Mitogenic modulation of Ca²⁺-activated K⁺ channels in proliferating A7r5 vascular smooth muscle cells

Abstract

1. Modulation of Ca²⁺-activated K⁺ channels (K_Ca) has been implicated in the control of proliferation in vascular smooth muscle cells (VSMC) and other cell types. In the present study, we investigated the underlying signal transduction mechanisms leading to mitogen-induced alterations in the expression pattern of intermediate-conductance K_Ca in VSMC.

2. Regulation of expression of IK_Ca/rK_Ca3.1 and BK_Ca/rK_Ca1.1 in A7r5 cells, a cell line derived from rat aortic VSMC, was investigated by patch-clamp technique, quantitative RT–PCR, immunoblotting procedures, and siRNA strategy.

3. PDGF stimulation for 2 and 48 h induced an 11- and 3.5-fold increase in rK_Ca3.1 transcript levels resulting in a four- and seven-fold increase in IK_Ca currents after 4 and 48 h, respectively. Upregulation of rK_Ca3.1 transcript levels and channel function required phosphorylation of extracellular signal-regulated kinases (ERK1/2) and Ca²⁺ mobilization, but not activation of p38-MAP kinase, c-Jun NH(2)-terminal kinase, protein kinase C, calcium-calmodulin kinase II and Src kinases.

4. In contrast to rK_Ca3.1, mRNA expression and functions of BK_Ca/rK_Ca1.1 were decreased by half following mitogenic stimulation. Downregulation of rK_Ca1.1 did not require ERK1/2 phosphorylation or Ca²⁺ mobilization.

5. In an in vitro-proliferation assay, knockdown of rK_Ca3.1 expression by siRNA completely abolished functional IK_Ca channels and mitogenesis.

6. Mitogen-induced upregulation of rK_Ca3.1 expression is mediated via activation of the Raf/MEK- and ERK-signaling cascade in a Ca²⁺-dependent manner. Upregulation of rK_Ca3.1 promotes VSMC proliferation and may thus represent a pharmacological target in cardiovascular disease states characterized by abnormal cell proliferation.

Introduction

In the course of disease, excessive proliferation of vascular smooth muscle cells (VSMC) plays an important pathophysiological role during arteriosclerosis, neointimal proliferation following angioplastic intervention (Racusen et al., 1999), transplant vasculopathies (McBride et al., 1988), and vascular remodeling as a consequence of arterial hypertension (Endemann & Schiffrin, 2004). In several studies, numerous growth-stimulating molecules such as ET1, PDGF and EGF to name some have been proposed to promote the observed proliferation, migration, and subsequent dedifferentiation of VSMC in such disease states (Newby & Zaltsman, 2000). In addition, alterations in ion channel function have also been proposed to control proliferation of several cell types (Nilius & Droogmans, 2001) including VSMC by enhancing intracellular Ca²⁺ signaling and affecting cell cycle progression (Afroze & Husain, 2001). For instance, upregulation of receptor/second messenger-operated channels of the TRP gene family has been related to increased Ca²⁺ signaling in proliferating VSMC in vitro (Golovina et al., 2001; Yu et al., 2004). Cl⁻ channels and K⁺ channels also seem to play a role during these modulations as these channels set the membrane potential which in turn controls the driving force for Ca²⁺ entry (Nilius & Droogmans, 2001). Consequently, inhibition of such Cl⁻ and K⁺ channels have been shown to prevent mitogenesis in vitro (Eggermont et al., 2001; Jager et al., 2004; Grgic et al., 2005).

Nonproliferating VSMCs predominantly express the calcium-activated large-conductance K⁺ channel (BK_Ca or maxi K), a product of the K_Ca1.1 gene (Atkinson et al., 1991) (as per the new IUPHAR nomenclature: http://www.iuphar.org/compendium2.htm; a.k.a. Slo). BK_Ca plays a pivotal role in VSMC relaxation by dampening depolarization-dependent activation of Ca²⁺-channels and Ca²⁺-influx via membrane hyperpolarization (Waldron & Cole, 1999). In a recent study, we demonstrated that neointimal, proliferating VSMCs downregulate BK_Ca and instead express the intermediate-conductance K_Ca channel (IK_Ca) (Kohler et al., 2003) encoded by the K_Ca3.1 gene (as per the new IUPHAR nomenclature; a.k.a. IKCa1, KCNN4, IK1, hSK4). This IK_Ca channel has been proposed to be an important regulator of proliferation in several cell types (Khanna et al., 1999; Pena et al., 2000; Wulff et al., 2000). We could additionally show that selective inhibition of this IK_Ca channel substantially reduced neointima formation in rat carotid arteries in vivo and cultured rat VSMC in vitro (Kohler et al., 2003).

In the present study, we aimed to investigate the underlying signal transduction pathways of growth factor-induced reorganization of K_Ca gene expression in A7r5 cells. In particular, we tested whether IK_Ca deficiency induced by silencing of rK_Ca3.1 expression with K_Ca3.1-specific siRNA inhibits A7r5 cell proliferation. Our studies demonstrated that PDGF-BB induced a rapid and long-lasting upregulation of rK_Ca3.1 expression and IK_Ca channel function which required intracellular calcium and an intact Raf/MEK- and extracellular signal-regulated kinases (ERK)-signaling cascade. In contrast, BK_Ca/Slo expression was significantly downregulated. Strikingly, during mitogen-induced proliferation, siRNA-induced silencing of rK_Ca3.1 abolished A7r5 cell proliferation in-vitro.

