Upregulated ClC3 Channels/Transporters Elicit Swelling-Activated Cl− Currents and Induce Excessive Cell Proliferation in Idiopathic Pulmonary Arterial Hypertension

Taiki Amano; Aya Yamamura; Moe Fujiwara; Seiji Hirai; Rubii Kondo; Yoshiaki Suzuki; Hisao Yamamura

doi:10.1248/bpb.b22-00513

Abstract

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling of the pulmonary artery, which is mainly attributed to the excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) comprising the medial layer of pulmonary arteries. The activity of ion channels associated with cytosolic Ca²⁺ signaling regulates the pathogenesis of PAH. Limited information is currently available on the role of Cl⁻ channels in PASMCs. Therefore, the functional expression of ClC3 channels/transporters was herein investigated in the PASMCs of normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH). Expression analyses revealed the upregulated expression of ClC3 channels/transporters at the mRNA and protein levels in IPAH-PASMCs. Hypoosmotic perfusion (230 mOsm) evoked swelling-activated Cl⁻ currents (I_Cl-swell) in normal-PASMCs, whereas 100 µM 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) exerted the opposite effects. The small interfering RNA (siRNA) knockdown of ClC3 did not affect I_Cl-swell. On the other hand, I_Cl-swell was larger in IPAH-PASMCs and inhibited by DIDS and the siRNA knockdown of ClC3. IPAH-PASMCs grew more than normal-PASMCs. The growth of IPAH-PASMCs was suppressed by niflumic acid and DIDS, but not by 9-anthracenecarboxylic acid or T16A_inh-A01. The siRNA knockdown of ClC3 also inhibited the proliferation of IPAH-PASMCs. Collectively, the present results indicate that upregulated ClC3 channels/transporters are involved in I_Cl-swell and the excessive proliferation of IPAH-PASMCs, thereby contributing to the pathogenesis of PAH. Therefore, ClC3 channels/transporters have potential as a target of therapeutic drugs for the treatment of PAH.

INTRODUCTION

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and vascular remodeling, resulting in distal pulmonary arteries that are narrowed and occluded. This pathogenesis progressively increases pulmonary vascular resistance, followed by sustained elevations in pulmonary arterial pressure. Chronically elevated pulmonary arterial pressure leads to right ventricular hypertrophy and, ultimately, right heart failure.¹⁾ Prostacyclin and its derivatives (epoprostenol, treprostinil, iloprost, and beraprost), endothelin receptor antagonists (bosentan, ambrisentan, and macitentan), and phosphodiesterase type 5 inhibitors (sildenafil and tadalafil) are mainly used to treat PAH. Furthermore, selexipag, a prostaglandin I₂ agonist, and riociguat, a soluble guanylate cyclase stimulator, have recently been approved for the treatment of PAH.¹⁾ However, the five-year survival rate of PAH patients is only 65% in the U.S.A. and 69% in Europe.¹⁾ Therefore, there remains an unmet medical need and novel therapeutic options are needed.

Cytosolic Ca²⁺ signaling is an important regulator of the physiological and pathological responses of pulmonary arterial smooth muscle cells (PASMCs). An elevated cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) under physiological conditions mediates the contraction and proliferation of PASMCs. The activities of Ca²⁺-permeable ion channels, including voltage-dependent Ca²⁺ channels, receptor-operated Ca²⁺ channels, and store-operated Ca²⁺ channels, have been shown to regulate [Ca²⁺]_cyt.^2,3) The membrane potential, which is influenced by the activity of K⁺ channels, including voltage-dependent K⁺ channels, background K⁺ channels, and Ca²⁺-activated K⁺ channels, also modulates [Ca²⁺]_cyt.^2,3) However, the enhancements that occur in Ca²⁺ signaling under pathological conditions result in abnormal vascular events, including pulmonary vasoconstriction and vascular remodeling. Although Ca²⁺ and K⁺ channels have been implicated in the pathological mechanisms underlying PAH, limited information is currently available on the role of Cl⁻ channels.

Cl⁻ channels are ubiquitously distributed in all cells and function in various physiological responses, such as Cl⁻ homeostasis, cell volume regulation, the secretion of epithelial fluids, neuronal excitation, and smooth muscle contraction. The Cl⁻ channel superfamily is structurally classified into the following four groups: voltage-dependent Cl⁻ channels, cystic fibrosis transmembrane conductance regulator channels, Ca²⁺-activated Cl⁻ channels, and ligand-gated Cl⁻ channels (γ-aminobutyric acid type A and glycine receptors).⁴⁾ Voltage-dependent Cl⁻ channels are predominantly formed by dimeric assembly of ClC-coding proteins. Among ClC family members, ClC1, ClC2, ClC-Ka, and ClC-Kb proteins are responsible for Cl⁻ channels in the plasma membrane, while ClC3, ClC4, ClC5, ClC6, and ClC7 proteins comprise Cl⁻ transporters in endosomal and lysosomal membranes.⁵⁾

ClC3 protein expression has been detected in canine,⁶⁾ rat,⁷⁾ and human⁸⁾ PASMCs, and is upregulated in pulmonary hypertensive rats.⁷⁾ However, expression and functional analyses of ClC3 channels/transporters have not yet been performed on PAH patients. Therefore, we herein examined the physiological and pathological roles of ClC3 channels/transporters in the PASMCs of normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH). The present results suggest the upregulated expression of ClC3 channels/transporters in IPAH-PASMCs, which contributes to swelling-activated Cl⁻ currents (I_Cl-swell) and excessive cell proliferation.

