LCMP-00191-2005.R6 FINAL ACCEPTED VERSION ... - CiteSeerX

Page 1Articles of 47 in PresS. Am J Physiol Lung Cell Mol Physiol (June 30, 2006). doi:10.1152/ajplung.00191.2005

LCMP-00191-2005.R6

FINAL ACCEPTED VERSION

Bone Morphogenetic Protein-2 Upregulates Expression and Function of Voltage-gated K+ Channels in Human Pulmonary Artery Smooth Muscle Cells

Ivana Fantozzi1, Oleksandr Platoshyn1, Ada H. Wong2, Shen Zhang1, Carmelle V. Remillard1, Manohar R. Furtado2, Olga V. Petrauskene2 and Jason X.-J. Yuan1*

1

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of

California San Diego, La Jolla, CA 92093; 2Applied Biosystems, Foster City, CA 94404

Running title: BMP-2 upregulates Kv channel expression and function

* Address correspondence to: Jason X.-J. Yuan, M.D., Ph.D. Division of Pulmonary and Critical Care Medicine Department of Medicine University of California, San Diego 9500 Gilman Drive, MC 0725 La Jolla, CA 92093-0725 Phone: (858) 822-6534 Fax: (858) 822-6531 E-mail: [email protected]

Copyright © 2006 by the American Physiological Society.

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Abstract Activity of voltage-gated K+ (Kv) channels in pulmonary artery smooth muscle cells (PASMC) plays an important role in the control of apoptosis and proliferation, in addition to regulating membrane potential and pulmonary vascular tone. Bone morphogenetic proteins (BMP) inhibit proliferation and induce apoptosis in normal human PASMC, whereas dysfunctional BMP signaling and downregulated Kv channels are involved in pulmonary vascular medial hypertrophy associated with pulmonary hypertension. The goal of this study was to evaluate the effect of BMP-2 on Kv channel function and expression in normal human PASMC. BMP-2 (100 nM for 18-24 hrs) significantly (>2-fold) upregulated mRNA expression of KCNA5, KCNA7, KCNA10, KCNC3, KCNC4, KCNF1, KCNG3, KCNS1, and KCNS3, whereas it downregulated (at least 2-fold) KCNAB1, KCNA2, KCNG2 and KCNV2. The most dramatic change was the >10-fold downregulation of KCNG2 and KCNV2, two electrically silent γ subunits that form heterotetramers with functional Kv channel α subunits (e.g., KCNB12). Furthermore, the amplitude and current density of whole-cell Kv currents were significantly increased in PASMC treated with BMP-2. It has been demonstrated that K+ currents generated by KCNB1 and KCNG1 (or KCNG2) or KCNB1 and KCNV2 heterotetramers are smaller than those generated by KCNB1 homotetramers, indicating that KCNG2 and KCNV2 (two subunits that were markedly downregulated by BMP-2) are inhibitors of functional Kv channels. These results suggest that BMP-2 divergently regulates mRNA expression of various Kv channel α/β/γ subunits and significantly increases whole-cell Kv currents in human PASMC. Finally, we present evidence that attenuation of c-Myc expression by BMP-2 may be involved in BMP-2mediated increase in Kv channel activity and regulation of Kv channel expression. The increased

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Kv channel activity may be involved in the proapoptotic and/or antiproliferative effects of BMP2 on PASMC.

Key Words: pulmonary arterial hypertension; patch clamp; membrane potential; bone morphogenetic protein

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Introduction Activity of voltage-gated K+ (Kv) channels in vascular smooth muscle cells regulates the resting membrane potential and excitation-contraction coupling (57). The current generated by K+ efflux through Kv channels, IK(V), is heavily influenced by numerous vasoactive agonists that control vascular tone (57). In pulmonary arterial smooth muscle cells (PASMC) from animals and humans, downregulated Kv channel expression and reduced Kv channel function have been linked to pulmonary vasoconstriction triggered by acute hypoxia (33, 70, 79, 104) and to the sustained pulmonary vasoconstriction and severe pulmonary vascular remodeling induced by chronic hypoxia (69, 81, 88). Persistent hypoxic pulmonary vasoconstriction and hypoxiamediated pulmonary vascular medial hypertrophy increase pulmonary vascular resistance, which contribute to the development of pulmonary hypertension and subsequent right heart failure in patients with chronic obstructive pulmonary disease and congenital cardiopulmonary diseases, as well as in residents living in high altitude areas. In addition to contribution to hypoxia-mediated pulmonary hypertension, intimal and medial hypertrophy of small and medium-sized pulmonary arteries are hallmarks of the pulmonary vascular remodeling processes that underlie the development and maintenance of high pulmonary arterial pressure in patients with familial and idiopathic pulmonary arterial hypertension (83). Overgrowth of PASMC in the pulmonary vascular media is particularly important in the development of pulmonary arterial hypertension because a) sustained PASMC contraction leads to vasoconstriction while excessive PASMC growth enhances the contractile force, and b) PASMC hypertrophy and proliferation cause narrowing of the pulmonary vascular lumen, thereby increasing pulmonary vascular resistance and pulmonary arterial pressure.

