Original Article

Functional Regulation of ClC-3 in the Migration of Vascular Smooth Muscle CellsNovelty and Significance

Sindura B. Ganapathi, Shun-Guang Wei, Angelika Zaremba, Fred S. Lamb, Stephen B. Shears
https://doi.org/10.1161/HYPERTENSIONAHA.112.194209
Hypertension. 2013;61:174-179
Originally published December 12, 2012

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Abstract

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 [Ca2+]. Raising intracellular [Ca2+] to 0.5 μM activated a Cl− current (ICl.Ca), approximately half of which was eliminated on inhibition by KN-93 of calmodulin-dependent protein kinase II. ICl.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 ICl.Ca. Gene disruption of ClC-3 reduced ICl.Ca by 50%. Moreover, ICl.Ca in the ClC-3 null VSMCs was not affected by either KN-93 or inositol-3,4,5,6-tetrakisphosphate. We conclude that ICl.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.

Introduction

Proliferation and migration of vascular smooth muscle cells (VSMCs) are critical steps in the process of vascular remodeling that is associated with hypertension, atherosclerosis, and restenosis.1,2 The identification of proteins that participate in VSMC proliferation and migration, and characterization of their regulation, can assist therapeutic intervention in vascular remodeling.1–3

Cell migration is underpinned by changes in cell shape that are driven by temporally and spatially separated phases of localized cell swelling and cell shrinkage.4,5 The latter are activities that require precise spatiotemporal control of ion channels and transporters so as to coordinate movements of ions and water across the plasma membrane. The regulation of plasmalemmal Cl− flux by the ClC-3 channel/transporter6 contributes to the migration of certain tumor cells.7,8 However, it has not previously been studied whether ClC-3 mediates the migration of VSMCs in vitro or in vivo.

The manner in which ClC-3 activity is controlled underscores its potential importance as a regulator of cellular biology. In some cell types, ClC-3–dependent Cl− flux is stimulated by calmodulin-dependent protein kinase II (CaMKII).8–10 Furthermore, the degree of activation of ClC-3 by CaMKII is physiologically constrained by inositol 3,4,5,6-tetrakisphosphate (Ins[3,4,5,6]P4), a member of the inositol phosphate signaling family.11 In the current study we have investigated whether ClC-3 and Ins(3,4,5,6)P4 regulate plasmalemmal Cl− flux and migration of VSMCs. To recapitulate the promigratory phenotype of VSMCs during vascular disease, we used primary cell cultures12; we obtained VSMCs from wild-type and ClC-3 null mice.

Materials and Methods

Detailed methods are available in the online-only Data Supplement. ClC-3 null and wild-type aortic VSMCs were obtained as described previously.13 Cells were grown in DMEM containing 5% FBS, 1% vitamins, 1% glutamine, 1% penicillin/streptomycin, 1% nonessential amino acids, and 25 mM HEPES (pH 7.4). Cells were maintained at 37°C in 5% CO2. Plasmalemmal Cl− currents were measured using the whole-cell patch clamp technique in conditions where Cl− was the only permeant anion. The migration assays were performed at 37°C in 5% CO2 in 24-well hanging cell-culture inserts with 8 µM pore size (Millipore, Billerica, MA). To each insert, 40 000 cells were added in 200 μL of DMEM. Each insert was placed in a well that contained 650 μL of DMEM. Migration assays were begun 90 minutes after seeding with the addition of either 40 ng/mL of platelet-derived growth factor-BB (R&D Systems, Minneapolis, MN) or 10% FBS, plus either a test reagent (added to both compartments of the transwell chamber) or a matched control containing an equivalent concentration of vehicle (dimethyl sulfoxide). Cells were incubated for 17 hours (with FBS) or 5 hours (with platelet-derived growth factor). In control experiments, total cell number and viability were checked with a Countess Cell Counter (Life Technologies). Migrated cells on the bottom surface of the insert were identified using a Diff-Quik Stain set (Siemens Healthcare Diagnostics Inc, Newark DE); 1 minute in fixative, 2 minutes in solution I, and 4 minutes in solution II followed by rinsing in distilled water. Six 132-mm2 sections of the inserts were imaged by bright field microscopy (×20 objective), and then the number of migrated cells was counted; values from the 6 different areas were averaged. All of the current/voltage data were statistically analyzed by ANOVA with a Bonferroni post hoc test. Other data were analyzed with a Student t test. In the figure legends, n indicates the number of experiments. Values of P<0.05 were considered significant.

