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(Circulation. 2001;104:1557.)
© 2001 American Heart Association, Inc.

Basic Science Reports

c-Jun Decreases Voltage-Gated K⁺ Channel Activity in Pulmonary Artery Smooth Muscle Cells

Ying Yu, MD, PhD^*; Oleksandr Platoshyn, MS^*; Jifeng Zhang, PhD^*; Stefanie Krick, MD; Ying Zhao, BS; Lewis J. Rubin, MD; Abraham Rothman, MD; Jason X.-J. Yuan, MD, PhD

From the Departments of Medicine (Y.Y., O.P., S.K., Y.Z., L.J.R., J.X.-J.Y.) and Pediatrics (J.Z., A.R.), University of California, San Diego.

Correspondence to Jason X.-J. Yuan, MD, PhD, Division of Pulmonary and Critical Care Medicine, University of California, San Diego, 200 W Arbor Dr, San Diego, CA 92103-8382. E-mail xiyuan{at}ucsd.edu

	Abstract

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Background— Activity of voltage-gated K⁺ (K_v) channels controls membrane potential (E_m) that regulates cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) by regulating voltage-dependent Ca²⁺ channel function. A rise in [Ca²⁺]_cyt in pulmonary artery smooth muscle cells (PASMCs) triggers vasoconstriction and stimulates PASMC proliferation. Whether c-Jun, a transcription factor that stimulates cell proliferation, affects K_v channel activity in PASMCs was investigated.

Methods and Results— Infection of primary cultured PASMCs with an adenoviral vector expressing c-jun increased the protein level of c-Jun and reduced K_v currents (I_K(V)) compared with control cells (infected with an empty adenovirus). Using single-cell reverse transcription–polymerase chain reaction, we observed that the mRNA level of Kv1.5 and the current density of I_K(V) were both attenuated in c-jun–infected PASMCs compared with control cells and cells infected with antisense c-jun. Overexpression of c-Jun also upregulated protein expression of Kvß₂ and accelerated I_K(V) inactivation. Furthermore, E_m was more depolarized and [³H]thymidine incorporation was greater in PASMCs infected with c-jun than in control cells and cells infected with antisense c-jun.

Conclusions— These results suggest that c-Jun–mediated PASMC proliferation is associated with a decrease in I_K(V). The resultant membrane depolarization increases [Ca²⁺]_cyt and enhances PASMC growth.

Key Words: transcription factors • ion channels • genes • lung • remodeling

	Introduction

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Pulmonary vascular remodeling due to proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMCs) is an important pathological feature in pulmonary hypertension.^1,2 A rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC growth.^3–5 [Ca²⁺]_cyt is increased primarily by Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx through Ca²⁺ channels in the plasmalemma.^5–8 Among various Ca²⁺-permeable channels, the voltage-dependent Ca²⁺ channels (VDCCs) that are opened by membrane depolarization are a major Ca²⁺ entry pathway in vascular smooth muscle cells.^9,10

Membrane potential (E_m) is controlled primarily by the activity of Na⁺,K⁺-ATPase and the permeability of K⁺ ions across the plasma membrane through K⁺ channels. When K⁺ channels close or K⁺ channel expression is downregulated, whole-cell K⁺ currents decline and E_m becomes less negative.¹¹ The membrane depolarization opens VDCCs, promotes Ca²⁺ influx, increases [Ca²⁺]_cyt,^13–16 and stimulates PASMC growth.^12,13 Membrane depolarization may also promote Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange, which is sufficient to trigger Ca²⁺ release from ryanodine-sensitive Ca²⁺ stores, and increase [Ca²⁺]_cyt.¹⁴ In vascular smooth muscle cells, voltage-gated K⁺ (K_v) channels play an important role in the regulation of resting E_m.^10–13 Blockade of K_v channels causes membrane depolarization, opens VDCCs, induces Ca²⁺-dependent action potentials, and increases [Ca²⁺]_cyt in PASMCs.^10–13

c-jun is an immediate-early gene whose mRNA expression increases rapidly and transiently when quiescent cells are stimulated to grow.¹⁵ c-Jun is a nuclear protein that serves as a nuclear intermediate of signal transduction in cellular growth and differentiation.¹⁶ How c-Jun mediates PASMC growth is unclear. This study was designed to test the hypothesis that c-Jun induces PASMC proliferation and hypertrophy partially by regulating expression and function of K_v channels. The subsequent decrease in whole-cell K_v currents (I_K(V)) induces membrane depolarization, increases [Ca²⁺]_cyt, and stimulates PASMC proliferation.