MATERIALS AND METHODS

Cell Cultivation

Medium 199 (Sigma-Aldrich, St. Louis, MO, U.S.A.) containing 10% fetal bovine serum (Gibco-Invitrogen, Grand Island, NY, U.S.A.), 20 µg/mL endothelial cell growth supplement (BD Biosciences, Franklin Lakes, NJ, U.S.A.), 50 µg/mL D-valine (Sigma-Aldrich), 100 U/mL penicillin G (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 100 µg/mL streptomycin (FUJIFILM Wako Pure Chemical Corporation) was used to cultivate the PASMCs (passages 5–10) of normal subjects (Lonza, Walkersville, MD, U.S.A.) and patients with IPAH⁹⁾ at 37 °C in a 5% CO₂ atmosphere.

Quantitative Real-Time PCR

The StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, U.S.A.) was used to quantify mRNA expression levels.¹⁰⁾ Specific PCR primers were obtained from OriGene Technologies (Rockville, MD, U.S.A.): human ClC3 (GenBank Accession Nos. NM_173872; HP234116) and β-actin (NM_001101; HP204660). mRNA levels were normalized to the expression of β-actin as the endogenous control.

Western Blot Analysis

A Western blot analysis was conducted using a rabbit monoclonal anti-ClC3 antibody (1 : 1000; #13359, Cell Signaling Technology, Danvers, MA, U.S.A.) and goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugate antibody (1 : 5000; #170-6515, Bio-Rad, Hercules, CA, U.S.A.).¹⁰⁾ Signals were detected using an ImmunoStar LD reagent (FUJIFILM Wako Pure Chemical Corporation) and analyzed by the ImageQuant LAS 4000 system (GE Healthcare, Pittsburgh, PA, U.S.A.). Protein expression levels were normalized using a mouse monoclonal anti-β-actin antibody (1 : 5000; A5316, Sigma-Aldrich) and goat anti-mouse IgG-horseradish peroxidase conjugate antibody (1 : 5000; #170-6516, Bio-Rad).

Small Interfering RNA (siRNA) Knockdown

Lipofectamine RNAiMax reagent (Invitrogen, Carlsbad, CA, U.S.A.) was used to transfect PASMCs with 100 nM siRNA of a negative control (Medium GC Duplex #3; Invitrogen) or ClC3 (Stealth RNAi, HSS101968; Invitrogen). Experiments on siRNA were conducted 48 to 72 h after transfection.

Electrophysiological Recordings

A whole-cell patch-clamp technique was employed for electrophysiological recordings of single PASMCs and was conducted at room temperature (23 to 25 °C).¹¹⁾ The extracellular solution (isosmotic 330 mOsm) contained the following: 92 mM NaCl, 5.9 mM KCl, 2.2 mM CaCl₂, 1.2 mM MgCl₂, 14 mM glucose, 10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), and 100 mM sucrose (pH 7.4 with NaOH). Sucrose was removed from isosmotic solution to obtain a hypoosmotic solution (230 mOsm). The pipette solution contained the following: 120 mM CsCl, 20 mM tetraethylammonium (TEA) chloride, 4 mM MgCl₂, 2 mM ATPNa₂, 10 mM HEPES, and 5 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (pH 7.2 with CsOH).

Cell Growth Assay

PASMCs at a density of 1–3 × 10³ cells/well (1 × 10⁴ cells/well for siRNA experiments) were cultured for 6 h in a 96-well plate. Culture medium containing vehicle or the drug was then added for 24 to 72 h. Cell growth was assessed using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), which is based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.¹²⁾

Cell Proliferation Assay

PASMCs at a density of 1 × 10⁴ cells/well were cultured for 48 h in a 96-well plate. The Cell Proliferation ELISA BrdU (colorimetric) kit (Roche Diagnostics, Mannheim, Germany), which is based on the bromodeoxyuridine (BrdU) incorporation assay, was used to assess cell proliferation.¹²⁾

Drugs

EGTA and HEPES were supplied by Dojindo Laboratories. All other pharmacological reagents were purchased from Sigma-Aldrich. Niflumic acid (NFA), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), 9-anthracenecarboxylic acid (9-AC), and T16A_inh-A01 were dissolved in dimethyl sulfoxide (DMSO) to concentrations ranging between 10 and 100 mM as stock solutions.