Results

Cell Migration of Wild-Type and ClC-3 Null Aortic VSMCs

We have investigated whether ClC-3 influences the migration of VSMCs in transwell assays. Cells were obtained from both wild-type and ClC-3 null mice; the success of the gene disruption is illustrated by Western analysis (Figure 1A). Cell migration in response to platelet-derived growth factor, a promigratory stimulus,14 was substantially reduced in ClC-3 null cells compared with wild-type cells (Figure 1B through 1D). That same phenotype was also observed in an experimental paradigm (serum-stimulated migration)15,16 that more closely mimics the complex growth factor environment in vivo (Figure 1E through 1G). The reduced migration of ClC-3 null cells was not an indirect consequence of there being fewer cells (see legend to Figure 1). Thus, the ClC-3 null cells did not proliferate more slowly. The latter observation is consistent with a previous2 conclusion that disruption of the ClC-3 gene did not affect VSMC proliferation per se, although it specifically halved the proliferative response to the proinflammatory cytokine, tumor necrosis factor-α.

Figure 1.
Figure 1.

Migration of wild-type (WT) and ClC-3 null aortic smooth muscle cells. A, Western analysis of WT and ClC-3 null cells using anti–ClC-3 and anti-GAPDH antibodies. B through G, Cell migration in response to either 40 ng/mL of platelet-derived growth factor (PDGF; B through D) or 10% FBS (D, E, and G) was recorded in transwell assays as described in the Methods section. Representative images of stained, migrated cells are shown (B, C, E, and F) or means and SEs from 8 (D) or 11 (G) experiments. *P<0.05 (Student t test). Final cell numbers (×104) and viability (%, in parentheses) were as follows: WT + PDGF, 3.8±0.1 (94±2); ClC-3 null + PDGF, 3.2±0.2 (92±2); WT + serum, 5.7±1.0 (95±1), ClC-3 null + serum, 5.3±1.0 (95±1); n=3 for PDGF, n=5 for serum. P>0.05 for ClC-3 null vs corresponding WT.

Swelling-Activated and Ca2+-Activated Cl− Currents in Wild-Type and ClC-3 Null Aortic VSMCs

Plasmalemmal Cl− fluxes mediated by ClC-3 may be enhanced by either cell swelling (IClswell) or by elevated [Ca2+]IN (ICl.Ca; see Duan17 and Cuddapah et al18). In VSMCs, IClswell was unaffected by elimination of ClC-3, irrespective of whether Ca2+ was present19 or absent (Figure S1, available in the online-only Data Supplement). We next measured ICl.Ca in wild-type and ClC-3 null cells using medium containing BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) to buffer [Ca2+]IN (inset to Figure 2A). Little Cl− current was observed at 0 [Ca2+]IN, but with 0.5 μM of [Ca2+]IN, whole-cell current was increased in both cell types (Figure 2A and 2B). ICl.Ca was outwardly rectifying (Figure 2A and 2B); kinetic analysis (Figure S2) revealed time-dependent activation of Cl− currents at depolarizing potentials. These are the prototypical characteristics of ICl.Ca.20–24 These kinetic properties were not significantly affected by disruption of the ClC-3 gene (Figure S2).

Figure 2.
Figure 2.

ICl.Ca in wild-type (WT) and ClC-3 null murine aortic smooth muscle cells. A and B, Cl- flux across the plasma membrane was measured in response to a defined [Ca2+]IN set by a BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)/Ca2+ buffer, which was perfused into the cell through the patch pipette (as illustrated in the inset to A). Current voltage relationships (measured 20 ms before the end of the voltage pulse) are shown for WT (A) and ClC-3 null (B) cells perfused with buffer in which [Ca2+]IN was set to either 0 (open circles, n=8), 0.5 μM (closed circles, n=19 or 20), or 0.5 μM plus 100 μM niflumic acid added to the pipette solution (NFA; triangles, n=5). Vertical bars depict SEM. C, A comparison of the current/voltage relationships of WT cells (closed bars) with ClC-3 null cells (open bars) at +40 mV and −100 mV (these voltages were selected because they are equidistant from the Cl- equilibrium potential of ≈30 mV). **P<0.002, WT vs corresponding ClC-3 null. D, Migration in response to serum, and the effect of 100 μM NFA added to the culture medium; n=4–8; vertical bars depict SEM. **p<0.005, WT vs corresponding ClC-3 null. Final cell numbers (×104) and viability (%, in parentheses) were as follows: WT, 4.2±0.4 (96±1); ClC-3 null, 4.2±0.6 (98±1); WT + NFA, 4.1±0.4 (94±2), ClC-3 null + NFA, 3.7±0.6 (96±2), n=4. P>0.05, for the addition of NFA.