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Idiopathic pulmonary arterial hypertension is pathologically or histologically characterized by severe pulmonary vascular remodeling (due to smooth muscle and endothelial cell proliferation), obliteration of small arteries, intimal fibrosis, small vessel thrombi, and sometimes formation of the plexiform lesion (76). It is generally believed that multiple genetic and environmental factors are involved in the pathogenesis of familial and idiopathic pulmonary arterial hypertension (99), such as mutations of bone morphogenetic protein (BMP) receptor type II (BMP-RII) (19, 45, 49, 58), aberrant regulation of matrix metalloproteins (48, 73), downregulation of K+ channels (98, 101, 105), upregulation of Ca2+-permeable channels (95, 97), intake of anorexigens (1, 87), increased production of serotonin and upregulated expression of serotonin receptors and transporter (4, 8, 24, 46), increased angiopoietin-1 production (21, 23, 84), elevated endothelin-1 expression and activity (31), and imbalanced cyclic AMP (5, 60) and cyclic GMP (20, 53) metabolism. A number of factors can modulate PASMC proliferation and apoptosis, including transcription factors (14, 40, 66), growth factors (47, 55, 97), and mitogenic agonists (35, 63, 89, 107). Increased elastase activity and deposition of the matrix metalloprotein tenascin-C also have been linked with the enhanced proliferation in pulmonary arterial hypertension (39, 73). In some cells, transforming growth factor-β (TGF-β) in particular has been shown to modulate the expression of transcription factors, such as c-Myc (29, 93), thereby regulating cell proliferation and/or survival. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily also regulate PASMC proliferation, apoptosis, and differentiation (36, 51, 52, 55, 56, 92) via autocrine and paracrine mechanisms. Studies have shown that BMP-2, BMP-4, and BMP-7 can increase the rate of apoptosis and decrease the rate of proliferation of vascular smooth muscle cells (56, 92), including human PASMC (55, 106). In PASMC from patients diagnosed with

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idiopathic pulmonary arterial hypertension, BMP (BMP-2 and BMP-7)-induced apoptosis was significantly impaired (55, 106). BMP-2-mediated apoptosis was associated with transient phosphorylation of Smad1, a protein signaling element activated upon binding of BMPs to their type I and II (BMP-RI and -RII) receptors (36, 52), and with marked downregulation of Bcl-2, an antiapoptotic protein (106). In an earlier study, Bcl-2 was shown to enhance cell survival or inhibit cell apoptosis by, at least in part, decreasing Kv channel activity and downregulating Kv channel α subunit expression in rat PASMC (25). This supports the mounting evidence that enhanced K+ activity is essential to the onset and progression of PASMC apoptosis (74). The goal of the current study was to test the hypothesis that BMP-2 increases whole-cell IK(V) by divergently regulating mRNA expression of various Kv channel α/β/γ subunits in human PASMC. In addition, we also examined the effect of BMP-2 on the protein expression of c-Myc, a transcription factor that is upregulated in proliferating cells and downregulated by TGF-β, and explored the possibility that BMP-2 regulates Kv channel expression and promotes cell apoptosis by modulating c-Myc expression.

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MATERIALS AND METHODS Cell culture and preparation. Human PASMC (passage 4-7) from normal subjects (Cambrex, Walkersville, MD) were derived from pulmonary arteries of three individuals. PASMC were cryopreserved at passage 3, replated onto flasks to amplify cell number for 2-3 passages, and then used for the experiments for 2-3 passages (i.e., at passages 5-8). Cells were seeded and cultured in smooth muscle grown medium (SMGM, Cambrex) in a humidified atmosphere of 5% CO2-95% air at 37°C. SMGM was composed of smooth muscle basal medium (SMBM) supplemented with 5% fetal bovine serum (FBS), 0.5 ng/ml human epidermal growth factor (hEGF), 2 ng/ml human fibroblast growth factor (hFGF), and 5 µg/ml insulin. Proliferating (cultured in SMGM) and growth-arrested (cultured in SMBM) cells were treated with BMP-2 for 18 or 24 hrs prior to experimentation. Identification of the cells in culture as smooth muscle cells was verified using the smooth muscle α-actin monoclonal antibody and the nuclear acid stain, 4’, 6’-diamidino-2-phenylindole (DAPI, 5 µM). The DAPI-stained cells also crossreacted with the smooth muscle cell α-actin antibody, indicating that the cultures were all smooth muscle cells.

