The Amiloride Derivative Phenamil Attenuates Pulmonary Vascular ...

MOLECULAR AND CELLULAR BIOLOGY, Feb. 2011, p. 517–530 0270-7306/11/$12.00 doi:10.1128/MCB.00884-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 31, No. 3

The Amiloride Derivative Phenamil Attenuates Pulmonary Vascular Remodeling by Activating NFAT and the Bone Morphogenetic Protein Signaling Pathway䌤 Mun Chun Chan,1 Alexandra S. Weisman,1 Hara Kang,1 Peter H. Nguyen,1 Tyler Hickman,2 Samantha V. Mecker,2 Nicholas S. Hill,3 Giorgio Lagna,1 and Akiko Hata1,2* Molecular Cardiology Research Institute1 and Pulmonary, Critical Care, and Sleep Division,3 Tufts Medical Center, Boston, Massachusetts 02111, and Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 021112 Received 29 July 2010/Returned for modification 16 September 2010/Accepted 22 November 2010

Pulmonary artery hypertension (PAH) is characterized by elevated pulmonary artery resistance and increased medial thickness due to deregulation of vascular remodeling. Inactivating mutations of the BMPRII gene, which encodes a receptor for bone morphogenetic proteins (BMPs), are identified in ⬃60% of familial PAH (FPAH) and ⬃30% of idiopathic PAH (IPAH) patients. It has been hypothesized that constitutive reduction in BMP signal by BMPRII mutations may cause abnormal vascular remodeling by promoting dedifferentiation of vascular smooth muscle cells (vSMCs). Here, we demonstrate that infusion of the amiloride analog phenamil during chronic-hypoxia treatment in rat attenuates development of PAH and vascular remodeling. Phenamil induces Tribbles homolog 3 (Trb3), a positive modulator of the BMP pathway that acts by stabilizing the Smad family signal transducers. Through induction of Trb3, phenamil promotes the differentiated, contractile vSMC phenotype characterized by elevated expression of contractile genes and reduced cell growth and migration. Phenamil activates the Trb3 gene transcription via activation of the calciumcalcineurin-nuclear factor of activated T cell (NFAT) pathway. These results indicate that constitutive elevation of Trb3 by phenamil is a potential therapy for IPAH and FPAH. the monocrotaline (MCT) model (37) and chronic hypoxia (5). Together, these results suggest that inhibition of TGF-␤ or BMP signaling plays an important role in the pathogenesis of proliferative and obliterative vascular diseases. Phenamil, along with benzamil, dimethyl amiloride (DMA), and ethyl isopropyl amiloride (EIPA), are derivatives of the diuretic amiloride, and inhibitors of ion transporters. It has been demonstrated that dimethyl amiloride, a potent and specific inhibitor of the Na⫹/H⫹ transporter, can inhibit pulmonary artery smooth muscle cell (PASMC) proliferation in vitro and chronic-hypoxia-induced pulmonary hypertension in rats (35, 36). Unlike DMA, phenamil, benzamil, and amiloride are potent blockers of Na⫹ channels, including epithelial sodium channels (ENaCs) and acid-sensing ion channels (ASICs). vSMCs express low levels of ENaCs, but ASIC (acid-sensing ion channel protein 1 [ASIC1] to ASIC3) expression and function has been demonstrated (14). ASICs are essential for vSMC migration both at basal and PDGF-stimulated conditions (14). ASIC1 protein level is also elevated in pulmonary arteries under chronic-hypoxia treatment (17). The nuclear factor of activated T cell (NFAT) family of transcription factors, which includes five NFAT proteins (NFAT1 to NFAT5) and their splice variants, are known to regulate a range of genes in response to increases in the intracellular free calcium levels. NFATs were originally identified as transcription regulators in lymphoid cells, but it has since been shown that they play a critical role in a variety of cells, including vSMCs (21). At steady state, NFATs are phosphorylated, excluded from the nucleus, and thus inactive. Upon increase of the intracellular calcium concentration, calcineurin, a calmodulin-dependent phosphatase, dephosphorylates NFAT1

In response to vascular injury, vascular smooth muscle cells (vSMCs) undergo a unique process known as “phenotype modulation,” the transition from a quiescent, “contractile” phenotype to a proliferative, “synthetic” state (31, 32). Phenotypic plasticity is essential for vascular development and vascular repair after injury. However, aberrant switching from the contractile to synthetic phenotype, characterized by increased vSMC proliferation, increased migration, and decreased ability to contract, underlies the formation of various proliferative vascular disorders, including atherosclerosis, postangioplasty restenosis, lymphangioleiomyomatosis (LAM), and pulmonary artery hypertension (PAH) (40, 45). The transforming growth factor ␤ (TGF-␤) and bone morphogenetic protein (BMP) pathways play essential roles in cardiac myogenesis (24, 39), vasculogenesis (25), and angiogenesis. In vSMCs, TGF-␤ and BMP have been shown to promote the contractile phenotype and inhibit switching to the synthetic phenotype (20). Both TGF-␤s and BMPs inhibit vSMC proliferation and migration and increase contractile vSMC gene expression (16, 20, 22). Loss-of-function mutations of the genes encoding receptors of TGF-␤s and BMPs have been linked to vascular disorders, such as idiopathic PAH (IPAH) and hereditary hemorrhagic telangiectasia (41). Furthermore, BMP and TGF-␤ signaling is reduced when phenotype switching is induced both in cell culture by platelet-derived growth factor (PDGF) stimulation (5) or in vivo by using * Corresponding author. Mailing address: Molecular Cardiology Research Institute, Tufts Medical Center, 800 Washington Street, Box 8486, Boston, MA 02111. Phone: (617) 636-0614. Fax: (617) 636-5649. E-mail: [email protected]. 䌤 Published ahead of print on 6 December 2010. 517

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to NFAT4 proteins, which promotes nuclear translocation and association with DNA (21). A recent study suggested that phenamil facilitates BMP2induced osteoblastic differentiation and mineralization through increased expression of Tribbles 3 (Trb3) protein in the mesenchymal stem cell line M2-10B4 cells (33). We previously identified Trb3 as a potent and crucial positive modulator of phenotype switch in vSMCs. Increased Trb3 was shown to promote the degradation of Smad ubiquitin regulatory factor 1 (Smurf1), a negative regulator of BMP Smad-dependent signaling (6). In comparison, the prosynthetic PDGF cytokine promotes downregulation of Trb3, which results in decreased Smad protein levels and vSMC contractile gene expression (5, 6). The pro-BMP signaling and thus procontractile function of Trb3 in vSMCs is inhibited when BMP type II receptor (BMPRII) is mutated in the same manner as in some IPAH patients. Furthermore, PASMCs from rats subjected to chronic hypoxia show decreased Trb3 protein expression and corresponding decreased BMP signaling. These results suggest that control of Trb3 expression and activity plays an important role in the careful control of the switch between the synthetic and contractile phenotypes. In this study, we are interested in determining whether a drug-induced increase in Trb3 expression can be used as a therapeutic strategy in models of PAH. We observed that phenamil induces Trb3 expression and attenuates hypoxia-induced PAH and vascular remodeling in rats. We demonstrate that in cultured pulmonary vSMCs, phenamil promotes transcriptional activation of Trb3 by activating the calcineurin-NFAT pathway through elevation of intracellular calcium concentration. Phenamil facilitates the procontractile effect of BMP signaling and the maintenance of a contractile phenotype. These data establish an ameliorating effect of elevated BMP signal and maintenance of the contractile phenotype in vSMCs during vascular remodeling via induction of Trb3. MATERIALS AND METHODS Cell culture. Human primary pulmonary artery smooth muscle cells (PASMCs) were purchased from Lonza (CC-2581) and were maintained in Sm-GM2 medium (Lonza) containing 5% fetal bovine serum (FBS). Early passage (passage 4 to 7) PASMCs were used for this study. PAC1 cells were purchased from ATCC and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) (Sigma). Aortic vascular smooth muscle Ao184 cells were obtained from M. E. Mendelsohn and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS (Sigma) as previously described (40a). Recombinant human BMP4 was purchased from R&D Systems. Phenamil, benzamil, amiloride, and cyclosporine (CsA) were purchased from Sigma and solubilized in dimethyl sulfoxide (DMSO). The cells were treated under starvation conditions (0.2% FBS) as described previously (20). Real-time reverse transcription-PCR (RT-PCR) and primers. Quantitative analysis of the change in expression levels was calculated by a real-time PCR machine (iQ5; Bio-Rad) as described previously (5, 6). Sequences of real-time PCR primers for human smooth muscle ␣-actin (SMA), calponin (CNN), SM22, rat SMA and CNN, human Trb3, mouse Trb3, and rat Trb3 were previously described (5, 6). The sequences of other primers are available on request. ChIP assay. The chromatin immunoprecipitation (ChIP) assay was performed as described previously (28). Briefly, soluble chromatin was prepared from Ao184 cells following cross-linking reaction with 5 mM dimethyl adipimidate and 1% formaldehyde. Chromatin was then incubated with either 10 ␮g anti-NFAT2 (also known as NFATc1) antibody (MA3-024; Thermo Scientific) or rabbit nonspecific IgGs as a negative control. PCR primers specific for Trb3 phenamil response element (PRE) are available on request, while the nonspecific SMA gene promoter was described previously (28).