ICl.Ca was ≈50% smaller in ClC-3 null VSMCs than in wild-type cells (Figure 2). It has not been demonstrated previously that ICl.Ca is decreased after knockout of the ClC-3 gene in any cell type. Thus, we highlighted this novel result by depicting steady-state currents at −100 mV and +40 mV (Figure 2C). We chose these voltages because they are equidistant from the ECl at −31 mV; although opposite in polarity, the 2 resultant currents are elicited by electrical driving forces that are equal in magnitude. We conclude that ICl.Ca in the wild-type cells is composed of approximately equal contributions from both ClC-3–dependent and ClC-3–independent components (Figure S3).

A generic Cl− channel blocker, niflumic acid,25 inhibited ICl.Ca in both cell types (Figure 2A and 2B). Niflumic acid also inhibited serum-stimulated migration in the wild-type cells (Figure 2D); cell numbers and viability were not affected by niflumic acid (Figure 2 legend). Niflumic acid did not alter migration in ClC-3 null cells (Figure 2D). Our data suggest that the ClC-3–dependent component of ICl.Ca contributes to VSMC migration (Figure S3).

Regulation of ICl.Ca by CaMKII Requires ClC-3

How does Ca2+ activate ICl.Ca in a ClC-3–dependent manner in aortic VSMCs? We are unaware of evidence that Ca2+ can stimulate ClC-3 directly, but Ca2+ can act indirectly by stimulating CaMKII, which in some cell types can activate ClC-3.8,9,26 However, other studies with smooth muscle cells from the pulmonary artery and the airways23,27–29 indicated that CaMKII does not stimulate ICl.Ca and instead inhibits it. We determined the impact of endogenous CaMKII on ICl.Ca in aortic smooth muscle cells using KN-93, a specific, cell-permeant CaMKII inhibitor.30 KN-93 reduced by ≈40% to 50% the degree to which 0.5 μM [Ca2+]IN activated ICl.Ca in wild-type cells (Figures 3A and S4). Significantly, KN-93 did not affect ICl.Ca in the ClC-3 null cells (Figure 3B). Control experiments indicated that there were similar degrees of CaMKII activity in the 2 cell types (Figure S5). Thus, in aortic VSMCs, endogenous CaMKII mediates Ca2+-dependent activation of ICl.Ca through a ClC-3–dependent process (Figure S3).

Figure 3.
Figure 3.

The effects of KN-93 and inositol-3,4,5,6-tetrakisphosphate (Ins[3,4,5,6]P4) on ICl.Ca in wild-type and ClC-3 null aortic smooth muscle cells. Either wild-type (A) or ClC-3 null cells (B) were perfused with buffer in which [Ca2+]IN was set to 0.5 μM (squares). In some experiments, either 100 μM KN-93 (circles) or 10 μM Ins(3,4,5,6)P4 (crosses) was added to the pipette solution. Current/voltage relationships for these Cl - currents are shown (n=9–20; vertical bars depict SEM). The insets to each graph depict the currents obtained at .100 and +40 mV. See Figure S6 for the complete current/voltage relationship for experiments in which KN-93 and Ins(3,4,5,6)P4 were added together. *P<0.05, **P<0.02, vs controls.

In certain cell types, the degree to which CaMKII activates ICl.Ca is supervised by Ins(3,4,5,6)P410,11,20; cellular levels of Ins(3,4,5,6)P4 are elevated downstream of the Ins(1,4,5)P3/Ca2+ cascade.11 Ins(3,4,5,6)P4 does not inhibit CaMKII activity, per se31 (as KN-93 does; see Figure S3). Instead, Ins(3,4,5,6)P4 specifically prevents CaMKII from activating ICl.Ca.11,20 This is a receptor-coupled mechanism by which Ins(3,4,5,6)P4 regulates salt and fluid secretion, insulin release, and neuronal excitability.11,20 A physiologically relevant11 concentration of 10 μM Ins(3,4,5,6)P4 reduced the size of ICl.Ca by 40% to 50% (Figure 3A). The inhibitory effects of KN-93 and Ins(3,4,5,6)P4 on ICl.Ca were not additive (Figures 3A and S6), consistent with the end point for both agents being inhibition of ClC-3 activity, albeit by different mechanisms (Figure S3). Our data (Figure 3A) represent the first demonstration that Ins(3,4,5,6)P4 regulates ICl.Ca in any VSMC.