	Methods

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Cell Preparation
Primary cultured PASMCs were prepared from Sprague-Dawley rats as previously described.^6,10,12 Briefly, adventitia and endothelium were carefully removed from the isolated pulmonary arterial branches (third to fourth division). The smooth muscle was digested with collagenase and elastase. The cells were plated onto coverslips or in flasks and cultured in 10% FBS-DMEM in a 37°C, 5% CO₂, humidified incubator.

Generation of Recombinant Adenoviral Vector and c-jun Infection Protocol
E1 region–deleted recombinant adenoviral vectors carrying either sense (+c-jun) or antisense (A-c-jun) c-jun cDNA were constructed. A 2.6-kb-pair fragment of full-length c-jun cDNA was then subcloned in sense or antisense orientation into the pACCMVpLpA shuttle vector to yield the sense and antisense constructs, pSR–sense-c-jun and pSR–antisense-c-jun. Both pSR–sense-c-jun and pSR–antisense-c-jun were then independently cotransfected with pJM17 into HEK-293 cells by calcium phosphate/DNA coprecipitation. For viral plaque assays, the cotransfected HEK-293 cells were overlaid with 0.65% agarose (prepared with 1x DMEM) every 3 to 4 days. The growth of these E1-deleted adenoviruses is limited to the HEK-293 cells. The polymerase chain reaction (PCR) assay was used for identification of the recombinant adenoviral vectors. The adenoviral vectors expressing sense and antisense c-jun were used to infect PASMCs.¹⁷ The cells were infected with the appropriate virus at 500 pfu/cell in DMEM containing 0.2% FBS and incubated with gentle swirling every 20 to 30 minutes for 3 hours. Cells were used 24 to 48 hours after adenoviral infection for experimentation.

Electrophysiological Measurements
Whole-cell K⁺ currents were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface by use of patch-clamp techniques.¹⁰ The extracellular (bath) solution contained (in mmol/L) NaCl 141, KCl 4.7, CaCl₂ 1.8, MgCl₂ 1.2, HEPES 10, and glucose 10 (pH 7.4). In Ca²⁺-free solution, CaCl₂ was replaced by equimolar MgCl₂, and 1 mmol/L EGTA was added. The internal (pipette) solution contained (in mmol/L) KCl 125, MgCl₂ 4, HEPES 10, EGTA 10, and Na₂ATP 5 (pH 7.2). 4-Aminopyridine (4-AP, Sigma) was directly dissolved into the solution on the day of use (pH 7.4).

Western Blot Analysis
The cell lysates were sonicated and centrifuged at 12 000 rpm for 10 minutes, and the insoluble fraction was discarded. Proteins (10 µg) were mixed and boiled in SDS-PAGE sample buffer for 5 minutes. The protein samples separated on 10% SDS-PAGE were transferred to nitrocellulose membranes. After incubation overnight at 4°C in a blocking buffer (0.1% Tween-20 in PBS) containing 5% nonfat dry milk powder, the membranes were incubated with the rabbit anti–c-Jun and anti-Kvß₂ polyclonal antibodies (Biosourse). The membranes were then washed and incubated with anti-mouse horseradish peroxidase–conjugated IgG for 90 minutes at 24°C. The bound antibody was detected with an enhanced chemiluminescence detection system (Amersham).