Methods

Materials

Cell culture media were obtained from Biochrom (Berlin, Germany). Unless otherwise stated, antibodies were from New England Biolabs (Ipswich, MA, U.S.A.). The MEK inhibitors PD98059 (20 μM) or U0126 (10 μM), the selective inhibitor of c-Jun N-terminal kinase (JNK) SP600125 (20 μM), the p38 MAP kinase inhibitor SB203580 (10 μM) were purchased from Tocris (Cologne, Germany), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate-acetoxymethyl ester (BAPTA-AM, 10 μM) and TMB-8 (100 μM, both intracellular calcium antagonists), chelerythrine (1 μM, protein kinase C (PKC) inhibitor), KN-93 (40 μM, calcium-calmodulin-dependent kinase II (CaMKII) inhibitor), wortmannin (0.1 μM, PI3 kinase inhibitor) or the Src tyrosine kinase inhibitor PP2 (2 μM) were from Sigma-Aldrich (Deisenhofen, Germany).

Cell culture

Transformed VSMC, cell line-A7r5, were cultured as previously described (Kohler et al., 2003). Prior to stimulation A7r5 cells were kept in serum-free medium for 48 h to induce growth arrest.

Patch-clamp experiments

Whole-cell patch-clamp experiments and data analysis were performed as described previously (Papassotiriou et al., 2000; Kohler et al., 2001). For activation of K_Ca currents, A7r5 cells were dialyzed with a KCl-pipette solution containing 3 μM [Ca²⁺]_free (mM): 140 KCl, 1 MgCl₂, 2 ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1.90 CaCl₂, and 5 N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES), pH 7.2. In another set of experiments, pipette solution contained 0.72 or 1.36 mM CaCl₂ and 2 mM EGTA yielding 0.1 and 0.4 μM [Ca²⁺]_free or 100 μM [Ca²⁺] without EGTA. The NaCl bath solution contained (mM): 137 NaCl, 4.5 Na₂HPO₄, 3 KCl, 1.5 KH₂PO₄, 0.4 MgCl₂ and 0.7 CaCl₂, pH 7.4.

RNA isolation and quantitative RT–PCR

Cells were harvested 2 or 48 h after stimulation, RNA was isolated using the high pure RNA isolation kit (Roche Diagnostics GmbH, Mannheim, Germany) and RNA was subsequently reverse transcribed using M-MLV reverse transcriptase (Life Technologies, Eggenstein, Germany). Expression levels were quantified with an ABI-Prism-7700 Sequence Detection System (ABI, Darmstadt, Germany) using intron-spanning primers and internal oligonucleotides labeled with 6-carboxy-fluorescein on the 5′ end and 6-carboxytetramethylrhodamine on the 3′ end. Identity of PCR products was verified by sequencing. Linearity of each PCR assay was confirmed by serial dilutions of cDNA.

Primer and internal oligonucleotides:

• rK_Ca3.1 (product size: 217 bp): F5′-CTGAGAGGCAGGCTGTCAATG-3′ (813–833);

• R5′-ACgTgTTTCTCCgCCTTgTT-3′ (1010–1029);

• P5′-AAGATTGTCTGCTTGTGCACCGGAGTC-3′ (926–952)

• rK_Ca1.1 (product size: 181 bp): F5′-TTTACCGGCTGAGAGATGCC-3′ (3926–3945);

• R5′-TGTGAGGAGTGGGAGGAATGA-3′ (4086–4106);

• P5′-ACCTCAGCACCCCCAGCCAGTG-3′ (3947–3968)

• rat glyceraldehyde-3-phosphate dehydrogenase (rGAPDH) (product size: 101 bp):

• F5′-CGGCACAGTCAAGGCTGAG-3′ (1014–1032);

• R5′-CAGCATCACCCCATTTGATGT-3′ (1094–1114);

• P5′-CCCATCACCATCTTCCAGGAGCGA-3′ (1060–1083)

GenBank™ accession: rK_Ca3.1: AF156554; rK_Ca1.1: AF135265; rGAPDH: AB017801.

Each 25 μl PCR reaction contained 500 nM forward and reverse primer, 150 nM probe, 3 μl cDNA, and 1 × TaqMan Universal Master Mix (ABI, Darmstadt, Germany). PCR parameters were 50°C × 2 min, 95°C × 10 min, and 50 cycles at 95°C × 15 s, 60°C × 1 min.