Statistical Analysis

Results are presented as means ± standard error (S.E.). The significance of differences between two groups and among groups was examined by the Student’s t-test and Scheffé’s test following ANOVA, respectively, using BellCurve for Excel software (version 3.22; Social Survey Research Information, Tokyo, Japan).

RESULTS

Upregulated Expression of ClC3 in IPAH-PASMCs

Quantitative real-time PCR and Western blotting analyses were performed to compare the expression levels of ClC3 channels/transporters between normal- and IPAH-PASMCs. ClC3 mRNA levels were higher in IPAH-PASMCs (0.033 ± 0.003 of β-actin, n = 8, p < 0.001) than in normal-PASMCs (0.008 ± 0.001, n = 8) (Fig. 1A). ClC3 protein expression levels were also higher in IPAH-PASMCs (1.69 ± 0.04-fold, n = 8, p < 0.001) than in normal-PASMCs (1.00 ± 0.04, n = 8) (Figs. 1B, C). Collectively, these results indicate the upregulated expression of ClC3 channels/transporters in IPAH-PASMCs.

Fig. 1. Upregulated Expression of ClC3 Channels/Transporters in IPAH-PASMCs

Quantitative real-time PCR and Western blotting analyses of ClC3 channel/transporter expression in normal- and IPAH-PASMCs were performed. A. ClC3 mRNA expression levels in normal- and IPAH-PASMCs (n = 8). mRNA levels were normalized by the expression of the endogenous control, β-actin. B. Representative blots showing ClC3 protein expression in normal-and IPAH-PASMCs. C. ClC3 protein expression in normal- and IPAH-PASMCs (n = 8). Protein levels were normalized to the expression of β-actin and normal-PASMCs. ** p < 0.01 vs. normal-PASMCs.

Effects of ClC3 siRNA on ClC3 Expression in Normal- and IPAH-PASMCs

The siRNA knockdown of ClC3 were performed to investigate the functional role of ClC3 channels/transporters in normal- and IPAH-PASMCs. We previously reported that the transfection of ClC3 siRNA specifically downregulated the expression of ClC3 mRNA without any off-target effects on other ClC genes.¹³⁾ Therefore, the expression efficiency of the siRNA knockdown of ClC3 was herein examined in normal- and IPAH-PASMCs at the protein level using a Western blotting analysis. Following the siRNA knockdown of ClC3, ClC3 protein expression levels were reduced in normal-PASMCs (0.52 ± 0.06-fold, n = 8, p < 0.001 vs. control siRNA, 1.00 ± 0.05, n = 8) and IPAH-PASMCs (0.28 ± 0.04-fold, n = 8, p < 0.001 vs. control siRNA, 1.48 ± 0.06-fold, n = 8) (Fig. 2). These results suggest that the transfection of ClC3 siRNA efficiently knocked down the expression of ClC3 in normal- and IPAH-PASMCs.

Fig. 2. Decreased ClC3 Expression Levels in Normal- and IPAH-PASMCs Transfected with ClC3 siRNA

A Western blotting analysis was performed to examine the effects of the siRNA knockdown of ClC3 on ClC3 expression levels in normal- and IPAH-PASMCs. Normal- and IPAH-PASMCs were transfected with control or ClC3 siRNA. A. Representative blots showing ClC3 protein expression in normal- and IPAH-PASMCs transfected with control or ClC3 siRNA. B. ClC3 protein expression levels in normal- and IPAH-PASMCs transfected with control or ClC3 siRNA (n = 8). Protein levels were normalized to the expression of β-actin and control siRNA/normal-PASMCs. ** p < 0.01 vs. control siRNA; ^## p < 0.01 vs. normal-PASMCs.

I_Cl-swell in Normal-PASMCs

Previous studies demonstrated that ClC3 channels/transporters in vascular smooth muscle cells were activated by a hypoosmotic stimulation^4,14); therefore, I_Cl-swell in normal-PASMCs was measured under voltage-clamp configurations. The addition of 120 mM Cs⁺ and 20 mM TEA to the pipette solution abolished K⁺ currents. Ca²⁺-activated Cl⁻ currents were also blocked by low [Ca²⁺]_cyt conditions with 5 mM EGTA in the pipette solution. Single PASMCs were stimulated for 200 ms from the holding potential of 0 mV to the selected test potentials (−80 to +80 mV) every 5 s. In normal-PASMCs transfected with control siRNA, whole-cell Cl⁻ currents increased after the hypoosmotic perfusion (230 mOsm) (28.4 ± 4.0 pA/pF at +80 mV, n = 6, p < 0.001 vs. isosmotic 330 mOsm solution, 1.5 ± 0.4 pA/pF, n = 6; −21.3 ± 3.1 pA/pF at −80 mV, n = 6, p < 0.001 vs. isosmotic solution, −0.9 ± 0.3 pA/pF, n = 6) (Figs. 3A, B, D). I_Cl-swell was inhibited by 100 µM DIDS (5.4 ± 1.1 pA/pF at +80 mV, n = 6, p < 0.001 vs. hypoosmotic solution; −9.3 ± 1.6 pA/pF at −80 mV, n = 6, p = 0.003).