MOL. CELL. BIOL. RNA interference. Synthetic small interfering RNA (siRNA) targeting human Trb3 or mouse Trb3 were synthesized at Dharmacon, and the sequences were previously described (6). siRNA targeting ASIC1 (si-ASIC1) (S909), si-ASIC2 (S908), si-ASIC3 (S11784), and si-ENaC␤ (S12546) were purchased from Ambion. A siRNA with a nontargeting sequence (scrambled siRNA; Dharmacon) was used as a negative control. The siRNAs were transfected at 50 nM using RNAi Max (Invitrogen) according to the manufacturer’s directions. Fluorescein isothiocyanate (FITC)-conjugated fluorescent oligonucleotides (Block-it,; Invitrogen) were used to evaluate transfection efficiency. Plasmid DNA transfection and cDNA expression constructs. Trb3 expression plasmid, BRE-Luc, and p1.9-luc (Luc and luc stand for luciferase) were previously described (6). p1.4-luc, p1.0-luc, p0.9-luc, and p0.8-luc were subcloned from p1.9-luc into empty pGL3 basic vector. Primer sequences are available on request. PRE(WT)-luc, PRE(Mut1)-luc, PRE(Mut2)-luc, and PRE(Mut3)-luc were subcloned into empty pGL3 basic vector using synthesized PCR primer duplexes. Primer sequences are available on request. Plasmid GFP-VIVIT (GFP stands for green fluorescent protein) was obtained from Addgene (plasmid 11106) (3). Immunoblotting. Cells were lysed in TNE buffer (5), and total cell lysates were separated on SDS-polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore), immunoblotted with antibodies, and visualized using an enhanced chemiluminescence detection system (Amersham Biosciences). Protein bands were quantitated by using the imaging analysis software program ImageJ (rsbweb.nih.gov/ij/). The antibodies used for immunoblotting are as follows: anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (clone 2E3-2E10; Abnova), anti-smooth muscle ␣-actin (SMA clone 1A4; Sigma) and anti-calponin 1 (catalog no. C2687; Sigma). Anti-human and rat Trb3 antibodies were previously described (6). Immunofluorescence staining. Cells were fixed and permeabilized in a 50% acetone–50% methanol solution and subjected to staining using anti-SMA antibody (clone 1A4; Sigma) conjugated with FITC and anti-human Trb3 (antihTrb3) conjugated with Cy3 and nuclear staining with 4⬘-6-diamidino-2-phenylindole (DAPI) (Invitrogen). Luciferase reporter assay. Luciferase reporter construct was transfected together with ␤-galactosidase (␤-gal) plasmid as an internal transfection control. Luciferase assays were carried out as described previously (20). Collagen matrix contraction assay. The collagen matrix contraction assay was performed as described previously (28; also unpublished data). Wound healing assay. PASMCs were plated into the wells of 12-well plates (12,000 cells/well) and stimulated with the BMP and/or phenamil. A single scratch wound was generated with a 200-␮l disposable pipette tip. Scratch wounds were photographed over 24 h with a Nikon inverted microscope, and the width of the wound was quantitated with the ImageJ software. Ca2ⴙ depletion/repletion assay. The calcium depletion/repletion assay was performed as described previously (17). Briefly, PAC1 cells were plated onto a 10-cm plate and stimulated with different concentrations of phenamil for 24 h. The cells were trypsinized and resolubilized in Ca2⫹-free physiological saline solution (PSS) (17) with 3 mM EGTA to chelate residual Ca2⫹, 50 ␮M diltiazem to inhibit L-type voltage gated Ca2⫹ channels, and the sarcoplasmic/endoplasmic reticulum Ca2⫹-ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) (10 ␮M). The cells were then loaded with Ca2⫹-sensitive fluorescent indicator fura-2 AM (2 ␮M) (Molecular Probes), excess dye was washed off, and the cells were resolubilized in PSS containing diltiazem and CPA at 1 ⫻ 106 cells/ml. Three hundred microliters of resolubilized cells was used for each experiment. Changes in the fura-2 emission ratios (ratio of fur-2 emission when fura-2 was excited at 340 and 380 nm [F340/F380]) were then determined upon repletion of extracellular Ca2⫹ (2.5 mM). After a second depletion with EGTA, the maximum Ca2⫹ load was determined by the addition of the digitonin detergent to permeabilize the cells and repletion with excess Ca2⫹ (10 mM). The response was calculated as the relative change in the fura-2 emission ratios (F340/F380) from baseline divided by the change in the maximum Ca2⫹ load after digitonin treatment from baseline. Each experimental condition was tested in three replicate experiments, and the average was calculated and graphed. Animal model. All animal experiments were performed in accordance with the guidelines and regulations of the Institutional Animal Care and Use Committee at Tufts Medical Center. Miniature osmotic infusion pumps (model 2004; Alzet, Palo Alto, CA) which deliver saline-DMSO (50% saline–50% DMSO) solution or phenamil in saline-DMSO were implanted subcutaneously in male SpragueDawley rats (age, 6 to 7 weeks; weight, 250 to 300 g) between the scapulae and connected via a catheter advanced into the left carotid artery. Four rats were used for each condition. Phenamil (15 or 30 mg/kg of body weight/day) was administered continuously for 21 days at an infusion rate of 1 ml/h. The rats were