Measurement of macroscopic IK(V). Whole-cell IK(V) were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments) using conventional voltage-clamp techniques. PASMC plated onto glass cover slips were superfused in a perfusion chamber placed on the microscope stage with Ca2+-free bath solution containing (in mM): 141 NaCl, 4.7 KCl, 3 MgCl2, 1 EGTA, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with 2 M NaOH). Whole-cell K+ currents were recorded using a pipette solution containing (in mM): 5 Na2ATP, 135 KCl, 4 MgCl2, 10 EGTA, and 10 HEPES (pH adjusted to 7.2 with 2 M KOH). When cells were superfused with the Ca2+-free bath solution and dialyzed with the 5 mM ATP- and 10 mM

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EGTA-containing (Ca2+-free) pipette solution, the contribution of outward K+ currents through Ca2+-activated K+ channels and ATP-sensitive K+ channels to the recorded whole-cell K+ currents was significantly minimized. It has been demonstrated that 1-3 mM ATP markedly blocked ATP-sensitive K+ channels in PASMC (16), while removal or chelation of extracellular and intracellular Ca2+ with high concentration of EGTA significantly reduced Ca2+-activated K+ channel activity (9, 100, 102, 103). Series resistance compensation was performed in all whole-cell recording experiments. Step-pulse protocols and data acquisition were performed using pCLAMP software (Axon Instruments). Leak and capacitative currents were subtracted using the P/4 protocol in pCLAMP software. Currents were filtered at 1-2 kHz (-3 dB) and digitized at 2-4 kHz. Whole-cell IK(V) were recorded during 300-ms voltage steps ranging between –60 and +80 mV (in 20 mV increments) from a holding potential of –70 mV. All experiments were performed at room temperature (22-24°C). Current-voltage (I-V) relationship curves report both current amplitude (in pA) and current density (normalized to cell capacitance; pA/pF).

Real-time RT-PCR. Total RNA was extracted from human PASMC using an RNeasy Mini Kit (Qiagen) according to previously published methods (106). Sense and antisense primers for the real-time RT-PCR experiments were specifically designed by scientists at Applied Biosystems (Foster City, CA) from the coding regions of each Kv channel gene; the fidelity and specificity of the sense and antisense primers were examined using a BLAST program. Due to proprietary issues and the policy of Applied Biosystems, the exact primer sequences used for the real-time RT-PCR experiments are not provided but can be requested from the company based on the

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information (e.g., Assay ID, the reference sequence, and the exon boundaries used to design the primers) shown in Table 1. Reverse transcription (RT) was performed using the High Capacity cDNA Archive kit (Applied Biosystems) that uses random primers. PCR amplification with real-time detection was performed using the TaqMan® Universal PCR Master Mix with AmpErase® (uracil Nglycosylase) and total RNA of 100 ng/µL. Initial PCR reaction mixtures were prepared in 96 well plates, then 10 µl aliquots of the reactions were distributed to each of 4 quadrants on 384 well plates using an automated method on a Biomek FX robotic workstation. Real-time PCR was performed using an ABI PRISM® 7900HT Sequence Detection System. Thermal cycling conditions comprised an initial UNG incubation at 50°C for 2 min, AmpliTaq Gold® DNA Polymerase activation at 95°C for 10 min, 40 cycles of denaturing at 95°C for 15 sec, and annealing and extension at 60°C for 1 min. Real-time PCR assays were designed using Applied Biosystems’ bioinformatics design pipeline. These assays were all part of the Applied Biosystems TaqMan AssayTM or Custom TaqMan AssayTM product lines. Fold change (FC) calculation was performed using the comparative Ct or the ∆∆Ct method, based on the formula FC = 2–∆∆Ct, to calculate normalized fold changes in gene expression in test samples relative to a calibrator sample (i.e., treated samples relative to untreated samples) (30). The first step in the FC analysis is normalization of target gene expression signal to endogenous control expression (∆Ct). In our experiments, both 18S and GAPDH were used as housekeeping genes. However, only 18S behaved consistently between samples. Therefore, it was used as the endogenous control signal. The second step is to calculate the difference between normalized target-gene expressions in BMP-2 treated and untreated

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samples (∆∆Ct). FC calculation, which represents the difference of gene expression levels between BMP-2 treated samples and control (or untreated) samples, was carried out for each gene individually using the formula mentioned above. Each measurement was repeated in three samples from different cell cultures. A limitation of the study is that only one apparently reliable housekeeping gene (18S) was used for data analysis, but despite this the data do show that there are clear changes in the relative abundance of Kv channel mRNAs.