Threshold cycles (C_t) were calculated using TaqMan®-software (ABI-User-Bulletin-#2). Real-time RT–PCR signals for rK_Ca3.1 and rK_Ca1.1 were standardized to rGAPDH by using the equation: C_tx−C_trGAPDH=ΔC_t. The equation, Δ C_tw/o−ΔC_tx=ΔΔC_t, was used to determine changes in expression, where the ΔC_tw/o-value (growth factor stimulated) was subtracted from the control Δ C_tw/o -value (w/o=without stimulus) of the same experiment. Fold increases in expression were calculated by the equation, 2^ΔΔC_t=fold change.

siRNA experiments and in vitro proliferation studies

Synthetic siRNA directed against rK_Ca3.1 (sense: 5′-GGAGGUCCAGCUGUUCAUGtt-3′, antisense: 5′-CAUGAACAGCUGGACCUCCtt-3′) and scrambled negative control siRNA (sense: 5′-CAUUCACUCAGGUCAUCAGtt-3′, antisense: 5′-CUGAUGACCUGAGUGAAUGtt-3′), which does not interfere with any known mRNA, were synthesized by Ambion (Cambridgeshire, U.K.). A7r5 cells were transfected for 4 h with 25 nM of the siRNAs using siPORT™ Amine (Ambion) in accordance with the manufacturer's protocol. At this concentration, functional expression of rK_Ca3.1 was effectively eliminated for up to 72 h. Lower (10 nM) or higher (50 nM) concentrations of siRNAs resulted in inefficient transfection or cell death, respectively. Prior to transfection with siRNA-Ctrl or siRNA-rK_Ca3.1, VSMC were kept in serum-free DMEM for 48 h. Stimulation of these cells with FCS (10%) or PDGF-BB (10 ng ml⁻¹) was performed directly following the transfection. At 10–20% confluence, photo-micrographs of cells were taken in fixed fields before and 48 h after stimulation and the percent increase in cell count was calculated.

Western blotting

Immunoblotting were performed as described previously (Reusch et al., 2001b) applying an anti-IK_Ca primary antibody (1 : 200, Sigma, Germany) and phospho-specific antibodies against ERK1/2, phospho-specific Akt and respective secondary antibodies (Cell Signaling Technology, Frankfurt, Germany).

Statistical analysis

Data are given as mean±s.e. If appropriate, the Student's t-test or ANOVA and Bonferroni post hoc test were used to assess differences between groups. P-values of P<0.05 were considered significant.

Results

Growth factor-induced upregulation of IK_Ca function and rK_Ca3.1 expression in A7r5 cells

Numerous growth factors such as PDGF and EGF induce VSMC proliferation in vitro and have also been demonstrated to play a role in neointima formation in vivo (Newby & Zaltsman, 2000). However, these complex remodeling processes are only incompletely understood. Growth factor-induced K⁺ channel activity like IK_Ca upregulation might, for example play a pivotal intermediate step during these processes. To test this hypothesis, we compared IK_Ca function and rK_Ca3.1 expression in A7r5 cells before, 2, 4 and 48 h after stimulation with PDGF-BB (10 ng ml⁻¹). Prior to stimulation, the amplitude of ‘total' K_Ca currents was rather small, whereas following PDGF stimulation, the amplitude of the K_Ca current increased five-fold at 4 h and remained upregulated for at least 48 h. Cell capacitance was not altered by either PDGF or FCS treatment (data not shown). Original traces of K_Ca currents, mean K_Ca currents normalized to cell capacitance, and time course of upregulation of K_Ca currents are shown in Figure 1a, left, middle and right panels.

Figure 1.

Mitogenic stimulation induces mRNA expression of rK_Ca3.1 and increases IK_Ca currents in A7r5 cells. (a) Left panel, representative whole cell recordings of K_Ca currents in unstimulated (w/o) A7r5 cells and after stimulation with PDGF (10 ng ml⁻¹) or FCS (10%). Middle panel, mean K_Ca currents (activated by cell dialysis with a 3 μM-free Ca²⁺ concentration and normalized to cell capacitance) in PDGF-stimulated or -unstimulated (w/o) A7r5 cells. Data points represent mean±s.e., ^*P<0.05; ^**P<0.01 (ANOVA); right panel, amplitude of K_Ca currents, at membrane potential of 0 mV before and 2, 4 and 48 h after PDGF stimulation, respectively. (b) Left panel, PDGF-induced K_Ca currents in the presence of low (4.5 mM) and high (140 mM) extracellular K⁺, respectively. Reversal potentials of ≈−89 and 0 mV indicate K⁺ selectivity of the Ca²⁺-activated current. Middle panel, original whole-cell recordings showing Ca²⁺ dependence of PDGF-induced K_Ca currents. Right panel, concentration-dependent increase of mean IK_Ca currents by Ca²⁺ (EC₅₀ 400 nM). (c) Mixed BK_Ca and IK_Ca currents in A7r5 cells after PDGF stimulation and blockade of IK_Ca currents by TRAM-34 (100 nM) and BK_Ca currents by IbTX (100 nM). (d) Western blotting analysis of the IK_Ca protein expression in A7r5 cells with (+) PDGF (10 ng ml⁻¹) stimulation for 48 h or without (−) PDGF stimulation. The blot shown in the upper panel was probed with an antibody against rIK_Ca. Equal loading is demonstrated with the antibody detecting β-actin in the lower panel.