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subjected to hypobaric hypoxia (10.5% oxygen) or normoxia (room air) for 21 days as described previously (34). Assessment of RV hypertrophy. At the end of the hypoxia treatment, the rats were weighed and anesthetized under normoxic conditions with ketamine (60 mg/kg given intramuscularly [i.m.]) and pentobarbital sodium (20 mg/kg given intraperitoneally [i.p.]). The trachea was cannulated with a blunt 20-gauge needle, and the lungs were ventilated with room air with an inspiratory pressure of 9 cm H2O and positive-end expiratory pressure of 2 cm H2O. The right ventricular peak pressure (RVPP) was measured by inserting a 26-gauge needle with 12 in. of P-50 tubing connected to a pressure transducer into the right ventricle (RV) as described previously (34). Continuous measurement of RV pressure was recorded on a polygraph, and RVPP was measured as the highest pressure recording over a 1- to 3-min period during which pressure recordings remained stable. After completion of hemodynamic measurements, blood was collected from the inferior vena cava for determination of hematocrit. Rats were sacrificed, and the heart was dissected into right and left atria, RV and left ventricle (LV) free walls, and the interventricular septum. Each section of the heart was blotted dry on sterile gauze and weighed. The left lung was frozen in liquid nitrogen and stored at ⫺80°C for RNA and protein measurements. Double fixation of the right lung was achieved in the distended state by infusion of 4% aqueous buffered formalin into the trachea at a H2O pressure of 25 cm and into the pulmonary trunk at a H2O pressure of 5 cm. The right lung preserved in 10% buffered formaldehyde was embedded in paraffin for analysis of lung histology. Lung morphometry. For paraffin embedding, the entire lung was dissected in tissue blocks from all lobes. Sectioning at 3 mm was performed from all paraffinembedded blocks. Hematoxylin-and-eosin (H&E) staining was performed according to common histological procedures. For pulmonary vascular morphometry, the thickness of the walls of distal pulmonary vessels (outside diameter, 10 to 50 ␮m) accompanying terminal bronchioles was measured with an image analysis program (NIH ImageJ). Vessels smaller than 10 ␮m were considered capillaries and excluded from further consideration. At least 18 vessels of comparable size per rat were measured from 4 rats per group. For SMA immunostaining, paraffin sections were deparaffinated and quenched for endogenous peroxidase activity. The sections were incubated with anti-SMA antibody and then with biotinylated secondary antibody. After incubation with horseradish peroxidase (HRP)-streptavidin, visualization was performed with diaminobenzidine (DAB) chromogen. The SMA/area ratio was determined by quantification of positive signals performed using Adobe PhotoShop. Five evenly stained areas of the same size were selected for each image. Anti-SMA antibody signal intensities were expressed in pixel numbers using a color range that represents positive staining and averaged. Statistical analysis. The results presented are averages of at least three experiments each performed in triplicate with standard errors. Statistical analyses were performed by analysis of variance, followed by Tukey’s multiple-comparison test or by Student’s t test as appropriate, using Prism 4 (GraphPAD Software Inc.). P values of ⬍0.05 were considered significant and are indicated with asterisks.

RESULTS Phenamil attenuates hypoxia-induced pulmonary hypertension and vascular remodeling. It has been demonstrated that cation channels, such as acid-sensing ion channels (ASICs), and transient receptor potentials (TRPs) are highly expressed in vascular smooth muscle cells (vSMCs) and promote vSMC contractility, proliferation, and migration in vitro (8, 12, 14, 30). Phenamil is a selective inhibitor of TRPs and ASICs and more potently inhibits these channels than other amiloride derivatives, such as dimethyl amiloride (DMA), benzamil, ethyl isopropyl amiloride (EIPA), and amiloride (8). We hypothesized that infusion of phenamil during chronic-hypoxia treatment in rats might effectively prevent remodeling of pulmonary arteries by inhibiting proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and inhibit elevation of pulmonary artery resistance. We infused phenamil (15 or 30 mg/ kg/day) or DMSO (control) into male Sprague-Dawley rats during 21 days of hypoxia (10% oxygen) or normoxia (room air) treatment (4 rats per condition). After 21 days of hypoxia

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treatment, the ratio of the weight of the right ventricule to the weight of the left ventricule weight plus the weight of the septum (all these weights are dry weights) (RV/LV⫹S), right ventricular peak pressure (RVPP), and hematocrit were measured. No sign of abnormality in body weight, food intake, or other behaviors was observed in phenamil-infused rats (unpublished data). As expected, significant ventricular hypertrophy developed in response to hypoxia, as indicated by an increase in the RV/LV⫹S ratio (24% ⫾ 1% for the normoxic control versus 40% ⫾ 2% for the hypoxic control) (Fig. 1A). Interestingly, the RV/LV⫹S ratio in phenamil-infused hypoxia-treated rats was significantly reduced in comparison with control rats (27% ⫾ 2% for hypoxia with phenamil versus 40% ⫾ 2% for the hypoxic control), which is similar to the ratio for control rats in normoxia (24% ⫾ 1%) (Fig. 1A). Infusion of phenamil did not alter the RV/LV⫹S ratio under normoxic conditions (24% ⫾ 4%for normoxia with phenamil versus 24% ⫾ 1% for the normoxic control) (Fig. 1A), suggesting that phenamil has little effect on pulmonary vasculature under physiological conditions. Consistent with the RV/LV⫹S ratio, hypoxia-induced elevation of RVPP was also significantly reduced in phenamilinfused rats (34 ⫾ 2.7 mm Hg for hypoxia with phenamil versus 41.5 ⫾ 4.96 mm Hg for the hypoxic control) (Fig. 1A). The hematocrit was also significantly increased after hypoxia treatment (55% ⫾ 1% for the normoxic control versus 42% ⫾ 1% for the hypoxic control), but it was reduced in phenamil-infused rats (47% ⫾ 1% for hypoxia with phenamil versus 55% ⫾ 1% for the hypoxic control) (Fig. 1A). Altogether, these results demonstrate that phenamil reduces chronic-hypoxia-induced pulmonary artery hypertension (PAH). To examine the potential effect of phenamil on pulmonary vascular remodeling induced by hypoxia, the thickness of the pulmonary artery (PA) wall was examined by measuring medial areas in rats treated with hypoxia or normoxia. Hypoxiatreated control rats exhibited 70% increase in the medial area under hypoxia, indicating that hypoxia mediates the increase in medial thickness, which is characteristic of PAs in human PAH patients (Fig. 1B). In contrast, in rats infused with a high concentration of phenamil under hypoxia treatment, the medial area was markedly reduced to levels not significantly different from the levels in samples from normoxia-treated rats (Fig. 1B). This result demonstrates that infusion of phenamil during hypoxia treatment is able to inhibit pulmonary vascular remodeling. The phenotype switch of vSMCs from a differentiated contractile phenotype to a dedifferentiated synthetic phenotype is associated with vascular remodeling, as synthetic vSMCs are highly proliferative and migratory. Phenotype switch can be monitored by measuring the level of expression of vSMC-specific contractile genes. Immunohistochemistry of the contractile gene marker smooth muscle ␣-actin (SMA) in PAs, followed by quantitation, indicates that hypoxia leads to a reduction in the intensity of SMA stain (SMA staining/medial area) as previously reported (5). However, infusion of phenamil rescues SMA intensity (Fig. 1C). This suggests that phenamil might promote maintenance of vSMC contractile gene expression and vSMC contractile phenotype. Therefore, phenamil inhibits the hypoxia-induced switch to the synthetic phenotype, including inhibiting increased cell proliferation and migration, which ultimately leads to thickening of the PA wall. In response to chronic hypoxia, mRNA expression of the

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FIG. 1. Phenamil reduces hypoxia-induced pulmonary hypertension and vascular remodeling. (A) Rats were subjected to 21 days of normoxia or hypoxia treatment with continuous infusion of dimethyl sulfoxide (DMSO) (control) or low or high doses of phenamil (15 and 30 mg/kg/day). The weights of isolated right ventricle, left ventricle, and septum were measured, and the RV/LV⫹S ratio (the ratio of the weight of the right ventricule to the weight of the left ventricule weight plus the weight of the septum [all weights are dry weights]) (left), right ventricular peak pressure (RVPP) (right), and hematocrit level (middle) are presented. Values are shown as means plus standard errors (error bars) (n ⫽ 4 rats per condition). Values that are significantly different (P ⬍ 0.05) are indicated by the bars and an asterisk. (B) Representative images of pulmonary vascular remodeling under normoxia or hypoxia are shown on the right. The rats were infused with DMSO (control) or phenamil (30 mg/kg/day). Bars, 20 ␮m. The thickness of the medial wall of pulmonary arteries was quantitated based on microscopy images of hematoxylin-and-eosin (H&E)-stained lung sections using ImageJ software and is shown on the left. The values represent averages of 18 pulmonary arterial medial areas per rat. *, P ⬍ 0.05. (C) Immunohistochemical analysis of smooth muscle ␣-actin (SMA) expression in pulmonary arteries of hypoxic or normoxic rats infused with DMSO or phenamil (30 mg/kg/day). Immunohistochemical microscopy images were used to quantitate SMA intensity by calculating total positive staining of SMA in media divided by medial wall areas (left panel). *, P ⬍ 0.05. (D) Levels of SMA, SM22, Id3, and Trb3 mRNAs in lung samples of rats infused with DMSO or phenamil (30 mg/kg/day) under hypoxia or normoxia were measured by qRT-PCR and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *, P ⬍ 0.05.