Fig. 3. Swelling-Activated and DIDS-Sensitive Cl⁻ Currents in Normal-PASMCs

I_Cl-swell elicited by a hypoosmotic stimulation was recorded in normal-PASMCs in the voltage-clamp mode. K⁺ and Ca²⁺-activated Cl⁻ currents were completely suppressed by the addition of 120 mM Cs⁺/20 mM TEA and 5 mM EGTA, respectively, to the pipette solution. Single cells were stimulated for 200 ms from the holding potential of 0 mV to the selected test potentials (−80 to +80 mV) every 5 s. Normal-PASMCs were transfected with control or ClC3 siRNA. A. Representative traces of whole-cell Cl⁻ currents in normal-PASMCs in isosmotic (330 mOsm) and hypoosmotic (230 mOsm) solutions with or without 100 µM DIDS. B. Current-voltage relationship of Cl⁻ currents in control siRNA-transfected normal-PASMCs in isosmotic and hypoosmotic solutions with or without DIDS (n = 6). C. Current-voltage relationship of Cl⁻ currents in isosmotic and hypoosmotic solutions with or without DIDS in ClC3 siRNA-transfected normal-PASMCs (n = 6). D. Summarized data of current density at +80 and −80 mV in normal-PASMCs (n = 6). ** p < 0.01 vs. isosmotic solution; ^## p < 0.01 vs. hypoosmotic solution.

The siRNA knockdown of ClC3 was performed to demonstrate the direct involvement of ClC3 activity in I_Cl-swell in normal-PASMCs. However, I_Cl-swell was detected in normal-PASMCs even after the transfection of ClC3 siRNA (27.2 ± 3.7 pA/pF at +80 mV, n = 6, p < 0.001 vs. isosmotic solution and p = 0.834 vs. control siRNA; −19.7 ± 3.1 pA/pF at −80 mV, n = 6, p < 0.001 vs. isosmotic solution and p = 0.727 vs. control siRNA) (Figs. 3A, C, D). I_Cl-swell was suppressed by 100 µM DIDS (3.4 ± 0.8 pA/pF at +80 mV, n = 6, p < 0.001 vs. hypoosmotic solution; −6.1 ± 1.3 pA/pF at −80 mV, n = 6, p < 0.001). Therefore, the activity of ClC3 channels/transporters did not appear to play a role in the regulation of I_Cl-swell in normal-PASMCs.

ClC3-Mediated I_Cl-swell in IPAH-PASMCs

I_Cl-swell due to a hypoosmotic stimulation was observed in IPAH-PASMCs. In IPAH-PASMCs transfected with control siRNA, whole-cell Cl⁻ currents were increased by a hypoosmotic stimulation (230 mOsm) (48.3 ± 1.9 pA/pF at +80 mV, n = 6, p < 0.001 vs. isosmotic solution, 1.2 ± 0.3 pA/pF, n = 6; −34.4 ± 2.0 pA/pF at −80 mV, n = 6, p < 0.001 vs. isosmotic solution, −1.3 ± 0.2 pA/pF, n = 6) (Figs. 4A, B, D). I_Cl-swell was larger in IPAH-PASMCs than in normal-PASMCs (p = 0.001 at +80 mV and p = 0.005 at −80 mV) (compare between Figs. 3, 4), and were inhibited by 100 µM DIDS (7.1 ± 2.0 pA/pF at +80 mV, n = 6, p < 0.001 vs. hypoosmotic solution; −14.7 ± 4.1 pA/pF at −80 mV, n = 6, p < 0.001).

Fig. 4. I_Cl-swell Mediated by Upregulated ClC3 Channels/Transporters in IPAH-PASMCs

I_Cl-swell elicited by a hypoosmotic stimulation was recorded in IPAH-PASMCs transfected with control or ClC3 siRNA in the voltage-clamp mode. Single cells were stimulated for 200 ms from the holding potential of 0 mV to the selected test potentials (−80 to +80 mV) every 5 s. A. Representative traces of whole-cell Cl⁻ currents in IPAH-PASMCs in isosmotic (330 mOsm) and hypoosmotic (230 mOsm) solutions with or without 100 µM DIDS. B. Current-voltage relationship of Cl⁻ currents in control siRNA-transfected IPAH-PASMCs in isosmotic and hypoosmotic solutions with or without DIDS (n = 6). C. Current-voltage relationship of Cl⁻ currents in ClC3 siRNA-transfected IPAH-PASMCs in isosmotic and hypoosmotic solutions with or without DIDS (n = 6). D. Summarized data of current density at +80 and −80 mV in IPAH-PASMCs (n = 6). ** p < 0.01 vs. isosmotic solution; ^## p < 0.01 vs. hypoosmotic solution; ^$$ p < 0.01 vs. control siRNA/hypoosmotic solution.