The PDGF-induced current was indeed a K⁺-selective and Ca²⁺-activated current since in the presence of low (4.5 mM) or high (140 mM) extracellular K⁺ concentrations, the current reversed near the K⁺ equilibrium potential of –89 or at 0 mV (Figure 1b, left panel). Cell dialysis with varying amounts of free Ca²⁺ (0.1–100 μM) resulted in a concentration-dependent increase in K⁺ currents with an EC₅₀ of ≈400 nM (Figure 1b, middle and right panel). In addition, the current was almost completely blocked by applying the selective IK_Ca inhibitor TRAM-34 (100 nM) (Wulff et al., 2000; Eichler et al., 2003) at negative membrane potential, leaving a small TRAM-34-insensitive K⁺ current component at positive membrane potentials (Figure 1c). The remaining K⁺ current was completely sensitive to the BK_Ca selective blocker IbTx (100 nM). Thus, the data with selective pharmacological inhibitors indicate that K_Ca currents in PDGF-stimulated A7r5 cells are largely mediated by IK_Ca currents with a small contribution of BK_Ca currents. Note that since the combination of TRAM-34 and IbTx treatment completely blocked total K⁺ currents, A7r5 cells appear to lack substantial other voltage-gated K⁺ currents irrespective of PDGF stimulation. This K_Ca-expression pattern in mitogen-stimulated A7r5 cells resembles that seen in proliferating neointimal VSMC in vivo rather than that observed in contractile nonproliferating VSMC (Kohler et al., 2003).

To test whether the high amplitude of IK_Ca functions after PDGF treatment is a consequence of new protein synthesis, we performed Western blot analysis using a specific IK_Ca antibody. This Western blot analysis revealed that PDGF stimulation for 48 h induced significant amounts of the IK_Ca protein while the IK_Ca protein was not detectable in the nonstimulated cells (Figure 1d).

Parallel studies applying quantitative RT–PCR technology revealed an about 11-fold increase in rK_Ca3.1 transcript levels following 2 h PDGF stimulation (Figure 2a), thus clearly preceding the observed upregulation in IK_Ca function. At 48 h following PDGF stimulation, rK_Ca3.1 transcript level was still 3.5-fold increased over unstimulated A7r5 cells (Figure 2a). Stimulation with 10% FCS for 48 h similarly upregulated IK_Ca function (Figure 1a, left and middle panels) and rK_Ca3.1 mRNA expression (Figure 2a). In contrast, stimulating A7r5 cells with thrombin (1 U ml⁻¹), another potent mitogen for A7r5 cells (McNamara et al., 1996), was ineffective in either augmenting IK_Ca current amplitude (0.4±0.1 pA pF⁻¹ at 0 mV, n=11) or upregulating expression of rK_Ca3.1 (−0.6±0.3 ΔΔC_t, n=7).

Figure 2.

Effects of MAP kinase blockers and other signal transduction modulators on mRNA expression levels of IK_Ca/rK_Ca3.1 in mitogen-stimulated A7r5 cells. (a) Semiquantitative RT–PCR analysis of rK_Ca3.1 mRNA expression levels following stimulation with PDGF for 2 and 48 h and with FCS (10%) for 48 h as well as following PDGF stimulation for 2 h after pretreatment with signal transduction inhibitors. (b) Pretreatment with the MEK-inhibitor PD98059 prevents PDGF-induced upregulation of IK_Ca/rK_Ca3.1 function in A7r5 cells. (c) Total K_Ca currents normalized to cell capacitance after PDGF stimulation with or without pretreatment with PD98059. Charts: left ordinate: ΔΔC_t values (ΔΔC_t=ΔC_tw/o−ΔC_tx) represent change in expression over control (unstimulated VSMC (n=19)). Right ordinate: x-fold change (2^ΔΔC_t) above control. Data are given as mean±s.e.; ^*P<0.01 vs w/o; ^#P<0.01 vs PDGF alone; ANOVA was used to compare respective ΔC_t values.

PDGF-induced upregulation of IK_Ca function and rK_Ca3.1 expression requires activation of the Raf/MEK and ERK-signaling cascade

The participation of the MAP kinases ERK1/2 has been shown to play an important role in VSMC proliferation in vitro and in vivo (Kingsley et al., 2002). Interestingly, the Ras/Raf/MEK/ERK-signaling cascade has also been involved in the mitogen-induced modulations of IK_Ca functions in rat fibroblasts (Pena et al., 2000) and of IK_Ca functions and K_Ca3.1 expression in human endothelial cells (Grgic et al., 2005). In T lymphocytes, promoter analysis of the K_Ca3.1 gene revealed that activation of K_Ca3.1 gene expression requires DNA binding of the transcription factor AP1 (Ghanshani et al., 2000) which has been shown to be a downstream target of ERK1/2 kinase activity (Park & Levitt, 1993). With respect to rK_Ca3.1 expression, treatment of A7r5 cells with the MEK-inhibitor PD98059 (20 μM) for 30 min prior to stimulation with PDGF abolished the growth factor-mediated upregulation of rK_Ca3.1 transcript levels at 2 h (Figure 2a) and of IK_Ca current amplitude at 48 h (Figure 2b and c). Similar data were obtained with the MEK inhibitor U0126 (10 μM, data not shown). Pretreatment of A7r5 cells with either the JNK inhibitor SP600125 or the p38-MAP kinase inhibitor SB203580 was without any effect on PDGF-induced upregulation of rK_Ca3.1 transcript levels (Figure 2a) or IK_Ca current amplitudes (data not shown).