contractile gene markers SMA and SM22, as well as Trb3 were dramatically decreased as previously reported (Fig. 1D, Trb3) (5). Trb3 was characterized as a protein that stabilizes the bone morphogenetic protein (BMP)-specific signal transducers Smad1/5/8 and facilitates BMP-Smad signaling (5, 6). Consistent with the decrease in Trb3, a target of the BMP-Smad signaling Id3 mRNA was reduced after hypoxia treatment (Fig. 1D, Id3). In phenamil-infused rats, however, contractile gene expression was elevated to about 80% (SMA) or 50% (SM22) of the levels in control normoxic rats (Fig. 1D, right), indicat-

ing that phenamil is able to partially inhibit the hypoxia-induced downregulation of contractile genes. In lungs from normoxic rats, phenamil induced Trb3 mRNA (⬃3-fold) (Fig. 1D, Trb3), similar to previous reports in mouse marrow stromal cells (33). In phenamil-infused rats under hypoxia, the levels of Trb3 and Id3 from control rats under hypoxia are restored to levels similar to those of control rats under normoxia (Fig. 1D). These results suggest that in vivo, phenamil is able to elevate Trb3 expression, facilitate BMP-Smad signaling, and promote contractile phenotype in a pathological condition. In conclu-

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sion, this animal study indicates that phenamil attenuates pulmonary vascular remodeling and elevation of PA resistance, presumably through promoting the contractile phenotype in vSMCs. Phenamil facilitates various activities of BMP signaling. It has been shown that in pulmonary vSMCs, the BMP signaling pathway is critical for maintenance of pulmonary vascular homeostasis by promoting and/or maintaining the contractile phenotype characterized by decreases in proliferation and migration and increase in contractile gene expression (5, 6, 11, 20, 28). Increased expression of Trb3 in PASMCs facilitates the induction of a contractile phenotype by BMP (6). On the basis of in vivo data showing that infusion of phenamil augments Trb3 expression and inhibits downregulation of the contractile gene SMA, we hypothesized that phenamil might reduce vascular remodeling in vivo by elevating Trb3 expression, leading to positive modulation and facilitation of procontractile BMP signaling. To examine whether phenamil mimics BMP4 and inhibits vSMC proliferation, human primary PASMCs were treated with BMP4 alone, phenamil alone, or with both, and proliferation of PASMCs was monitored by cell number counts after 48 h. Increasing concentrations of phenamil inhibited cell growth in a dose-dependent manner (⬃30% and ⬃50% inhibition), which is similar to the level of inhibition seen with 3 nM BMP4 treatment (⬃50% inhibition) (Fig. 2A). At higher doses, phenamil potentiated the BMP4-mediated inhibition of cell growth from 50% when stimulated with BMP4 alone to 62% inhibition when treated with both BMP4 and 10 ␮M phenamil (Fig. 2A). The effect of phenamil on PASMC migration was similarly examined by scratch wound assay. In mock-treated cells, the wound created on confluent cells was closed after 24 h (Fig. 2B). In comparison, a significant gap was observed in phenamil-treated cells, suggesting that phenamil significantly decreases cell migration (Fig. 2B). As previously reported, BMP is able to partially inhibit migration of PASMCs into the open wound (Fig. 2B, BMP4 versus mock). Similar to the results of the cell proliferation assay in Fig. 2A, phenamil could enhance and potentiate the effect of BMP in migration (Fig. 2B), and the stimulation with both phenamil and BMP4 showed the lowest rate of migration (Fig. 2B). This further supports the hypothesis of a link between phenamil and the BMP signaling pathway. Finally, we examined the effect of phenamil on the contractility of PASMCs using a collagen lattice contraction assay. A previous study demonstrates that BMP increases the contractility of PASMCs through actin remodeling, visualized by a decrease in collagen lattice (28), and elevated expression of Trb3 is able to potentiate the BMP effect (5). Similar to BMP4, phenamil was able to induce contraction in the collagen lattice (Fig. 2C), which is in agreement with results from migration (Fig. 2B) and proliferation (Fig. 2A) assays described above. Interestingly, unlike the migration and proliferation assays, cotreatment with phenamil and BMP4 did not further increase contraction compared to either treatment alone (Fig. 2C). We hypothesize that this is due to limitations of this assay, where maximum contraction is seen with either BMP4 or phenamil alone. Despite this difference, together these results confirm that phenamil facilitates BMP4-mediated procontractile activities and is capable of promoting the contractile phenotype in

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PASMCs characterized by lower proliferation and migration and higher contractility. Induction of Trb3 is critical for phenamil facilitation of BMP signaling. To examine whether phenamil is able to induce Trb3 and contractile gene expression, we examined the levels of contractile gene markers SMA and SM22 after treatment for 24 h with phenamil and/or BMP4 in cultured human primary PASMCs. Phenamil treatment alone exhibited only a weak effect on basal expression of contractile markers, but it potentiated BMP4-mediated induction of contractile genes in a dose-dependent manner (Fig. 3A, SMA and SM22) consistent with in vivo data (Fig. 1C and D). Elevation of Trb3 and SMA protein by phenamil was also confirmed by immunofluorescence staining in human aortic smooth muscle Ao184 cell line (unpublished data). Next we examined the potential effect of phenamil on a non-vSMC-specific target of the BMP-Smad pathway, the Id3 gene. As induction of Id3 mRNA is rapid (2 h) after BMP4 treatment, PASMCs were pretreated for 24 h with phenamil, followed by 2 h with BMP4, and Id3 mRNA was analyzed by quantitative RT-PCR (qRT-PCR) analysis. We found that phenamil induces Id3 in a dose-dependent manner in parallel with Trb3 levels (Fig. 3B). Similar results were obtained with other targets of the BMP-Smad pathway, Id2 and Smad6 genes (unpublished data). The effect of phenamil was abolished by treatment with the BMP receptor kinase antagonist LDN193189 (unpublished data), suggesting that phenamil treatment leads to induction of BMP-Smad signaling. To confirm that phenamil regulates gene regulation through Trb3, endogenous Trb3 was reduced by siRNA, followed by phenamil treatment. Transfection of siRNA directed against Trb3 (si-Trb3) reduced basal Trb3 expression to ⬍20% and inhibited phenamil induction of Trb3 expression (Fig. 3C). With si-Trb3 transfection, phenamil no longer had an effect on expression of SMA or Id3 (Fig. 3C), suggesting that induction of Trb3 is essential for phenamil-mediated regulation of BMP target genes. Furthermore, phenamil-mediated inhibition of cell growth and migration was attenuated by downregulation of Trb3 by siRNA (unpublished data), confirming that Trb3 is an essential downstream effecter of phenamil. Phenamil and other structurally similar amiloride derivatives exhibit differential specificity and potency of ion channel inhibition (8). To examine the ability of different amiloride derivatives to regulate Trb3 and contractile gene expression, PASMCs were treated with phenamil, benzamil, or amiloride at the concentration determined by the relative potency of inhibition of amiloride-sensitive Na⫹ channel (44). Benzamil weakly induced Trb3 (Fig. 3D), as well as SMA and SM22 (Fig. 3E); however, amiloride had no effect on any of these genes (phenamil ⬎⬎ benzamil ⬎ amiloride) (Fig. 3D and E). These results indicate that a specific ion channel(s) that is potently inhibited by phenamil but less sensitive to benzamil or amiloride may play a critical role in facilitating the BMP signaling pathway in vSMCs. Inhibition of ASICs plays a role in induction of Trb3. We next attempted to identify the ion channels in vSMCs which when inhibited by phenamil lead to increased induction of Trb3. We first examined the possible role of epithelial sodium channel (ENaC). ENaC is made up of a number of different subunits, of which the ␤-subunit (ENaC␤) is essential for for-