Single-Cell RT-PCR
Multiplex single-cell reverse transcription (RT)-PCR was performed to determine the mRNA expression of c-jun and Kv1.5 at the single-cell level.¹⁸ After I_K(V) had been recorded, the cell was carefully aspirated into a collection pipette that contained 12 µL of the pipette solution supplemented with 10 µmol/L dNTP and 0.5 U/µL RNase inhibitor. The content in the pipette was then expelled immediately into a 0.2-mL PCR tube that contained 8 µL of the solution composed of (mmol/L) Tris-HCl 10, KCl 50, MgCl₂ 2.5, dithiothreitol 10, oligo(dT) 1.25, and dNTPs 0.5, and 5 U AMV reverse transcriptase XL. RT was performed for 60 minutes at 42°C. Then, first-round PCR with 45 cycles was performed in the same tube by the addition of 80 µL of the premixed PCR buffer containing 10 mmol/L Tris-HCl, 50 mmol/L KCl, 2.5 mmol/L MgCl₂, 20 nmol/L each of sense and antisense primers (first primers) for all the genes of interest, and 5 U Taq polymerase (RNA PCR kit, Takara). Two-microliter aliquots of the first-round PCR products were reamplified by the second-round PCR with 25 to 30 cycles, which was carried out separately with fully nested gene-specific primers (nested primers) for each target gene. Second-round PCR-amplified products were separated on 1.5% agarose gel and visualized with GelStar gel staining. The cell-free samples were also used in PCR as a negative control. To semiquantify the PCR products, an invariant mRNA of ß-actin was used as an internal control. The sense and antisense primers were specifically designed from the coding regions of rat c-jun (X17215) and Kv1.5 (M27158) (Table).

View this table: [in this window] [in a new window]   Table 1. Oligonucleotide Sequences of the Primers Used for Single-Cell RT-PCR

Determination of DNA Synthesis
DNA synthesis was evaluated by [³H]thymidine incorporation. Cells were first cultured in serum-free DMEM for 24 hours and then infected with the adenoviral vector carrying +c-jun or A-c-jun for 3 hours in 0.2% FBS-DMEM. [³H]Thymidine (1 µCi/well) was added after 48 hours, and the incorporated radioactivity was determined by a liquid scintillation counter 12 hours later. The results are represented as mean counts per minute from 9 to 12 experiments. For 4-AP experiments, the cells were incubated for 20 minutes, 3 times intermittently during a period of 24 hours, in 0.2% FBS-DMEM containing 1.25 mmol/L 4-AP before [³H]thymidine was added.

Statistical Analysis
The composite data are expressed as mean±SEM. Statistical analyses were performed by use of unpaired Student’s t test or 1-way ANOVA and Fisher’s protected least significant difference (PLSD) tests where appropriate. Differences were considered to be significant at a value of P<0.05.

	Results

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Overexpression of c-Jun Decreases I_K(V) in PASMCs
The protein level of c-Jun was significantly higher in rat PASMCs infected with the adenovirus expressing +c-jun than in cells infected with control adenovirus that does not carry the c-jun gene (Cont) and cells infected with the adenovirus expressing A-c-jun (Figure 1A). Overexpression of c-Jun was associated with a significant decrease in amplitude of I_K(V) (Figure 1B). The averaged current amplitudes at -40 mV were 25±4 pA in control cells and 12±2 pA (P<0.01) in the c-jun–infected cells (Figure 1C). Infection of c-jun negligibly affected membrane capacitance (C_m) (Figure 2A) but markedly reduced current density (Figure 2C) of I_K(V). The relationships of current-density and voltage show that overexpression of c-Jun decreased the current-density of I_K(V) by 62% at +80 mV (from 56.6±7.3 to 21.3±1.8 pA/pF) (Figure 2C, inset).

View larger version (28K): [in this window] [in a new window]   Figure 1. c-Jun decreases IK(V). A, Western blot analysis of c-Jun protein (43 kDa) levels in control cells (Cont) and cells infected with +c-jun or A-c-jun. B, Representative current records elicited by test potentials from -40 to +80 mV in 20-mV increments (holding potential, -70 mV) in control cells and cells infected with +c-jun or A-c-jun. C, Current-voltage relationships (I-V curves, mean±SEM) obtained from control cells (n=54) and cells infected with +c-jun (n=63) and A-c-jun (n=32, from 6 rats). P<0.001 between Cont and +c-jun curves.