To determine whether the observed upregulation of rK_Ca3.1 expression in A7r5 cells is mediated via activation of the Raf/MEK/ERK- or other MAP kinase-signaling cascades, we measured PDGF-induced phosphorylation of MAP kinases ERK1/2, c-Jun NH(2)-terminal kinase (JNK), or p38 MAP kinase. PDGF treatment resulted in pronounced MEK and ERK1/2 phosphorylation, which was maximal at 5–10 min and declined within 100 min to basal levels (Figure 3a). Other mitogens, that is EGF (10 ng ml⁻¹) and thrombin (1 U ml⁻¹)-induced ERK phosphorylation similar to PDGF. While the time course and degree of ERK phosphorylation after stimulation with EGF and PDGF were similar, thrombin-induced ERK phosphorylation appeared weaker and more transient (please refer to online Supplementary information).

Figure 3.

Effect of MAP-kinase inhibitors on the phosphorylation pattern of MEK1/2, ERK1/2 (a) and Akt (b) in PDGF-stimulated A7r5 cells. A7r5 cells were treated with different inhibitors as indicated. After 30 min, cells were stimulated with PDGF-BB (10 ng ml⁻¹) for the indicated times (in min). For each time point, aliquots of cell lysates were separated on SDS–PAGE and probed for the phosphorylated forms of MEK (phospho-S217/221), ERK1/2 (phospho T202/Y204) and Akt (phospho-S473). Equal loading is demonstrated with antibodies detecting the respective total proteins. The experiment shown is representative of three experiments showing similar results. QM=serum-free quiescent medium.

Phosphorylation of terminal JNK or p38 MAP kinase was not detectable after PDGF stimulation whereas challenging A7r5 cells with FCS induced phosphorylation of all three MAP kinases in these cells (data not shown). Incubation with MEK inhibitors PD98059 or U0126 for 30 min prior to PDGF stimulation dose dependently inhibited ERK-1/2 phosphorylation (Figure 3a and b), whereas the p38-MAP kinase inhibitor SB203580 (Figure 3a) and the JNK inhibitor SP600125 (data not shown) were without any effects. Previous studies confirmed subsequent translocation of phosphorylated ERK-1/2 into the nucleus where numerous transcription factors could become phosphorylated or activated (data not shown, Schauwienold et al., 2003). Interestingly, PDGF stimulation also activated the phosphatidylinositol-3-kinase (PI-3 kinase)-Akt pathway in these cells. As shown in Figure 3b applying a phospho-S473-Akt-specific antiserum, PDGF induced a robust phosphorylation of Akt in Western blots that was sensitive to inhibition with PI-3 kinase inhibition by wortmannin (Mizutani et al., 2002) or LY294002 (data not shown).

Calcium dependence of PDGF-induced upregulation of rK_Ca3.1 expression

Since PDGF-stimulation resulted in a Ca²⁺ release from internal stores in A7r5 (not shown) and cultured native VSMC (Reusch et al., 2001a, 2001b), we tested whether inhibition of phospholipase C (PLC) or Ca²⁺ chelation/application of intracellular calcium antagonists modulate ERK phosphorylation and consequently rK_Ca3.1 expression. Chelation of intracellular Ca²⁺ with BAPTA-AM (10 μM) or application of the intracellular calcium antagonist, TMB-8 (100 μM) as well as pretreatment with the PLC-selective inhibitor U73122 (10 μM) prevented ERK phosphorylation in a dose-dependent manner (Figure 4) and pretreatment with BAPTA-AM (not shown) or TMB-8 prevented upregulation of rK_Ca3.1 mRNA levels (Figure 2a) following stimulation with PDGF.

Figure 4.

Calcium dependence of PDGF-induced ERK1/2 phosphorylation. A7r5 cells were treated with inhibitors as indicated. After 30 min, cells were stimulated with PDGF (10 ng ml⁻¹) for 5 min. Cellular lysates were probed with phospho-specific ERK1/2 antisera. Equal loading is demonstrated with antibodies detecting the respective total proteins.

In additional screening experiments, PKC inhibitors like chelerythrine (10 μM) or bisindolylmaleimide (data not shown), an inhibitor of CaMKII, KN-93 (30 μM), blockage of Src kinases by PP2 (1 μM) or of PI3 kinase by wortmannin (100 nM) did not prevent PDGF-induced augmentation of rK_Ca3.1 expression (Figure 2a). Inhibition of all these signaling molecules did not alter basal rK_Ca3.1 mRNA expression levels in unstimulated cells, except inhibition of MEK by PD98059, which resulted in a significant reduction of basal rK_Ca3.1 mRNA expression levels (not shown). A similar Ca²⁺ mobilization was observed after stimulation with the other mitogens, EGF (10 ng ml⁻¹) and thrombin (1 U ml⁻¹) (not shown).

Taken together, these results suggest that PDGF-induced rK_Ca3.1 expression and augmented IK_Ca functions are mediated via activation of the Raf/MEK- and ERK-signaling cascade in a Ca²⁺-dependent manner.