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FIG. 2. Phenamil facilitates bone morphogenetic protein (BMP)-mediated contractile phenotype and inhibition of cell growth. (A) Pulmonary artery smooth muscle cells (PASMCs) were pretreated with increasing concentrations of phenamil as indicated for 24 h prior to treatment with 3 nM BMP4. The number of cells was counted 48 h after BMP4 treatment. The relative numbers of cells compared to untreated cells were plotted as means plus standard errors of the means (SEMs) (n ⫽ 3). *, P ⬍ 0.01. (B) PASMCs were treated with DMSO (mock), 20 ␮M phenamil, 3 nM BMP4, or both for 24 hours. A single scratch wound was generated with a 200-␮l disposable pipette tip. Scratch wounds were photographed over 24 h (shown on the right), and the widths of the wounds were quantitated with the ImageJ software. Data were plotted as the distance of migration over time (left panel) and are presented as the means of triplicate measurements per condition in three independent experiments. *, P ⬍ 0.05. (C) PASMCs were treated with DMSO (mock) or 20 ␮M phenamil for 24 h, followed by 3 nM BMP4 treatment for 24 h. The cells were then embedded in collagen gel lattices on the 12-well plate. Twenty-four hours after the collagen lattices were dissociated from the well, the gel lattices were photographed (left panel). The relative lattice area was quantitated by dividing the area of gel lattice by the initial area of the well (right panel). Experiments were performed three times. Data represent means plus SEMs. *, P ⬍ 0.01.

mation of a functional channel. Therefore, siRNA directed against ENaC␤ (si-ENaC␤) was transfected to PASMCs. si-ENaC␤ reduced the expression of ENaC␤ to ⬃15% (Fig. 4A). Despite efficient knockdown of functional ENaC, phenamil stimulation still led to induction of Trb3 and contractile gene SM22 similar to the level observed in cells transfected with nontargeting control siRNA (si-Control) (Fig. 4A). This suggests that inhibition of ENaC does not play a critical role in phenamil induction of Trb3. This is consistent with the observation that amiloride analogs, such as amiloride and benzamil,

which are more potent inhibitors of ENaC than phenamil is, exhibited little effect on Trb3 or contractile genes, as shown in Fig. 3D and E. Next, we examined the possible involvement of acid-sensing ion channels. ASICs are voltage-insensitive, pH-sensitive cationic channels permeable to Ca2⫹, Na⫹, and K⫹. Phenamil is known to inhibit ASICs more potently than other amiloride analogs (17). Four different ASIC genes have been identified, and ASIC proteins form homomeric or hetereomeric cation channels that are proton gated. ASIC1,

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FIG. 3. Phenamil promotes contractile gene expression through induction of Trb3. (A) PASMCs were pretreated with 10 or 20 ␮M phenamil for 24 h prior to stimulation with 3 nM BMP4 for 24 h. The relative levels of expression of SMA, SM22, and Trb3 mRNA normalized to GAPDH were determined by qRT-PCR and are presented as means plus SEMs, with each experiment conducted in triplicate (n ⫽ 3). *, P ⬍ 0.05. (B) PASMCs were pretreated with 10 or 20 ␮M phenamil for 24 h prior to stimulation with 3 nM BMP4 for 2 h. The relative levels of expression of Id3 and Trb3 mRNA normalized to GAPDH were determined by qRT-PCR, and the results are presented as means plus SEMs. Experiments were conducted in triplicate (n ⫽ 3). *, P ⬍ 0.05. (C) PASMCs were transfected with 50 nM nontargeting control siRNA (si-Control) or siRNA against human Trb3 (si-Trb3) for 24 h, followed by stimulation with 10 ␮M phenamil for 24 h and 3 nM BMP4 for another 24 h. The relative level of expression of Trb3, SMA, or Id3 normalized to GAPDH was plotted, and the results are shown as means plus SEMs (n ⫽ 3). *, P ⬍ 0.01. (D) PASMCs were treated with 10 or 20 ␮M phenamil, 40 or 80 ␮M benzamil, or 200 or 400 ␮M amiloride for 24 h. The relative level of expression of Trb3 mRNA was determined by qRT-PCR. Results are presented as fold induction in comparison with DMSO (mock) treatment and presented as means plus SEMs. Each experiment was conducted in triplicate (n ⫽ 3). (E) PASMCs were treated with 10 ␮M phenamil, 10 ␮M benzamil, or 200 ␮M amiloride for 24 h, followed by stimulation with 3 nM BMP4 for an additional 24 h. The relative levels of expression of SMA and SM22 mRNA normalized to GAPDH were determined by qRT-PCR, and the results are presented as means plus SEMs. Experiments were conducted in triplicate (n ⫽ 3). *, P ⬍ 0.05.

ASIC2, and ASIC3 are expressed in vSMCs, while ASIC4 is not (14). We found that compared to ASIC2, ASIC1 and ASIC3 are highly expressed in PASMCs, as ASIC2 expression is about 10-fold less than ASIC1 expression (Fig. 4B). It is worth noting that mRNA levels of ASIC1 and ASIC3 are both over 100-fold higher than ENaC␤ mRNA levels,

suggesting significant expression and role of these channels in PASMCs. Furthermore, expression of ASIC mRNA is not affected by phenamil stimulation. Downregulation of ASIC1 alone or ASIC3 alone by siRNA transfection in PASMCs elevated Trb3 mRNA (Fig. 4C) and protein (unpublished data) levels to a degree similar to the effect of phenamil.

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FIG. 4. Inhibition of acid-sensing ion channel (ASIC) family of ion channels by phenamil leads to Trb3 induction. (A) PASMCs were transfected with 40 nM nontargeting control siRNA (si-Control) or siRNA against human epithelial sodium channel ␤ (ENaC␤) (si-ENaC␤) for 24 h, followed by stimulation with 10 ␮M phenamil for 24 h. The relative levels of expression of Trb3, SM22, and ENaC␤ normalized to GAPDH were plotted, and the results are shown as means plus SEMs (n ⫽ 3). *, P ⬍ 0.001. (B) PASMCs were stimulated with 10 ␮M phenamil for 24 h. The relative levels of expression of ASIC1, ASIC2, and ASIC3 normalized to GAPDH were plotted, and the results are shown as means plus SEMs (n ⫽ 3). (C) PASMCs were transfected with 40 nM control siRNA (si-Control), siRNA against human ASIC1 (si-ASIC1), ASIC2 (si-ASIC2), or ASIC3 (si-ASIC3) individually or a combination of si-ASIC1 and si-ASIC3 (si-ASIC1&3). After 10 ␮M phenamil treatment for 24 h, the relative levels of expression of Trb3 and SM22 mRNA normalized to GAPDH were plotted, and the results are shown as means plus SEMs (n ⫽ 3) in the top graph. *, P ⬍ 0.001. The relative levels of expression of ASIC1, ASIC2, are ASIC3 normalized to GAPDH were also plotted, and the results are shown as means plus SEMs (n ⫽ 3) in the bottom graph. *, P ⬍ 0.001. (D) PASMCs were transfected with 40 nM si-Control siRNA (si-Control) or si-ASIC1, followed by 24 h of stimulation with 20 ␮M phenamil (⫹). The cells were harvested and subjected to immunoblot analysis with anti-human Trb3 antibody, anticalponin antibody (CNN), or anti-GAPDH antibody as a loading control. The results shown are from one representative experiment of three independent experiments.