View larger version (29K): [in this window] [in a new window]   Figure 2. Effects of c-Jun on Cm and current amplitude and density of IK(V). Cm (A) and IK(V) amplitude at +80 mV (B) in control cells (Cont) and cells infected with +c-jun or A-c-jun (mean±SEM). ***P<0.001 vs Cont. C, Averaged families of current density elicited by test potentials from -40 to +80 mV (holding potential, -70 mV) in control cells (n=54) and +c-jun–infected cells (n=63, from 6 rats). Inset: Relationships of current density and voltage in control cells and +c-jun–infected cells (mean±SEM). P<0.001 between Cont and +c-jun curves.

mRNA Level of c-Jun Is Inversely Proportional to the Amplitude of I_K(V) in Single PASMCs
The level of c-Jun mRNA was much higher (a) and the amplitude of whole-cell I_K(V) (b and c) was markedly lower in a c-jun–infected cell than in a control cell (Figure 3A). Furthermore, the level of c-Jun mRNA was much lower and the amplitude of I_K(V) was much higher in a cell infected with antisense c-jun than in a cell infected with c-jun (Figure 3B). The same results were reproduced in 5 pairs of control and c-jun–infected cells.

View larger version (16K): [in this window] [in a new window]   Figure 3. Overexpression of c-Jun correlates with decrease in IK(V) in single PASMCs. A, Representative single-cell RT-PCR–amplified products in agarose gels (a) for c-Jun (262 bp) and ß-actin (244 bp), families of currents (b, elicited by test potentials from -40 to +80 mV) and corresponding current-voltage relationships (c) in control cell (Cont) and cell infected with c-jun (+c-jun). B, Representative single-cell RT-PCR–amplified products in agarose gels (a), current records (b), and corresponding current-voltage relationships (c) in a cell infected with sense c-jun and a cell infected with A-c-jun.

Overexpression of c-Jun Downregulates the mRNA Expression of the K_v Channel -Subunit
After recording of I_K(V), the PASMC was collected to determine mRNA expression of Kv1.5 by RT-PCR. As shown in Figure 4, the Kv1.5 mRNA level in a cell infected with c-jun was much lower than in a control cell, whereas the ß-actin mRNA level was similar (Figure 4A). Furthermore, in the c-jun–infected cell, the decreased mRNA expression of Kv1.5 correlated with the diminished amplitude of whole-cell I_K(V) (Figure 4B). The same results were reproduced in 5 pairs of the cells. These results suggest that c-Jun may decrease I_K(V) by affecting both the function and expression of K_v channels in PASMCs.

View larger version (28K): [in this window] [in a new window]   Figure 4. Decreased Kv1.5 mRNA expression correlates with decreased IK(V) in c-jun–infected PASMCs. Representative single-cell RT-PCR–amplified products in agarose gels (A) for Kv1.5 (196 bp) and ß-actin (244 bp), superimposed current records (B, elicited by test potentials from -40 to +80 mV), and current density-voltage relationships (C) in a control cell (Cont) and a cell infected with +c-jun.

Overexpression of c-Jun Stimulates Kvß₂ Protein Expression and Accelerates I_K(V) Inactivation
The K_v channel ß-subunit is a regulatory subunit that confers inactivation on the K_v channel -subunits (eg, Kv1.5) and blocks K_v channels as an open-channel blocker.^19–21 Therefore, an increase in ß-subunit expression should decrease I_K(V).

In cells infected with c-jun, the current inactivation was accelerated, whereas the current activation appeared to be unaffected, compared with control cells. The time constants for the current inactivation (_inact) at +80 mV were 247±34 and 125±36 ms (P<0.01) in controls cells and c-jun–infected cells, respectively (Figure 5A to 5C). Furthermore, the protein level of Kvß₂ was significantly greater in the c-jun–infected cells than in control cells (Figure 5D), suggesting that c-Jun upregulates protein expression of the K_v channel ß-subunit.