PDGF downregulates BK_Ca function and BK_Ca/rK_Ca1.1 expression in A7r5 cells

Our previous studies on K_Ca functions in proliferating neointimal VSMC in situ revealed that upregulation of rK_Ca3.1 functions was paralleled by a loss of BK_Ca function and rK_Ca1.1 expression (Kohler et al., 2003). We therefore hypothesized that this loss of BK_Ca function and expression may also be a consequence of mitogenic stimulation in VSMC.

Patch-clamp experiments were conducted in the presence of 100 nM TRAM-34 to block IK_Ca currents and unmask small BK_Ca currents in A7r5 cells. Following 48 h treatment with PDGF the current amplitude of BK_Ca present in unstimulated cells was reduced (Figure 5a, upper and lower panels). With respect to BK_Ca, parallel RT–PCR studies revealed that mRNA levels of rSlo encoding the pore-forming α-subunit of the BK_Ca channel complex were significantly decreased by half after PDGF stimulation for 2 h (Figure 5b). Similar to PDGF, FCS stimulation for 48 h significantly diminished BK_Ca/rK_Ca1.1 expression levels by half (Figure 5b).

Figure 5.

Mitogenic stimulation with PDGF for 48 h resulted in decreased functional expression of BK_Ca/rK_Ca1.1 in A7r5 cells. (a) Upper panel, Representative whole-cell recordings of BK_Ca currents and, lower panel, mean BK_Ca currents (normalized to cell capacitance) in PDGF-stimulated and -unstimulated (w/o) A7r5 cells in the presence of the IK_Ca-blocker TRAM-34 (100 nM). Data points represent mean±s.e., ^*P<0.05, Student's t-test. (b) Effects of MAPK blockers and other signal transduction modulators on mRNA expression levels of BK_Ca/rK_Ca1.1 in mitogen-stimulated A7r5 cells. Semiquantitative RT–PCR analysis of BK_Ca/rK_Ca1.1 mRNA expression levels following stimulation with PDGF for 2 h and with FCS (10%) for 48 h as well as following 2 h-PDGF stimulation after pretreatment with signal transduction inhibitors. Charts: left ordinate: ΔΔC_t values (ΔΔC_t=ΔC_tw/o−ΔC_tx) represent change in expression over control (unstimulated VSMC (n=13)). Right ordinate: x-fold change (2^ΔΔC_t) above control. Data are given as mean±s.e.; ^*P<0.01 vs w/o; ^#P<0.01 vs PDGF alone (ANOVA).

PDGF-induced downregulation of BK_Ca/rK_Ca1.1 expression does not require the activation of the Raf/MEK and ERK-signaling cascade

To characterize the signal transduction mechanisms underlying the PDGF-induced downregulation of BK_Ca/rK_Ca1.1 expression we analyzed BK_Ca/rK_Ca1.1 expression in the same sets of experiments as stated above. The modest downregulation of BK_Ca/rK_Ca1.1 expression at 2 h following mitogenic stimulation was not prevented by inhibiting ERK1/2 phosphorylation with PD98059, p38 MAP kinase with SB203580, or of JNK with SP600125 (Figure 5b). Additionally, pretreatment of A7r5 cells with BAPTA-AM (not shown) and TMB-8, inhibition of PKC with chelerythrine, Src kinases with PP2, or PI3-K with wortmannin had no effects on PDGF-induced reduction of BK_Ca/rK_Ca1.1 expression (Figure 5b), whereas blockade of CaMKII with KN-93 prevented downregulation of BK_Ca/rK_Ca1.1 expression (Figure 5b). Overall, these results suggest that, in contrast to rK_Ca3.1, the PDGF-induced downregulation of BK_Ca/rK_Ca1.1 expression in A7r5 cells is most likely independent of the activation of MAP kinases and Ca²⁺ mobilization, but may require CaMKII activity.

Knockdown of rK_Ca3.1 expression by siRNA inhibits mitogenesis in A7r5 cells in vitro

As described earlier, pharmacological inhibition of IK_Ca channel activity by TRAM-34 largely attenuated epidermal growth factor (EGF)-induced mitogenesis in VSMC in vitro and neointima formation in vivo (Kohler et al., 2003). To further support and specify the importance of IK_Ca channels for mitogenesis in A7r5 cells, a siRNA approach was chosen. For this purpose, we transfected A7r5 cells with either siRNA directed towards rK_Ca3.1 mRNA or suitable siRNA controls of equal length with no known mRNA interference. Following siRNA transfection using the siPort Amine™ transfection reagent, cells were further cultivated in medium containing 10% FCS or 10 ng ml⁻¹ PDGF and cell number as well as K_Ca functions were determined after 48 h.

IK_Ca functions were absent in all rK_Ca3.1-siRNA-transfected cells (n=11) which were investigated by patch clamping. In contrast, Ctrl-siRNA-transfected cells (n=14) exhibited normal IK_Ca functions similar to the increased IK_Ca functions observed in PDGF- or FCS-stimulated cells. Original current traces and mean K_Ca currents normalized to cell capacitance are shown in Figure 6a and b.

Figure 6.