When both ASIC1 and ASIC3 were reduced simultaneously (Fig. 4C, siASIC1&3), Trb3 induction by phenamil was abolished. Downregulation of ASIC2 had no effect on the level of Trb3 (Fig. 4C), suggesting that inhibition of ASIC2 activity is not involved in phenamil induction of Trb3 expression. Furthermore, knockdown of ASIC1 promoted both Trb3 protein expression and protein expression of the con-

tractile marker calponin (CNN) in PASMCs (Fig. 4D). Together, these results suggest that inhibition of ASIC1 and ASIC3 by phenamil plays a critical role in the induction of Trb3 and the procontractile activity of the BMP pathway. Transcriptional activation of the Trb3 gene by phenamil. Next, we examined the molecular mechanism by which phenamil stimulation leads to induction of Trb3. PASMCs were

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FIG. 5. Putative NFAT binding site on Trb3 promoter is critical for induction of Trb3 by phenamil. (A) PASMCs were treated with 10 ␮M phenamil for various lengths of time as indicated. The relative expression of Trb3 mRNA normalized to GAPDH was determined by qRT-PCR, and the relative mRNA levels are presented as means plus SEMs. Experiments were conducted in triplicate (n ⫽ 3). *, P ⬍ 0.01. (B) A rat PASMC cell line, PAC1, was transfected with a Trb3 luciferase reporter construct containing ⬃1.9-kb human Trb3 promoter region (p1.9-luc). The cells were then stimulated with increasing concentrations of phenamil for 24 h as indicated, and the luciferase assay was conducted. Data were plotted as relative luciferase (Luc) activity normalized to ␤-galactosidase activity and are shown as means plus SEMs (n ⫽ 3). (C) PAC1 cells were transfected with different Trb3 promoter deletion mutants, and then the luciferase assay was conducted. Values are means plus SEMs (n ⫽ 3). *, P ⬍ 0.001. (D) Schematic representations of Trb3-promoter luciferase constructs and summary of the luciferase results obtained in Fig. 6B. The location of the 19-bp phenamil response element (PRE) is indicated by the black box. (E) Sequences of the PREs. The putative STAT or NFAT binding site is underlined. PAC1 cells were transfected with luciferase constructs containing three copies of wild-type PRE sequence [PRE(WT)luc], or 2-bp mutation within PRE [PRE(Mut1 to -3)-luc]. p1.0-luc was transfected as a control. Results were plotted as fold induction of luciferase activities after phenamil treatment over control (DMSO) treatment; values are means plus SEMs (n ⫽ 3). *, P ⬍ 0.001.

treated with phenamil for various lengths of time (2 to 48 h), and Trb3 mRNA level was examined by qRT-PCR. Weak (⬃1.6-fold) but significant elevation of Trb3 mRNA was observed at the 8-h time point, which was further increased after 24 h (⬃5-fold) and 48 h (7.7-fold) (Fig. 5A). Consistent with the level of mRNA (Fig. 5A), the amount of Trb3 protein was also increased by phenamil (unpublished data). The effect of

phenamil is specific to Trb3, as other members of the Trb family, Trb1 and Trb2, were not affected (unpublished data). To test the potential effect of phenamil on Trb3 gene transcription, the previously described luciferase reporter construct containing an ⬃1.9-kb human Trb3 promoter region (p1.9-luc) (29) was transfected into the rat PASMC line PAC1 cells, followed by treatment with increasing concentrations of

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phenamil. Phenamil was able to induce the luciferase activity in a dose-dependent manner (Fig. 5B). An increase of about 2- to 3-fold in the reporter activity at 10 to 15 ␮M phenamil is similar to the level of induction of endogenous Trb3 by phenamil (Fig. 3D). The relative potency of induction of the p1.9-luc activity by different amiloride derivatives (phenamil ⬎ benzamil ⬎ amiloride) (unpublished data) was consistent with the result for endogenous Trb3 mRNA (Fig. 3D). Therefore, the 1.9-kb Trb3 promoter region recapitulates the response of the full-length Trb3 promoter to phenamil. To identify a region within this 1.9-kb construct responsible for the phenamil response, a series of deletion mutants were generated as indicated in Fig. 5D. Deletion of ⬃1 kb from the 5⬘ end of the promoter showed no dramatic effect on the response to phenamil (Fig. 5B and C, p1.4-luc and p1.0-luc). However, deletion of an additional 100 bp (p0.9-luc) abolished the responsiveness to phenamil (Fig. 5C). Upon close examination of this 100-bp region, we found a 19-bp sequence that is highly conserved between the human and mouse Trb3 gene sequences, which we tentatively termed the “phenamil response element (PRE)” (Fig. 5D and E). To examine whether PRE is sufficient to convey responsiveness to phenamil, we cloned three copies of the PRE sequence into a luciferase vector upstream of an exogenous transcription initiation site [Fig. 5E, PRE(WT)-luc]. Luciferase activity of the PRE-luc construct was induced about 2-fold upon phenamil treatment, similar to the p1.0-luc construct (Fig. 5E). PRE contains several potential binding sites for transcription factors, such as signal transducers and activators of transcription (STAT) binding sequence (TCATAGGTG) and nuclear factor of activated T cell (NFAT) binding sequence (GGAAA) (Fig. 5E, left panel). To examine the potential involvement of these factors in the induction by phenamil, 2-bp mutations were introduced into the 5⬘ end of PRE (Mut1), in the STAT site (Mut2), or in the NFAT site (Mut3) (Fig. 5E). While PRE(Mut1)-luc and PRE(Mut2)-luc both responded to phenamil, mutation in the putative NFAT binding site of PRE [PRE(Mut3)-luc] resulted in loss of phenamil induction (Fig. 5E). These results suggest an essential role of NFAT binding site in the transcriptional activation of Trb3 by phenamil. Phenamil mediates activation of NFAT. We next examined the potential role of NFAT on phenamil induction of Trb3. An NFAT-luciferase reporter construct (NFAT-Luc), which contains 3 copies of the NFAT binding site (GGAAA) isolated from the interleukin-2 (IL-2) gene promoter (a transcription target of NFAT) (21), was transfected into PAC1 cells, and luciferase activity was measured after phenamil treatment for 24 h. NFAT-Luc activity was induced by phenamil (Fig. 6A) by ⬃2.5-fold, but not by benzamil or amiloride (unpublished data), similar to the result obtained with endogenous Trb3 (Fig. 3D). Downregulation of ASIC1 and ASIC3 attenuated the activation of NFAT-Luc by phenamil similar to the result of Trb3 and SM22 in Fig. 4C (unpublished data), confirming that phenamil activates the NFAT pathway via inhibition of ASICs (ASIC1 and ASIC3). Activation of NFAT-Luc was abolished by cotreatment with cyclosporine (CsA), an inhibitor of calcineurin (Fig. 6A). Calcineurin is a Ca2⫹/calmodulindependent serine/threonine phosphatase that, upon activation, dephosphorylates NFATs. Dephosphorylation of NFAT leads to increased nuclear translocation and thus activation of tran-