Ins(3,4,5,6)P4 did not affect swelling-activated Cl− currents in VSMCs (Figure S1), which are not dependent on ClC-3 (Figure S1). Moreover, Ins(3,4,5,6)P4 did not alter ICl.Ca in the ClC-3 null cells (Figure 3B). These results provide important information concerning the specificity of Ins(3,4,5,6)P4 action; it has not been shown previously that ClC-3 is absolutely required for Ins(3,4,5,6)P4 to regulate ICl.Ca. Overall, our data demonstrate that ClC-3 provides VSMCs with a specific signaling mechanism by which Ins(3,4,5,6)P4 and CaMKII can regulate one component of ICl.Ca (Figure S3).

Regulation of ClC-3 and the Migration of Aortic VSMCs

CaMKII is known to regulate VSMC motility.3 We next investigated whether the regulation of migration by CaMKII requires ClC-3. We again used serum as a promigratory stimulus; serum drives VSMC migration in vitro by elevating intracellular [Ca2+] and activating CaMKII.3,14–16,32 When we added increasing concentrations of KN-93 (10–100 μM) to inhibit CaMKII, we found dose-dependent inhibition of migration of wild-type cells (maximal effect, 45%; Figure 4A); KN-93 did not alter the number or the viability of wild-type cells at the highest dose used (Figure 4 legend). Significantly, 10 to 50 μM KN-93 did not affect migration of ClC-3 null cells (Figure 4B). These data substantiate our conclusion that CaMKII regulates cell migration through ClC-3 (Figure S3).

Figure 4.
Figure 4.

The effects of KN-93 and inositol-3,4,5,6-tetrakisphosphate (Ins[3,4,5,6]P4) on the migration of wild-type (WT) and ClC-3 null aortic smooth muscle cells. The migration of wild-type (A and C) or ClC-3 null cells (B and D) was recorded in transwell assays as described in the Methods section. The concentrations of KN-93 and cell-permeant Ins(3,4,5,6)P4 are indicated in the figures. n=4; *P<0.05 vs controls. Final cell numbers (×104) and viability (%, in parentheses) were as follows: WT, 4.0±41.0 (93±1); WT + 100 μM KN-93, 3.9±2.0 (94±1); WT + 10 μM inositol-3,4,5,6-tetrakisphosphate (IP4), 3.8±1.0 (90 ± 2); ClC-3 null, 3.6±5.0 (93±2); ClC-3 null + 100 μM KN-93, 2.8±5.0* (87±3). n=4, *P<0.05 vs cell numbers for ClC-3 null vs ClC-3 null + KN-93.

At its maximal dose of 100 μM, KN-93 slightly inhibited the migration of ClC-3 null cells (Figure 4B), an effect that was possibly related to the 22% decrease in cell number at this concentration (Figure 4 legend). We next added Ins(3,4,5,6)P4, which does not inhibit CaMKII per se, but instead selectively prevents the activation of ClC-3 by CaMKII.10,20 A total of 10 μM Ins(3,4,5,6)P4 inhibited migration of wild-type cells by 48% (Figure 4C), without affecting cell number or viability (legend to Figure 4). Ins(3,4,5,6)P4 did not inhibit migration in ClC-3 null cells (Figure 4D), further supporting our conclusion that ClC-3 regulates VSMC migration.

Discussion

Our study makes a number of new observations concerning the importance of ClC-3 and Ins(3,4,5,6)P4 for plasmalemmal Cl− flux (ICl.Ca) and cell motility in VSMCs. Our results provide the first demonstration, in any cell type, that the size of ICl.Ca is substantially decreased after disruption of the ClC-3 gene (Figure 2). It is also a novel finding that endogenous CaMKII acts through ClC-3 to stimulate ICl.Ca in a smooth muscle cell line (Figure S3). This new information concerning ClC-3 is important because spatio-temporal control of ion channels and transporters is key to the process of cell migration.4,5 Indeed, we report in this study that ClC-3 contributes to VSMC migration in a CaMKII-dependent manner (Figures 3 and 4). Our study also adds to the known repertoire of biological functions of Ins(3,4,5,6)P410 by showing that it regulates VSMC migration.

Previous work21 with parotid acinar cells indicated that ICl.Ca was not affected by elimination of ClC-3. That result has been frequently interpreted as an indication that ClC-3 does not contribute to ICl.Ca in any cell.33–37 That assumption is now proved to be incorrect by our new data (Figure 2). Clearly, the regulation of ICl.Ca in aortic smooth muscle cells differs from that in parotid acinar cells, and we believe that CaMKII is the distinguishing factor: in our aortic smooth muscle cells, ClC-3–dependent activation of ICl.Ca requires CaMKII (Figure 3). In parotid acinar cells, CaMKII does not activate ICl.Ca.38 These differences between parotid cells and VSMCs in the role of CaMKII can explain why ICl.Ca is differentially impacted by the elimination of ClC-3. Moreover, the activation of ICl.Ca by CaMKII in aortic smooth muscle cells also uncovers a new aspect of signaling specialization in vascular biology. Previous work23,27,29 has shown that CaMKII inhibits ICl.Ca in smooth muscle cells from the pulmonary artery.