Bioinformatics Design of the Real-Time PCR Assays. Applied Biosystems developed a bioinformatics assay design pipeline that consists of two parts: an assay design engine and an assay evaluation tool.

The assay design engine selects primers and probes based on

thermodynamic parameters (Tm, nucleotide composition, self-complementarity, etc.) to ensure 100% efficiency of the assays. The assay evaluation component of the pipeline scores the potential hybridization of the assay to closely related genes to produce high specificity assays. To assess cross-hybridization, the concept of forbidden targets (anti-targets) and a target sequence table were implemented. The anti-targets represent all sequences that should not be detected by an assay. The target sequence table groups redundant sequences for the same target. Validation of more than a thousand assays has demonstrated that the design pipeline generates primers/probe sets that perform with: i) near 100% PCR amplification efficiency, and ii) a high degree of specificity for the selective amplification of a targeted gene in the presence of closely related targets (30).

Regular RT-PCR. Total RNA was isolated from human PASMC cultured in SMBM (for 24 hrs) using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). cDNA was synthesized using

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SuperScriptJ reverse transcriptase (Invitrogen, Carlsbad, CA). RT-PCR was performed by a GeneAmp PCR System (Perkin Elmer, Boston, MA) using a Platinum PCR Supermix (Gibco). The sequences of sense and antisense primers (Table 2) for the regular RT-PCR experiments were specifically designed from the coding regions of K+ channel α and β subunit genes. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal control to semiquantify the PCR products and to normalize the PCR products of Kv channel subunits. Amplified PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining. Results presented are representative from experiments performed in three different PASMC cultures treated or not with BMP-2.

Western blot analysis. Human PASMC were first cultured in media containing PDGF for 6 hrs to stimulate proliferation. Protein expression of c-Myc was evaluated under control conditions and after treatment with BMP-2 for 1-6 hrs. Human PASMC were gently washed twice in cold PBS, scraped into lysis buffer (1% Nonidet P-4 (Amaresco), 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, and 30 µl/l aprotinin), and incubated on ice for 30 min. Cell lysates were then sonicated and centrifuged at 12,000 rpm for 10 min, and the insoluble fraction was discarded. In some experiments, cell lysates were treated with the peptide N-glycosidase F (20 U; New England Biolabs) overnight at 4ºC. The protein concentration in the supernatant was determined by the bicinchoninic acid protein assay using bovine serum albumin as a standard. Ten to twenty five microliter aliquots of protein were mixed and boiled in SDSPAGE sample buffer for 5 min. The protein samples separated on 10% SDS-PAGE were then transferred to nitrocellulose membranes by electroblotting in a MINI Trans-Blot cell transfer apparatus according to the manufacturer’s instruction (Bio-Rad Laboratories). After incubation

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overnight at 4ºC in a blocking buffer (0.1% Tween 20 in PBS) containing 5% nonfat dry milk powder, the membranes were incubated with polyclonal antibodies against c-Myc (Santa Cruz Biotechnology, Inc.) and α-actin (Sigma). Finally, the membranes were washed and incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG for 90 min at room temperature. The bound antibody was detected with an enhanced chemiluminescence detection system (Amersham). Band intensity is reported in arbitrary units for both c-Myc and α-actin, as well as the intensity ratio (c-Myc/α-actin). To compare protein expression levels of c-Myc and α-actin, the intensity of light transmission through the blot along a line drawn vertically across the c-Myc and α-actin bands was determined using the Image-Pro Plus analysis software. The intensity of c-Myc band was then divided by the intensity of α-actin band to produce the normalized intensity ratio for comparing relative changes of c-Myc protein expression under control conditions and during treatment with BMP-2.

Chemicals. Chemicals for electrophysiological measurements were purchased from Sigma unless indicated otherwise. 4-aminopyridine (4-AP, Sigma) and charybdotoxin (ChTX, Sigma) were directly dissolved in the bath solution on the day of use; pH value of the solution was measured after addition of the drugs and re-adjusted to 7.4 when necessary. Recombinant human BMP-2 (R&D Systems) was prepared in a stock solution in sterile phosphate-buffered saline (with 0.1% BSA); aliquots of the stock solution were then diluted into the appropriate culture media (SMGM or SMBM) on the day of use to the final concentration.

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Statistical Analysis. Data are expressed as means ±SE. Statistical analysis was performed using paired Students’ t-test or analysis of variance. Differences were considered to be significant when P