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scription (13) (Fig. 6A). The result obtained with CsA was confirmed by cotransfection of a plasmid expressing a smallpeptide-specific inhibitor of calcineurin (VIVIT), which blocks the NFAT-calcineurin interaction (3). Expression of VIVIT inhibited activation of NFAT-Luc by phenamil as well (Fig. 6A). Consistent with the NFAT-reporter assay, endogenous IL-2 mRNA was also potently induced by phenamil (⬃9-fold at 48 h). Similar to the results seen with the Trb3 promoter, other amiloride derivatives were much less effective in inducing IL-2 mRNA expression. (⬃3-fold at 48 h with 10 ␮M benzamil; no significant change with amiloride stimulation) (Fig. 6B). It is important to note that phenamil stimulation did not affect expression of NFAT1 to NFAT4 (unpublished data). As activation of NFAT is achieved through an increase in the intracellular Ca2⫹ concentration ([Ca2⫹]i), we examined whether phenamil affects store-operated Ca2⫹ entry (SOCE) and elevates [Ca2⫹]i in PASMCs. SOCE was measured by depletion of [Ca2⫹]i with EDTA in Ca2⫹-free physiological saline solution (PSS) in the presence of cyclopiazonic acid (CPA) and diltiazem in PAC1 cells, followed by measurement of [Ca2⫹]i using the fura-2 fluorescence indicator dye. Phenamil weakly elevated SOCE (Fig. 6C), while it produced no significant change in [Ca2⫹]i at the calcium-depleted state (not shown). This result indicates that phenamil is able to increase [Ca2⫹]i in PASMCs, which may then activate the calcineurinNFAT pathway. We next examined whether calcineurin activation of NFAT plays a role in induction of Trb3 by phenamil. Trb3-Luc reporter constructs responsive to phenamil [p1.0-luc, PRE(WT)luc, and PRE(Mut2)-luc] were treated with phenamil, CsA alone, or CsA and phenamil. Induction of these reporters by phenamil was blunted by cotreatment with CsA or coexpression of VIVIT (Fig. 6D). These results demonstrate that phenamil mediates activation of NFAT, which then leads to transcriptional activation of the Trb3 promoter via PRE. We next examined by chromatin immunoprecipitation (ChIP) assay in Ao184 cells the recruitment of NFAT2 (also known as NFATc1) to the endogenous Trb3 gene promoter upon phenamil treatment. Robust enrichment of endogenous NFAT2 at the Trb3 promoter was observed 8 h after stimulation with phenamil (Fig. 6E). Recruitment of NFAT2 to Trb3 coincides with the earliest time point at which induction of Trb3 by phenamil is detected (Fig. 5A). Dose-dependent increase in NFAT2 recruitment was also observed with increasing concentrations of phenamil treatment (unpublished data). Furthermore, the ability to recruit NFAT2 among the three amilorides (phenamil ⬎⬎ benzamil ⬎ amiloride) was consistent with the induction of endogenous Trb3 mRNA (Fig. 3D and unpublished data). These results confirm that phenamil mediates Ca2⫹-dependent activation of calcineurin, the nuclear translocation of NFAT2, and that the association of NFAT2 with Trb3 promoter leads to transcriptional activation. Finally, to confirm that activation of the NFAT pathway is essential for phenamil-mediated induction of contractile genes, PASMCs were treated with BMP4 in the presence of phenamil and/or NFAT inhibitor CsA. Induction of contractile genes (SMA and SM22) by phenamil or phenamil/BMP4 was inhibited by cotreatment with CsA to a level similar to that of BMP4 treatment alone, indicating that activation of the NFAT pathway is essential for mediating contractile gene induction

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FIG. 6. Phenamil mediates activation of the NFAT pathway. (A) PAC1 cells were transfected with NFAT reporter construct containing 3 copies of the NFAT binding site from the IL-2 promoter (NFAT-luc) and then treated with 10 ␮M phenamil alone or in the presence of the calcineurin inhibitor cyclosporine (CsA) (1 ␮M) or cotransfection with plasmid expressing the VIVIT peptide. The cells were harvested, and the luciferase assay was conducted. The results were normalized to ␤-galactosidase activity, and the results are shown as means plus SEMs (n ⫽ 3). (B) PASMCs were stimulated with 200 ␮M amiloride, 20 ␮M benzamil, or 20 ␮M phenamil for 8, 24, or 48 h. The relative levels of expression of IL-2 and Trb3 mRNA normalized to GAPDH were determined by qRT-PCR, and the results are presented as means plus SEMs. Each experiment was conducted in triplicate (n ⫽ 3). (C) PAC1 cells were treated with increasing concentrations of phenamil (0, 5, and 10 ␮M) for 24 h. The cells were then trypsinized, and fura-2 AM Ca2⫹ depletion/repletion assay was performed in the presence of cycloplazonic acid (10 ␮M) and diltiazem (50 ␮M). The intracellular concentration of Ca2⫹ ([Ca2⫹]i) is represented as the change (⌬) in the fura-2 emission ratios (F340/F380) from baseline. The experiment was performed twice in triplicate. *, P ⬍ 0.01. (D) PAC1 cells were transfected with either p1.0-luc, PRE(WT)-luc, PRE(Mut2)-luc, or PRE(Mut3)-luc alone or with 0.3 ␮g of pVIVIT (plasmid expressing the VIVIT peptide). After 24 h, cells transfected with luciferase constructs alone were treated with either DMSO (mock), 10 ␮M phenamil alone, 1 ␮M CsA alone, or both 10 ␮M phenamil and 1 ␮M CsA. The cells transfected with both luciferase construct and pVIVIT were stimulated with either DMSO (VIVIT alone) or 10 ␮M phenamil (Phenamil⫹VIVIT) as indicated for 24 h, and then the luciferase assay was performed. Results were plotted as relative luciferase activity normalized to ␤-galactosidase activity. The results are shown as means plus SEMs (n ⫽ 3). *, P ⬍ 0.001. (E) Human aortic smooth muscle (Ao184) cells were stimulated with 10 ␮M phenamil for 0, 4, 8, or 24 h. The cells were then harvested, and ChIP assay was performed using anti-NFAT2 antibody. ChIP with nonspecific control IgG was used as a negative control (control IgG). qRT-PCR was performed to measure enrichment of DNA fragment containing the PRE region of the Trb3 promoter. The results were plotted as relative enrichment to input (means plus SEMs [n ⫽ 3]). (F) Ao184 cells were stimulated with 20 ␮M phenamil alone or with phenamil with 5 ␮M CsA for 24 h. The cells were then stimulated with 3 nM BMP4 for 24 h as indicated and subjected to qRT-PCR analysis of SMA, SM22, or Trb3 mRNA normalized to GAPDH expression. The results are means plus SEMs of three independent experiments (n ⫽ 3). *, P ⬍ 0.001.

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FIG. 7. Schematic diagram of the mechanism for phenamil-mediated activation of procontractile BMP signaling. Upon phenamil-mediated inhibition of ASIC1 and ASIC3 on the surfaces of vSMCs, total free calcium in the cell increases. Calcium-activated calcineurin dephosphorylates NFATs, which then enter the nucleus and bind to the promoter region of the Trb3 gene. NFAT promotes transcription of Trb3, leading to increased Trb3 protein expression. Trb3 promotes BMP signaling through inhibition of Smurf1. Increased BMP signaling promotes the contractile phenotype, leading to decreased proliferation, decreased migration, and increased expression of vSMC markers.