SiRNA against rK_Ca3.1 abolishes functional expression of IK_Ca/rK_Ca3.1 and mitogenesis in A7r5 cells. (a) Original whole-cell recording showing absence of functional IK_Ca/rK_Ca3.1 in rK_Ca3.1-siRNA-treated A7r5 cells (n=11; from three independent experiments), but not in Ctrl-siRNA-treated cells (n=14). Note that BK_Ca/rK_Ca1.1 currents were not affected by this treatment. (b) Mean K_Ca currents normalized to cell capacitance in rIK_Ca1-siRNA- and Ctrl-siRNA-transfected A7r5 cells. (c) Knockdown of rK_Ca3.1 expression by IK_Ca3.1-selective siRNA but not Ctrl-siRNA abolishes A7r5 cell proliferation (% increase of Cell No.) induced by FCS (10%) or PDGF (10 ng ml⁻¹). Values are given as mean±s.e.; ^*P<0.01; Student's t-test.

In contrast to the effect on IK_Ca functions, transfection with rK_Ca3.1-siRNA had no impact on BK_Ca functions (Figure 6a and b) which were similar to the small BK_Ca functions observed in mitogen-stimulated cells (Figure 5a). This indicates that the IK_Ca currents in A7r5 cells are indeed encoded by the rK_Ca3.1 gene and that the rK_Ca3.1-siRNA did not interfere with BK_Ca/rK_Ca1.1 mRNA. Moreover, the deficiency in IK_Ca functions does not seem to be compensated by an upregulation of BK_Ca functions.

The proliferation studies revealed that Ctrl-siRNA-transfected cells were still able to proliferate in response to FCS and PDGF stimulation, whereas a significant proliferation of rK_Ca3.1-siRNA-transfected cells was not observed (Figure 6c). A similar suppression of proliferation was observed in the presence of the Ca²⁺-chelator TMB-8 or the MEK-inhibitor PD98059 (not shown) as also described previously (Iwasaki et al., 1998). These results suggest that the presence of IK_Ca channels and upregulation of rK_Ca3.1 gene expression are required for mitogen-induced VSMC proliferation as has been reported in neointimal VSMC (Kohler et al., 2003), and mitogenesis of cultured VSMC, fibroblasts, and lymphocytes in vitro.

Discussion

The results of the present study demonstrate that binding of PDGF-BB to its respective receptors leads to an upregulation of rK_Ca3.1 mRNA expression and of IK_Ca functions in the rat cell line A7r5 which was dependent on the activation of the Raf/MEK and ERK signaling cascade and most likely required [Ca²⁺]_i. In contrast to IK_Ca, low levels of BK_Ca/rK_Ca1.1 mRNA expression and function in unstimulated A7r5 cells further decreased following mitogenic stimulation. Evaluation of participating signaling pathways revealed, that neither an activation of the Raf/MEK and ERK MAPK signaling or [Ca²⁺]_i transients were required for this events. The upregulation of IK_Ca seems to be an important mechanism in the control of mitogen-induced A7r5 cell proliferation since deficiency in rK_Ca3.1 abolished A7r5 cell proliferation similar to the mitogenesis-suppressing effects of selective and nonselective IK_Ca blockers in cultured VSMC as well as in neointima formation in vivo (Kohler et al., 2003).

There is growing evidence that several ion channels are of importance in the control of cell proliferation (Ghanshani et al., 2000; Pena et al., 2000; Nilius & Droogmans, 2001; Kohler et al., 2003; Jager et al., 2004; Ouadid-Ahidouch et al., 2004; Grgic et al., 2005). Among the large family of K⁺ channels, especially the IK_Ca channel has been shown to regulate cell cycle progression and mitogenesis in T lymphocytes, fibroblasts, and several tumor cell lines in vitro (Ghanshani et al., 2000; Jager et al., 2004; Ouadid-Ahidouch et al., 2004; Grgic et al., 2005). Upregulation of this channel has also been found in tumor tissues and in proliferating, EGF-stimulated VSMC in vitro and in neointimal VSMC in vivo (Kohler et al., 2003). IK_Ca seems to participate in cell proliferation by increasing the ability of the cell to hyperpolarize and thereby to promote Ca²⁺ influx. Increased intracellular Ca²⁺ has been demonstrated to be required for progression through the Ca²⁺-dependent cell cycle control point, that is from G1 to S (Berridge, 2001; Jager et al., 2004; Ouadid-Ahidouch et al., 2004). In addition, K⁺ efflux through IK_Ca channels and the concomitant loss of water may mediate regulatory volume decrease (RVD) to counteract cell swelling which often occurs in the early phase of proliferation (Jakab et al., 2002). Such a role in RVD during cell proliferation has also been proposed for volume-regulated chloride channels (Jager et al., 2004). Correspondingly, blockers of both, K⁺ and Cl⁻ channels have been shown to induce growth arrest in several cell types (Eggermont et al., 2001).

Thus far, little is know about the underlying signal transduction mechanism leading to an upregulation of this channel in VSMC. In T lymphocytes, studies revealed that stimulation of PKC resulted in an upregulation of K_Ca3.1 mRNA expression in an AP-1 transcription factor-dependent manner (Ghanshani et al., 2000), and in fibroblasts, transforming growth factor-β-induced upregulation of IK_Ca function was sensitive to MEK inhibitors (Pena et al., 2000). Interestingly, in pancreatic cancer cell lines with ‘overactive' Ras/Raf/MEK/ERK activity, IK_Ca functions and K_Ca3.1 mRNA expression levels were dramatically increased (Jager et al., 2004). Together these studies suggest that the activation of Ras/Raf/MEK/ERK is critically linked to the regulation K_Ca3.1 mRNA expression following stimulation with growth factors and activation of their respective receptors.