The effects of the siRNA knockdown of ClC3 on I_Cl-swell after the hypoosmotic stimulation were assessed in IPAH-PASMCs. In contrast to normal-PASMCs, the transfection of ClC3 siRNA markedly inhibited I_Cl-swell in IPAH-PASMCs (23.7 ± 6.0 pA/pF at +80 mV, n = 6, p = 0.002 vs. isosmotic solution and p = 0.008 vs. control siRNA; −14.5 ± 3.5 pA/pF at −80 mV, n = 6, p = 0.003 vs. isosmotic solution and p < 0.001 vs. control siRNA) (Figs. 4A, C, D). I_Cl-swell was suppressed by 100 µM DIDS (4.4 ± 1.2 pA/pF at +80 mV, n = 6, p = 0.005 vs. hypotonic solution; −6.1 ± 1.6 pA/pF at −80 mV, n = 6, p = 0.056). Collectively, these results indicate that the upregulated expression of ClC3 induced I_Cl-swell in IPAH-PASMCs.

Effects of ClC Channel Blockers and ClC3 siRNA on the Growth of Normal- and IPAH-PASMCs

The MTT assay was performed to compare cell growth between normal- and IPAH-PASMCs. The growth of IPAH-PASMCs (2.18 ± 0.06 at 24 h, n = 9, p = 0.006; 2.95 ± 0.12 at 48 h, n = 9, p < 0.001; 3.68 ± 0.18 at 72 h, n = 9, p < 0.001) was faster than that of normal-PASMCs (1.93 ± 0.06 at 24 h, n = 9; 2.32 ± 0.08 at 48 h, n = 9; 2.37 ± 0.06 at 72 h, n = 9) (Fig. 5A), which is consistent with previous findings.¹⁵⁾

Fig. 5. Growth Inhibitory Effects of ClC Channel Blockers and ClC3 siRNA on Normal- and IPAH-PASMCs

The MTT assay was performed to assess the effects of conventional blockers of ClC channels (NFA, DIDS, and 9-AC) and Ca²⁺-activated Cl⁻ channels (T16A_inh-A01) on the growth of normal- and IPAH-PASMCs. A. Time-dependent cell growth of normal- and IPAH-PASMCs (n = 9). B. Effects of 100 µM NFA, DIDS, 9-AC, and 10 µM T16A_inh-A01 for 72 h on the growth of normal- and IPAH-PASMCs (n = 6). Data were normalized by vehicle groups (0.1% DMSO, 1.0). C. Effects of the siRNA knockdown of ClC3 on the growth of normal- and IPAH-PASMCs for 48 h (n = 24). Normal- and IPAH-PASMCs were transfected with control or ClC3 siRNA. * p < 0.05, ** p < 0.01 vs. vehicle control or control siRNA.

The MTT assay was also conducted to assess the effects of ClC channel blockers (NFA, DIDS, and 9-AC) on the growth of normal- and IPAH-PASMCs. The growth of normal-PASMCs was inhibited by the treatment with 100 µM NFA for 72 h (0.77 ± 0.03, n = 6, p = 0.013 vs. vehicle, 1.00 ± 0.07, n = 6) (Fig. 5B). In contrast, the addition of 100 µM DIDS (0.98 ± 0.06, n = 6, p = 0.862) and 9-AC (0.98 ± 0.09, n = 6, p = 0.906) to normal-PASMCs did not alter their growth. The addition of 10 µM of the Ca²⁺-activated Cl⁻ channel blocker, T16A_inh-A01 also did not affect their growth (0.99 ± 0.06, n = 6, p = 0.838). However, the growth of IPAH-PASMCs was suppressed by NFA (0.83 ± 0.04, n = 6, p = 0.027 vs. vehicle, 1.00 ± 0.05, n = 6) and DIDS (0.78 ± 0.03, n = 6, p = 0.003), but not by 9-AC (0.91 ± 0.04, n = 6, p = 0.375) or T16A_inh-A01 (0.93 ± 0.05, n = 6, p = 0.169).

Knockdown experiments using ClC3 siRNA were performed to demonstrate the direct involvement of the activity of ClC3 channels/transporters in the growth of normal- and IPAH-PASMCs. The siRNA knockdown of ClC3 inhibited the growth of normal-PASMCs for 48 h (0.55 ± 0.02, n = 24, p < 0.001 vs. control siRNA, 0.70 ± 0.02, n = 24) and IPAH-PASMCs (0.94 ± 0.02, n = 24, p < 0.001 vs. control siRNA, 1.20 ± 0.02, n = 24) (Fig. 5C). Therefore, the activity of ClC3 channels/transporters appeared to contribute to the growth of PASMCs.

The siRNA Knockdown of ClC3 Inhibited the Proliferation of IPAH-PASMCs

The BrdU incorporation assay was performed to examine the effects of the siRNA knockdown of ClC3 on the proliferation of normal- and IPAH-PASMCs. The siRNA knockdown of ClC3 attenuated the proliferation of normal-PASMCs for 48 h (0.84 ± 0.04, n = 16, p = 0.021 vs. control siRNA, 1.00 ± 0.04, n = 16) as well as that of IPAH-PASMCs (0.67 ± 0.02, n = 32, p < 0.001 vs. control siRNA, 1.00 ± 0.03, n = 32) (Fig. 6). These results indicate that upregulated ClC3 channels/transporters contributed to the excessive proliferation of IPAH-PASMCs.