There is a long-standing debate as to whether ClC-3 is either directly responsible for plasmalemmal ICl.Ca26 or instead indirectly regulates Cl− flux.34 Elsewhere39 it has been explained why it is difficult to unequivocally prove whether a protein is either directly responsible for an ion current or instead contributes to a process that regulates another channel. It is beyond the scope of our study to resolve this complex question. Nevertheless, our results indicate that the halving of ICl.Ca on disruption of the ClC-3 gene (Figure 2) reflects the loss of a specific signaling pathway, namely, CaMKII-dependent ICl.Ca. Consistent with that conclusion, ClC-3–dependent ICl.Ca was blocked by the addition of a CaMKII inhibitor, KN-93 (Figure 3) or by Ins(3,4,5,6)P4, an intracellular signal that specifically attenuates ClC-3 activation by CaMKII (Figures 3 and S3).

Our study shows that total ICl.Ca in VSMCs includes a component that is CaMKII and ClC-3 dependent and another component that is independent of both CaMKII and ClC-3 (Figure S3). The latter may reflect that portion of ICl.Ca that is directly activated by Ca2+.35,40,41 Unlike Ca2+, CaMKII is particularly suited for regulating longer-term responses, because the kinase can remain active (autonomous) even in a poststimulation context, at a time when [Ca2+]IN has returned to resting levels.42 That CaMKII memory could explain why the CaMKII-dependent portion of ICl.Ca, which is mediated by ClC-3, is important to a sustained biological activity such as cell migration. Nevertheless, there may be additional targets by which CaMKII regulates migration.3

Finally, our data are very relevant to an earlier study showing that ClC-3 null mice are less susceptible to neointima formation after carotid ligation.2 That phenotype was attributed to a reduced rate of tumor necrosis factor-α–dependent proliferation of VSMCs.2 Our new data suggest that reduced VSMC motility may be an important contributing factor. Both migration and proliferation of VSMCs contributes to vascular remodeling,1 so our study provides new insight into the mechanisms underlying this condition.

Perspectives

The regulation of ClC-3 influences migration (see above) of VSMCs, which is a causal factor for diseases of vascular remodeling, such as the formation of neointima2 and atherosclerosis.1 ClC-3 may also drive glioblastoma pathophysiology8 and cardiac hypertrophy.17 Thus, genetic or pharmacological intervention in cell signaling by ClC-3 may ultimately benefit multiple aspects of human health. To this end, Ins(3,4,5,6)P4 specifically prevents the activation of ClC-3 by CaMKII (see above) without altering CaMKII activity per se.20 Moreover, the inositol phosphate did not affect either IClswell (Figure S1) or the ClC-3 independent portion of ICl.Ca (Figure 2). We suggest that Ins(3,4,5,6)P4 is a potential lead compound for synthesizing a drug that might inhibit vascular remodeling by specifically targeting ClC-3.

Sources of Funding

This work was supported by the Intramural Research Program of the National Institutes of Health/National Institute of Environmental Health Sciences and an National Institutes of Health RO1 grant to F.S.L (HL62483).

Disclosures

None.

Footnotes

  • Received February 28, 2012.
  • Revision received March 17, 2012.
  • Accepted October 22, 2012.

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Novelty and Significance

What Is New?

  • Determination that a chloride channel/transporter, ClC-3, regulates plasmalemmal chloride flux and migration of VSMCs.

  • Regulation of VSMC migration is a new biological function for an inositol phosphate (IP4).

  • Identification of a promigratory substrate for calmodulin-dependent protein kinase II in VSMCs.

What Is Relevant?

  • Identification of cellular factors that regulate migration of VSMCs, a key step in the vascular remodeling associated with hypertension.

  • Increased understanding of neointima formation.

  • Identification of a lead compound for drugs targeting vascular remodeling.

Summary

We describe novel, cell-signaling roles of IP4 and ClC-3 in VSMC migration, thereby revealing new therapeutic approaches to vascular remodeling diseases.

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  • Functional Regulation of ClC-3 in the Migration of Vascular Smooth Muscle CellsNovelty and Significance
    Sindura B. Ganapathi, Shun-Guang Wei, Angelika Zaremba, Fred S. Lamb and Stephen B. Shears
    Hypertension. 2013;61:174-179, originally published December 12, 2012
    https://doi.org/10.1161/HYPERTENSIONAHA.112.194209
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