In A7r5 cells which we used in the present study as well as other VSMC (Reusch et al., 2001b), stimulation of the PDGF receptors resulted in ERK1/2 phosphorylation and an increase in [Ca²⁺]_i transients. As expected, ERK phosphorylation was suppressed by the use of MEK inhibitors. Additionally, a concomitant increase in [Ca²⁺]_i seemed to be required for ERK phosphorylation since suppression of [Ca²⁺]_i release from internal stores by either blocking PLC activity or manipulations of [Ca²⁺]_i abolished ERK phosphorylation. These results correlate well with our findings regarding the regulation of rK_Ca3.1 mRNA expression. Minimizing [Ca²⁺]_i and inhibition of ERK phosphorylation completely abolished the early upregulation of K_Ca3.1 mRNA expression and inhibition of ERK phosphorylation prevented the increase in IK_Ca functions following PDGF stimulation. Further MAP kinases do not seem to be involved in the regulation of K_Ca3.1 mRNA expression, since neither inhibitors of JNK nor p38 MAP kinase revealed any effects on mRNA expression. Similar to PDGF, a stimulation with thrombin (1 U ml⁻¹) induced an increase in [Ca²⁺]_i transients and ERK phosphorylation, although the degree of ERK phosphorylation appeared to be much weaker and more transient in nature than following PDGF stimulation. However, thrombin failed to upregulate rK_Ca3.1 mRNA expression and IK_Ca function. Thus, activation of the Ras/Raf/MEK/ERK-signaling cascade is required but not sufficient to upregulate rK_Ca3.1 mRNA expression.

In contrast to K_Ca3.1, BK_Ca/rK_Ca1.1 mRNA expression and BK_Ca functions tended to decrease following mitogenic stimulation of A7r5 cells. Such a diminished or even absent BK_Ca/rK_Ca1.1 mRNA expression and BK_Ca function has also been observed in proliferating neointimal VSMC in situ and in cultured proliferating VSMC in vitro (Kohler et al., 2003). In differentiated VSMC, BK_Ca channels mediate repolarization and the spontaneous transient outward currents resulting in a more negative membrane potential attenuate the depolarization-mediated activation of voltage-gated Ca²⁺-channels. Therefore, the loss of BK_Ca functions together with an upregulation of K_Ca3.1 may be indicative of dedifferentiation and a change in functional plasticity as the cells switch from a contractile to a proliferating phenotype. In contrast to K_Ca3.1, the downregulation of BK_Ca/rK_Ca1.1 mRNA expression in A7r5 cells appeared to occur independently of a participation of the Raf/MEK- and ERK- and JNK-, or p38-signaling cascade. With respect to upstream modulators of MAPK signaling, neither inhibition of PKC, Src kinases, Ca²⁺ signaling, nor PI-3 kinase was effective in preventing the observed downregulation of BK_Ca/rK_Ca1.1 mRNA expression.

IK_Ca channels have been proposed to participate in the control of mitogen-induced proliferation of several cell types such as fibroblast, VSMC in vitro, cancer cell lines (Grgic et al., 2005), and activated T lymphocytes. Correspondingly, during initial experiments, inhibition of IK_Ca channels inhibited cell proliferation in vitro as well as neointima formation following angioplasty in vivo. To further support this pivotal role of IK_Ca channels for mitogenesis in VSMC, we used a siRNA strategy in which a deficiency of IK_Ca channels should inhibit VSMC proliferation. The results show that A7r5 cells, in which the IK_Ca channel functions were successfully eliminated, do not proliferate. This finding underlines the importance of IK_Ca channels for the observed A7r5 cell proliferation and demonstrates that IK_Ca currents in A7r5 cells are indeed encoded by the rK_Ca3.1 gene. Since rK_Ca3.1-siRNA-transfected cells are devoid of any voltage-independent K_Ca currents, a deficiency in IK_Ca channels does not seem to be rapidly compensated by an increased functional expression of closely related K_Ca channels such as SK_Ca or by the low functional expression of BK_Ca/rK_Ca1.1 channels present in these cells.

In summary, the present study demonstrated that stimulation of A7r5 cells with PDGF-BB results in a significant and long-lasting upregulation of IK_Ca channels. This upregulation is mediated via an activation of the Raf/MEK- and ERK-signaling cascade and most likely requires calcium fluxes. Deficient IK_Ca channels dramatically affected growth factor-induced A7r5 cells proliferation in vitro. These findings together with results of our earlier studies indicate that IK_Ca channels may participate during the pathogenic processes underlying cardiovascular disease states such as hypertension, arteriosclerosis, or neointima formation following angioplasty characterized by abnormal VSMC proliferation and vascular remodeling.

Abbreviations

BK_Ca

large-conductance K_Ca

IbTx

Iberiotoxin

IK_Ca

intermediate-conductance

K_Ca

Ca²⁺-activated K⁺ channel

TRAM-34

1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole

Supplementary Information accompanies the paper on British Journal of Pharmacology website (http://www.nature.com/bjp)