Fig. 6. Reduced Proliferation of ClC3-Transfected IPAH-PASMCs

The BrdU incorporation test was performed to examine the effects of the siRNA knockdown of ClC3 on the proliferation of normal- and IPAH-PASMCs for 48 h. Normal- and IPAH-PASMCs were transfected with control or ClC3 siRNA (n = 16 to 32). Data were normalized by control siRNA. * p < 0.05, ** p < 0.01 vs. control siRNA.

DISCUSSION

The present results revealed i) the upregulated expression of ClC3 in IPAH-PASMCs, ii) I_Cl-swell elicited by upregulated ClC3 channels/transporters with a hypoosmotic stimulation in IPAH-PASMCs, and iii) the contribution of upregulated ClC3 channels/transporters to the excessive proliferation of IPAH-PASMCs.

ClC channels/transporters have been shown to play important roles in the regulation of myogenic tone, the membrane potential, and cell volume in vascular smooth muscle cells.^2,4) ClC subtypes have distinct electrophysiological properties.⁵⁾ ClC1 currents are activated by membrane depolarization. ClC2 currents are slowly activated by hyperpolarization with an inward rectification. ClC3 currents are activated by hyperpolarizing/depolarizing stimulations with a weak outward rectification. ClC4 and ClC5 currents are activated by depolarization at >+20 mV with a strong outward rectification. ClC7 currents are activated by depolarization with a strong outward rectification. ClC-Ka and ClC-Kb with barttin protein generate weak voltage-dependent currents. Alternatively, ClC subtypes have a characteristic pharmacological profile.⁵⁾ NFA broadly blocks ClC currents. DIDS reduces ClC3, ClC4, and ClC7 currents and weakly inhibits ClC2 and ClC5 currents. 9-AC weakly attenuates ClC1 and ClC2 currents, but not ClC3 or ClC5 currents. In addition to the results of expression analyses, electrophysiological and pharmacological data obtained in the present study suggest that voltage-dependent Cl⁻ currents in IPAH-PASMCs, which are activated by membrane depolarization with a weak outward rectification and sensitive to DIDS, are responsible for ClC3-coding proteins.

In addition to these electrophysiological and pharmacological data, ClC3 currents were shown to be increased by cell swelling following hypoosmolarity.^4,5,14,16) The present results clearly demonstrated increases in whole-cell Cl⁻ currents following the hypoosmotic perfusion (230 mOsm) and their activation was inhibited by DIDS and the siRNA knockdown of ClC3 in IPAH-PASMCs. Therefore, I_Cl-swell may comprise ClC3 channels/transporters in IPAH-PASMCs, consistent with previous findings on canine PASMCs.⁶⁾ However, in normal-PASMCs, the siRNA knockdown of ClC3 did not reduce I_Cl-swell that was sensitive to DIDS. Liang et al. reported that the activity of ClC3 channels/transporters did not contribute to I_Cl-swell in human PASMCs.⁸⁾ Furthermore, I_Cl-swell was detected in the PASMCs of ClC3-deficient mice.¹⁷⁾ These findings are consistent with the present results on normal-PASMCs. The ClC3 protein is ubiquitously expressed in most cell types, is a molecular candidate for a swelling-activated Cl⁻ channel, and functions as an intracellular Cl⁻/H⁺ transporter instead of a plasmalemmal Cl⁻ channel.⁵⁾ However, the ClC3 protein in the plasma membrane has been suggested to regulate I_Cl-swell in response to a hypoosmotic stimulation in several cell types, such as human chondrocytes.¹³⁾ Although further studies are needed to identify the molecular components of I_Cl-swell in PASMCs, we concluded that upregulated ClC3 channels/transporters in IPAH-PASMCs generated I_Cl-swell under our experimental conditions.

Since changes in the expression and activity of Ca²⁺ and K⁺ channels have been implicated in the pathogenesis of PAH, the up-/down-regulation of these channels has been demonstrated in PASMCs from PAH patients and animal PH models.²⁾ However, limited information is currently available on the involvement of Cl⁻ channels in the pathogenesis of PAH. Dai et al. previously reported the upregulated expression of ClC3 in the PASMCs of monocrotaline-induced pulmonary hypertensive rats, which was induced by endothelin and/or platelet-derived growth factor (PDGF)⁷⁾; however, changes in the expression of ClC channels/transporters in the PASMCs of PAH patients have not yet been demonstrated. The present data showed the upregulated expression of ClC3 channels/transporters at the mRNA and protein levels in IPAH-PASMCs. Elevated plasma concentrations of endothelin-1¹⁸⁾ and PDGF¹⁹⁾ in PAH patients may be involved in the mechanisms responsible for the upregulation of ClC3 channels/transporters. The expression of ClC3 channels/transporters in vascular smooth muscle cells was previously shown to be upregulated in diabetic rats²⁰⁾ and neointimal hyperplasia mice.²¹⁾ The cytosolic Cl⁻ concentration was previously reported to be 30 to 50 mM in PASMCs²²⁾; therefore, the reversal potential of Cl⁻ was calculated to be −40 to −25 mV. Since the resting membrane potential is −60 to −50 mV in PASMCs,^2,23) the activation of Cl⁻ channels (due to the upregulation of Cl⁻ channels) is supposed to cause membrane depolarization. We herein demonstrated the novel pathophysiological significance of ClC3 channels/transporters in PAH.