View larger version (24K): [in this window] [in a new window]   Figure 5. c-Jun accelerates IK(V) inactivation and upregulates Kvß2 expression. A, Currents (at +80 mV) averaged from 53 control cells (Cont) and 63 cells infected with (+c-jun). Normalized average currents (at +80 mV) showing current inactivation (B) and summarized data (mean±SEM) showing time constants (C) for current activation (act) and inactivation (inact) in control cells and cells infected with +c-jun. D, Western blot analysis of Kvß2 protein and summarized data (mean±SEM) showing protein levels of Kvß2 (normalized to level of -actin) in control and c-jun–infected cells. **P<0.01 vs Cont.

Overexpression of c-Jun and Blockade of K_v Channels Cause Membrane Depolarization and Stimulate PASMC Proliferation
To test the effects of c-Jun on resting E_m and cell proliferation, we compared E_m and thymidine incorporation in PASMCs infected with an empty adenovirus (Cont) and with adenoviral vectors carrying +c-jun and A-c-jun. As shown in Figure 6, [³H]thymidine incorporation was markedly increased (similar results were reproduced 3 times in cells isolated from 3 rats), and resting E_m was much depolarized in cells infected with +c-jun, compared with control cells and cells infected with -c-jun.

View larger version (21K): [in this window] [in a new window]   Figure 6. Effects of c-Jun and 4-AP on Em and PASMC proliferation. A, Summarized data (mean±SEM) showing [3H]thymidine incorporation (n=6 cell samples for each group) and resting Em (n=16 cells from 6 rats) in control cells (Cont) and cells infected with +c-jun or A-c-jun. B, Summarized data (mean±SEM) showing [3H]thymidine incorporation (n=12 cell samples for each group) and resting Em (n=20 cells from 3 rats) in control cells treated with (4-AP) or without (Cont) 4-AP (1.25 mmol/L). ***P<0.001 vs Cont.

Furthermore, pharmacological blockade of K_v channels with 4-AP (1.25 mmol/L) caused E_m depolarization and significantly increased [³H]thymidine incorporation in control cells (similar results were reproduced 3 times in cells isolated from 3 rats). These results suggest that the c-Jun–induced decrease in I_K(V) stimulates DNA synthesis in PASMCs by causing membrane depolarization and increase in [Ca²⁺]_cyt.

	Discussion

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Subsequent to the activation of immediate-early genes (eg, c-jun), the cellular signaling pathways that cause proliferation and hypertrophy of PASMCs are not well understood. In primary cultured rat PASMCs, we observed that overexpression of c-Jun reduced I_K(V) by downregulating Kv1.5 expression and upregulating Kvß₂ expression and induced membrane depolarization. It has been shown that in PASMCs, opening of VDCCs by membrane depolarization causes increased [Ca²⁺]_cyt and induces cell contraction^3,9,10 and proliferation.^4,12 In this study, the c-Jun–mediated decreases in I_K(V) and membrane depolarization were also associated with an increase in PASMC proliferation. These observations suggest that c-Jun–mediated PASMC growth may result from the regulation of K_v channel expression and function, the activity of K_v channels serving as an effector to prompt cell proliferation by modulating E_m and [Ca²⁺]_cyt.

An increase in c-Jun mRNA level was associated with thrombin-induced hypertrophy and PDGF-mediated proliferation of rat PASMCs.²² c-Jun is a transcription activator that upregulates responsive genes associated with proliferation, differentiation, and apoptosis.^16,23 c-Jun may indirectly regulate transcription of Kv1.5 gene by activating expression of an intermediate gene product that can subsequently downregulate K_v channel expression and decrease I_K(V). The augmenting effect of c-Jun on Kvß₂ expression implies that c-Jun modulates K_v channel - and ß-subunit gene expression by different mechanisms.

