotting and MTT assays were performed at least three times, and Fluo4 fluorescence intensity was examined in approximately 100 cells.
Student's t-test was performed for comparison of two groups, and one-way analysis of variance (ANOVA) followed by post hoc Fisher's test for comparison of three or more groups, with p < 0.05 being considered significant.

| The IK Ca channel mediates serum-induced cell proliferation via promoting TRPC1-mediated Ca 2+ entry
The TRPC1 channel is known to play an important role in regulating cell proliferation via mediating Ca 2+ entry 24 Figure 6A). Interestingly, such ERK activation was highly dynamic, reaching the maximal level at 30 min and declining upon prolonged stimulation ( Figure 6A). Serum-induced VSMC proliferation, examined using the MTT assay ( Figure 6B) or western blotting to compare the PCNA expression level ( Figure S3

| DISCUSSION
The IK Ca channel in VSMCs has been well known for its important role in regulating cell proliferation, a functional phenotype strongly implicated in the pathogenesis of multiple vascular diseases, but the underlying signalling mechanism is incompletely understood. In this study, we provide evidence to suggest that the IK Ca channel cooperates with the TRPC1 channel to raise the [Ca 2+ ] i that in turn activates the ERK1/2 signalling pathway to drive serum-induced VSMC proliferation (Figure 7).
In the present study, we demonstrated the importance of the IK Ca channel expressed in VSMCs in serum-induced cell proliferation via raising the [Ca 2+ ] i (Figures 1 and 2). The IK Ca channel has been hypothesized, albeit being still short of supporting evidence, to increase the [Ca 2+ ] i via mediating K + efflux to induce membrane Accumulating evidence indicates that up-regulated TRPC1 expression is associated with increased cell proliferation and the pathogenesis of various vascular diseases, including atherosclerosis, pulmonary hypertension and restenosis, as well as lung cancer. 25,26,44,45 The present study showed that the TRPC1 expression remained unchanged during serum-induced VSMC proliferation ( Figure 3A) but genetic depletion of the TRPC1 expression reduced serum-induced VSMC proliferation ( Figure 3C) and increase in [Ca 2+ ] i ( Figure 3D). These results strongly indicate that the TRPC1 channel is important in regulating serum-induced Ca 2+ signalling and cell proliferation. Nonetheless, further investigation is required to understand how the TRPC1 channel is activated during this process. The TRPC1 channel is widely viewed to regulate SOCE or act as a constituent of the mechanism mediating SOCE. Importantly, in this study, we provide strong evidence to support that the IK Ca channel cooperates with the TRPC1 channel expressed in VSMCs to raise the [Ca 2+ ] i or more specifically promotes TRPC1-mediated Ca 2+ entry (Figures 3-5). A similar molecular mechanism has been proposed in the regulation of breast cancer cell proliferation. 28 These findings clearly extend our understanding of the mechanisms engaging the IK Ca and TRPC1 channels in regulating cell proliferation. One interesting and important question that arises and needs further research is whether the IK Ca and TRPC1 channel interact with each other to form a signalling complex to coordinate seruminduced Ca 2+ signalling that stimulates VSMC proliferation. . Cell proliferation is presented relative to that in cells in FBS-free medium without PD98059 (B), and p-ERK level relative to that in cells cultured in FBS-free medium (A), without TRAM-34 (C), with siCTL transfection (D) or without BAPTA-AM (E). Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 In this study, we have further shown the importance of IK Ca / TRPC1-coordinated Ca 2+ -dependent activation of the ERK signal pathway in driving serum-induced cell proliferation. We have recently reported a similar mechanism by which the IK Ca channel mediates stiff substrate-induced Ca 2+ signalling and cell proliferation in VSMCs. 41 Interestingly, our study revealed serum-induced ERK activation was highly dynamic, declining and even lost upon prolonged exposure to FBS ( Figure 6A). This fast kinetic of ERK activation is consistent with the fast kinetic of Ca 2+ response to serum, which shows a fast initial strong Ca 2+ response followed by a relative steady Ca 2+ response ( Figure S5). We showed that serum-induced ERK activation in VSMCs was inhibited by treatment with TRAM-34 to block the IK Ca channel ( Figure 6C), siRNA-mediated knockdown of TRPC1 ( Figure 6D) and, importantly, by treatment with BAPTA-AM to chelate intracellular Ca 2+ increase ( Figure 6E). Collectively, these results are strong in supporting the concept that the IK Ca and TRPC1 channels coordinate serum-induced Ca 2+ signalling that in turn activates the ERK pathway and thereby stimulates VSMC proliferation (Figure 7).

| CONCLUSIONS
In summary, our present study shows that the IK Ca channel cooperates with the TRPC1 channel in serum-induced VSMC proliferation, via promoting TRPC1-mediated Ca 2+ entry to raise the [Ca 2+ ] i that in turns activates the ERK signalling pathway. Such a finding provides novel insights into the role of the IK Ca and TRPC1 channels in VSMC proliferation and the pathogenesis of related cardiovascular diseases.