The enhanced proliferation and attenuated apoptosis of PASMCs play important roles in the pathogenesis of PAH. PAH requires elevated [Ca²⁺]_cyt, which is regulated by the influx of Ca²⁺ through Ca²⁺-permeable channels and the membrane potential through the activities of K⁺ and Cl⁻ channels in PASMCs.²⁾ Cell proliferation is accompanied by cell volume swelling, whereas apoptosis results in cell shrinkage in the early stage. Therefore, the regulatory mechanism via swelling-activated Cl⁻ channels is highlighted in cardiovascular diseases associated with vascular remodeling. In vascular smooth muscle cells, the activity of ClC3 channels/transporters is reportedly involved in cell growth, proliferation, and apoptosis as well as myogenic contractions and volume regulation.^4,14) The upregulated expression of TMEM16A Ca²⁺-activated Cl⁻ channels²⁴⁾ was recently demonstrated in the PASMCs of IPAH patients²⁵⁾ and facilitated vascular remodeling. However, the results of the cell growth assay in the present study revealed that a selective inhibitor for TMEM16A channels, T16A_inh-A01, did not affect the growth of IPAH-PASMCs. On the other hand, the activity of ClC3 channels/transporters was involved in the excessive proliferation of IPAH-PASMCs, as previously reported in normal-PASMCs.⁸⁾ Collectively, these findings and the present results indicate that the proliferation of normal- and IPAH-PASMCs was regulated by the activity of ClC3 channels/transporters instead of that of TMEM16A channels under our experimental conditions.

In conclusion, upregulated ClC3 channels/transporters are involved in I_Cl-swell under hypoosmotic conditions and the excessive proliferation of the PASMCs of IPAH patients. Vascular remodeling mediated by the enhanced proliferation of PASMCs has been implicated in the pathogenesis of PAH. Therefore, ClC3 channels/transporters have potential as a therapeutic molecular target for the treatment with PAH.

Acknowledgments

We would like to thank Prof. Jason X.-J. Yuan (University of California, San Diego, U.S.A.) for PASMCs from IPAH patients. The present study was supported by Grants-in-Aid for Scientific Research (B) (22H02773 to Y. Suzuki; 22H02787 to H. Yamamura) and Scientific Research (C) (20K07092 to A. Yamamura) from the Japan Society for the Promotion of Science. We also received Grants-in-Aid from the Japan Research Foundation for Clinical Pharmacology (2018A19 to H. Yamamura) and the Institute of Drug Discovery Science at Nagoya City University (2018-04 and NCU-IDDS-A202105 to A. Yamamura and H. Yamamura). M. Fujiwara has a Ph.D. fellowship from the Japan Science and Technology Agency (JPMJSP2130) and a Nagai Memorial Research Scholarship from the Pharmaceutical Society of Japan. We are grateful for the assistance of the Research Equipment Sharing Center at Nagoya City University.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