Native K_v channels are homomeric or heteromeric tetramers that are composed of 2 structurally distinct subunits: the pore-forming -subunits and the regulatory ß-subunits. Kv1.5 is a delayed rectifier K_v channel -subunit that has been described in PASMCs from animals and humans.²⁴ The homomeric Kv1.5 channels, which are activated at potentials ranging from -30 to -60 mV, appear to be responsible for regulating resting E_m, whereas the heteromeric Kv1.5/Kv1.2 channels are involved in mediating membrane depolarization during hypoxia in PASMCs.²⁵ The K_v channel ß-subunit has been demonstrated to block the K_v channel -subunits as an open-channel blocker,²¹ confer fast inactivation on delayed rectifier K_v channel -subunits,^19,20 and confer oxygen and redox sensitivity on K_v channel -subunits.^20,25 Therefore, the c-Jun–mediated decrease in Kv1.5 expression and increase in Kvß₂ expression would reduce the number of homomeric Kv1.5 channels and increase the number of heteromeric Kv1.5/Kvß₂ channels, thus decreasing the amplitude and current density of I_K(V).

E_m in PASMCs is regulated primarily by K⁺ permeability, which is determined by sarcolemmal K⁺ channel activity.¹¹ Under resting conditions, K⁺ permeability through K_v channels is partially responsible for determining E_m in smooth muscle cells.^10–13 Thus, E_m is directly related to the whole-cell I_K(V). PASMCs have a very large membrane input resistance (1 to 10 G)¹¹; therefore, a modest change in I_K(V) should cause a large change in E_m. Indeed, overexpression of c-Jun reduced I_K(V) by 50% to 70% (at -40 and +80 mV) and caused a 15-mV depolarization. These results indicate that the c-Jun–mediated decrease in I_K(V) is sufficient to cause substantial membrane depolarization in PASMCs.

Studies on the kinetics of L-type VDCCs and its relationship with [Ca²⁺]_cyt have demonstrated that prolonged membrane depolarization at a range of -35 to -20 mV (a voltage range at which the Ca²⁺ channel inactivation is incomplete while the channel activation begins) can open Ca²⁺ channels sufficiently to cause a sustained increase in [Ca²⁺]_cyt.¹³ Because of the minimal resistance of the nuclear membrane to Ca²⁺ ions,²⁶ the sustained elevation of [Ca²⁺]_cyt would rapidly increase nuclear [Ca²⁺]. Furthermore, a very small increase in [Ca²⁺]_cyt would also result in a large increase in [Ca²⁺] in the sarcoplasmic reticulum, a cytoplasmic organelle involved in protein processing and lipid synthesis.⁶ In the cell cycle, Ca²⁺ is necessary for transitions from the resting state (G₀) to DNA synthesis and mitosis.⁴ Thus, increases in cytosolic, nuclear, and sarcoplasmic reticulum [Ca²⁺] may all contribute to stimulate PASMCs.

It has been demonstrated that an increase in [Ca²⁺]_cyt due to Ca²⁺ influx through VDCCs spatially stimulates transcription of c-fos/c-jun by activation of cAMP response element binding protein in the cytosol and nucleus.^27,28 Overexpression of c-Jun decreased I_K(V) and depolarized PASMCs. The resultant increase in [Ca²⁺]_cyt due to opening of VDCCs should further stimulate c-fos/c-jun transcription. Therefore, this may serve as a positive-feedback mechanism in regulating Ca²⁺-sensitive genes, which are required for cell proliferation and hypertrophy.

In summary, the results from this study demonstrate that overexpression of c-Jun downregulates expression of the K_v channel -subunit (Kv1.5) and upregulates expression of the ß-subunit (Kvß₂) in PASMCs. The resultant decrease in I_K(V) causes membrane depolarization and stimulates cell proliferation by raising [Ca²⁺]_cyt. Similar increases in [Ca²⁺]_cyt and membrane depolarization due to decreased K_v channel activity in PASMCs have been implicated in hypoxic pulmonary vasoconstriction^29–31 and primary pulmonary hypertension.³² Further studies are necessary to determine whether upregulated c-jun transcription and increased c-Jun function are responsible for the inhibited expression of K_v channels in patients with primary pulmonary hypertension and the downregulation of K_v channel -subunits in chronic hypoxia.

	Acknowledgments

 
This work was supported by NIH grants HL-54043 and HL-64549. Dr Yuan is an Established Investigator of the American Heart Association.

	Footnotes

 
*The first 3 authors contributed equally to this work.

Received February 15, 2001; accepted June 28, 2001.

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