1) McLaughlin VV, Shah SJ, Souza R, Humbert M. Management of pulmonary arterial hypertension. J. Am. Coll. Cardiol., 65, 1976–1997 (2015).
2) Olschewski A, Papp R, Nagaraj C, Olschewski H. Ion channels and transporters as therapeutic targets in the pulmonary circulation. Pharmacol. Ther., 144, 349–368 (2014).
3) Imaizumi Y. Reciprocal relationship between Ca²⁺ signaling and Ca²⁺-gated ion channels as a potential target for drug discovery. Biol. Pharm. Bull., 45, 1–18 (2022).
4) Bulley S, Jaggar JH. Cl⁻ channels in smooth muscle cells. Pflugers Arch., 466, 861–872 (2014).
5) Jentsch TJ, Pusch M. CLC chloride channels and transporters: structure, function, physiology, and disease. Physiol. Rev., 98, 1493–1590 (2018).
6) Yamazaki J, Duan D, Janiak R, Kuenzli K, Horowitz B, Hume JR. Functional and molecular expression of volume-regulated chloride channels in canine vascular smooth muscle cells. J. Physiol., 507, 729–736 (1998).
7) Dai YP, Bongalon S, Hatton WJ, Hume JR, Yamboliev IA. ClC-3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery. Br. J. Pharmacol., 145, 5–14 (2005).
8) Liang W, Huang L, Zhao D, He JZ, Sharma P, Liu J, Gramolini AO, Ward ME, Cho HC, Backx PH. Swelling-activated Cl⁻ currents and intracellular CLC-3 are involved in proliferation of human pulmonary artery smooth muscle cells. J. Hypertens., 32, 318–330 (2014).
9) Yu Y, Fantozzi I, Remillard CV, Landsberg JW, Kunichika N, Platoshyn O, Tigno DD, Thistlethwaite PA, Rubin LJ, Yuan JX. Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension. Proc. Natl. Acad. Sci. U.S.A., 101, 13861–13866 (2004).
10) Miyaki R, Yamamura A, Kawade A, Fujiwara M, Kondo R, Suzuki Y, Yamamura H. SKF96365 activates calcium-sensing receptors in pulmonary arterial smooth muscle cells. Biochem. Biophys. Res. Commun., 607, 44–48 (2022).
11) Noda S, Suzuki Y, Yamamura H, Imaizumi Y. Single molecule fluorescence imaging reveals the stoichiometry of BKγ1 subunit in living HEK293 cell expression system. Biol. Pharm. Bull., 43, 1118–1122 (2020).
12) Kondo R, Kawata N, Suzuki Y, Yamamura H. Ca²⁺ signaling and proliferation via Ca²⁺-sensing receptors in human hepatic stellate LX-2 cells. Biol. Pharm. Bull., 45, 664–667 (2022).
13) Yamada S, Suzuki Y, Bernotiene E, Giles WR, Imaizumi Y, Yamamura H. Swelling-activated ClC-3 activity regulates prostaglandin E₂ release in human OUMS-27 chondrocytes. Biochem. Biophys. Res. Commun., 537, 29–35 (2021).
14) Guan YY, Wang GL, Zhou JG. The ClC-3 Cl⁻ channel in cell volume regulation, proliferation and apoptosis in vascular smooth muscle cells. Trends Pharmacol. Sci., 27, 290–296 (2006).
15) Yamamura A, Guo Q, Yamamura H, Zimnicka AM, Pohl NM, Smith KA, Fernandez RA, Zeifman A, Makino A, Dong H, Yuan JX. Enhanced Ca²⁺-sensing receptor function in idiopathic pulmonary arterial hypertension. Circ. Res., 111, 469–481 (2012).
16) Yamamura H, Suzuki Y, Imaizumi Y. Physiological and pathological functions of Cl⁻ channels in chondrocytes. Biol. Pharm. Bull., 41, 1145–1151 (2018).
17) Yamamoto-Mizuma S, Wang GX, Liu LL, Schegg K, Hatton WJ, Duan D, Horowitz TL, Lamb FS, Hume JR. Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3^−/− mice. J. Physiol., 557, 439–456 (2004).
18) Fedorowicz A, Mateuszuk Ł, Kopec G, Skórka T, Kutryb-Zając B, Zakrzewska A, Walczak M, Jakubowski A, Łomnicka M, Słomińska E, Chlopicki S. Activation of the nicotinamide N-methyltransferase (NNMT)-1-methylnicotinamide (MNA) pathway in pulmonary hypertension. Respir. Res., 17, 108 (2016).
19) Selimovic N, Bergh CH, Andersson B, Sakiniene E, Carlsten H, Rundqvist B. Growth factors and interleukin-6 across the lung circulation in pulmonary hypertension. Eur. Respir. J., 34, 662–668 (2009).
20) Fu J, Fu J, Gao B, Wang L, Zhang N, Li X, Ji Q. Expression patterns of ClC-3 mRNA and protein in aortic smooth muscle, kidney and brain in diabetic rats. Clin. Invest. Med., 33, E146–E154 (2010).
21) Chu X, Filali M, Stanic B, Takapoo M, Sheehan A, Bhalla R, Lamb FS, Miller FJ Jr. A critical role for chloride channel-3 (CIC-3) in smooth muscle cell activation and neointima formation. Arterioscler. Thromb. Vasc. Biol., 31, 345–351 (2011).
22) Kitamura K, Yamazaki J. Chloride channels and their functional roles in smooth muscle tone in the vasculature. Jpn. J. Pharmacol., 85, 351–357 (2001).
23) Kuhr FK, Smith KA, Song MY, Levitan I, Yuan JX. New mechanisms of pulmonary arterial hypertension: role of Ca²⁺ signaling. Am. J. Physiol. Heart Circ. Physiol., 302, H1546–H1562 (2012).
24) Yamamura A, Yamamura H, Zeifman A, Yuan JX. Activity of Ca²⁺-activated Cl⁻ channels contributes to regulating receptor- and store-operated Ca²⁺ entry in human pulmonary artery smooth muscle cells. Pulm. Circ., 1, 269–279 (2011).
25) Papp R, Nagaraj C, Zabini D, et al. Targeting TMEM16A to reverse vasoconstriction and remodelling in idiopathic pulmonary arterial hypertension. Eur. Respir. J., 53, 1800965 (2019).

Corresponding author

Register with J-STAGE for free!