by phenamil (Fig. 6F). It is important to note that induction of contractile genes by BMP4 was not inhibited by CsA (Fig. 6F), suggesting that although BMP signaling in vSMCs is modulated by the CsA-sensitive NFAT-Trb3 pathway described above, BMP induction of contractile genes is itself CsA insensitive, and therefore NFAT independent. The mechanism by which BMP induces expression of contractile genes is more closely reported in previous publications (6, 11, 20). DISCUSSION In this study, we demonstrate that phenamil mediates transcriptional activation of Trb3 via activation of the NFAT family of transcription factors, facilitates the procontractile effects of the bone morphogenetic protein (BMP) signal, and promotes a highly contractile phenotype in vascular smooth muscle cells (vSMCs), which is antagonistic to the effect of platelet-derived growth factor (PDGF) (Fig. 7). Aberrant regulation of the vSMC phenotype, in particular a switch from a highly contractile phenotype to a less-contractile, synthetic phenotype is a critical phenomenon underlying the pathogenesis of a variety of vascular proliferative diseases, including pulmonary artery hypertension (PAH). Increased expression of both PDGF ligands and receptors has been reported in PAH animal models, as well as in human patients (2). An antagonist of the PDGF receptor, imatinib mesylate, is able to reverse the phenotype of experimental PAH in animal models and improve symptoms in human idiopathic PAH (IPAH) patients (2), suggesting that increased PDGF signaling in vSMCs contributes to the development of IPAH. Our recent study indicates that PDGF treatment in PASMCs induces microRNA-24 (miR-24), which targets the 3⬘ untranslated region of the Trb3 mRNA and promotes specific degradation of Trb3 mRNA in PASMCs (5). Smurf1, an E3 ubiquitin ligase which targets BMP signal transducers Smad1/5/8 for degradation (1), is elevated about 3-fold in PAs in chronic-hypoxia-treated rats or rats injected with

monocrotaline (MCT) (27). MCT injection has also been shown to reduce BMP signaling in PAs (37). These results are consistent with our observation that PAs of rats under chronichypoxia treatment exhibit a significant reduction in Trb3, which targets Smurf1 for proteosomal degradation (5), and Smad 1/5/8 expression. Therefore, we speculate that downregulation of Trb3 is a critical event during hypoxia or MCTinduced PA remodeling and development of pulmonary hypertension. Conversely, a constitutive increase in Trb3 expression in vSMCs might ameliorate vascular remodeling, as well as pulmonary hypertension through increase in BMP signaling. In support of this hypothesis, we showed evidence that constant infusion of phenamil during hypoxia treatment in rats effectively inhibits hypoxia-induced PA remodeling and pulmonary hypertension. Therefore, phenamil could be an effective therapy for pulmonary vascular remodeling in PAH. In vitro studies indicate that the amiloride analog phenamil is a highly specific inhibitor of Na⫹ channels, such as epithelial sodium channels (ENaCs) and acid-sensing ion channels (ASICs) (38). Our results show that downregulation of ASIC protein 1 (ASIC1) or ASIC3 by siRNA elevates Trb3 expression and facilitates BMP-mediated contractile activities, while downregulation of ENaC has no effect, which suggests that inhibition of ASIC1 and ASIC3 by phenamil is critical for induction of Trb3. It is interesting to note that knockdown of ASIC1 leads to reduced cell growth and migration in glioblastoma cells (43), similar to our results in PASMCs. Thus, it is plausible that phenamil might be able to inhibit cell growth and migration through the Trb3-BMP pathway in various cell types. Similar to phenamil, it has been reported that infusion of the amiloride analog DMA or EIPA, both of which are known as specific and potent inhibitors of the Na⫹/H⫹ antiporter, weakly inhibited pulmonary hypertension and pulmonary vascular remodeling (36). As dimethyl amiloride (DMA) and ethyl isopropyl amiloride (EIPA) are less potent inhibitors of ASICs than phenamil, it is likely that the beneficial effect of DMA or EIPA likely occurs through a mechanism independent of the ASIC-NFAT-Trb3 pathway. Unlike phenamil, infusion of DMA or EIPA only partially inhibited hypoxia-induced increase in PAH and vascular remodeling. It has been found that amiloride analogs attenuate mitogenic response to growth factors in rat aortic vSMCs and decrease neointima formation in carotid arteries after balloon injury (19, 23). However, not all studies demonstrate that stimulation of the Na⫹/H⫹ antiporter by alkalization is necessary for cell proliferation, and some Na⫹/H⫹ antiporter-deficient cells proliferate normally (15), suggesting a significant role of amiloride analogs in other contexts besides inhibition of the Na⫹/H⫹ antiporter. We demonstrate that phenamil treatment in PASMCs leads to activation of the NFAT pathway, resulting in transcriptional activation of the Trb3 gene. However, it has been reported that infusion of CsA, a calcineurin/NFAT inhibitor, in rats during MCT-induced PAH decreases PA pressure and medial hypertrophy (4), which contradicts our observation that phenamil attenuates hypoxia-induced PAH and vascular remodeling by activating NFAT. It is known that CsA also inhibits the activity of hypoxia-inducible factor 1␣ (HIF-1␣), a transcription factor involved in hypoxia-mediated gene regulation (10). Thus, the therapeutic effect of CsA in a PAH animal model might be

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primarily due to inhibition of HIF-1␣-dependent gene regulation (18). The VIVIT peptide is a more specific inhibitor of calcineurin than CsA, as it blocks the association of calcineurin with NFAT. Our observation that VIVIT is able to block the induction of Trb3 and contractile activities mediated by phenamil confirm an essential role of NFAT activity in the regulation of Trb3 by phenamil. The activity of NFATs is critically regulated by intracellular Ca2⫹ level, as it is essential for activation of calcineurin, which dephosphorylates NFATs and promotes their nuclear translocation. We observed that (i) inhibitors of calcineurin block the effect of phenamil on Trb3, and (ii) phenamil elevates intracellular free Ca2⫹ in PASMCs. Thus, we speculate that the primary mechanism of activation of NFATs by phenamil in PASMCs is through activation of calcineurin by elevation of intracellular calcium. The ASIC ion channels are store-operated calcium channels (SOCs) that mediate calcium entry when the Ca2⫹ store in the endoplasmic reticulum (ER) is depleted. However, ASIC1b (a splice variant of ASIC1) and ASIC3 are not permeable to Ca2⫹ (7, 17). As our results indicate the functional significance of ASIC1 and ASIC3 as downstream effectors of phenamil, we speculate that activation of the calcineurin-NFAT pathway by phenamil might be a result of sodium imbalance. We also speculate that inhibition of ASICs might trigger an activation of phospholipase C-␥ (PLC-␥), which hydrolyzes phosphatidylinositol-4,5,-bisphosphate to produce inositol-1,4,5triphosphate, and induces the release of calcium from intracellular stores. Alternatively, phenamil might activate calcineurin, at least in part, in a Ca2⫹-independent manner. Association of calcineurin and A-kinase-anchoring protein 79/ 150 (AKAP79/150) with ASIC1a has been demonstrated (9). As AKAP79/150 is a negative regulator of calcineurin, it is plausible that inactivation of ASIC1 by phenamil might allow dissociation of calcineurin from AKAP79/150 leading to activation of calcineurin. It is also possible that inhibition of ASICs might directly lead to an activation of calmodulin. Proteolytic cleavage of calcineurin by caspases also leads to activation (26). It has been reported that ASIC3 inhibits caspase 3 activity and prevents apoptosis in nucleus pulposus cells by an unknown mechanism (42). Therefore, it is plausible that inhibition of ASIC3 by phenamil activates caspase 3 and leads to proteolytic activation of calcineurin. This mechanism might be important for sustained activation of the NFAT pathway and elevation of Trb3 mRNA. NFAT proteins contain amino-terminal transactivation domain and carboxyl-terminal DNA binding domains, which preferentially bind to the sequence 5⬘-GGAAA-3⬘. NFAT proteins frequently bind DNA as a complex with different DNA binding proteins, such as GATA or AP-1, which bind their own binding site adjacent to the NFAT binding site (21). We repeatedly observed a reduced response to phenamil of the mutant of the first 2 bp of the PRE sequence [PRE(Mut1)]. This result suggests that a transcription factor that recognizes the 5⬘ end of the PRE sequence may play a role in recruitment and/or binding of NFAT to the Trb3 promoter. The identity of this transcription factor would be a subject of a future study. In summary, our findings that phenamil is able to facilitate the contractile actions of the BMP signal through induction of Trb3 and can inhibit pulmonary vascular remodeling mean that phenamil has promise as a novel therapy for PAH.

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ACKNOWLEDGMENTS We thank L. Covic and N. Nguyen for assistance with the intracellular calcium measurements and K. D. Bloch and P. B. Yu for sharing LDN-193189. We also thank all members of the Hata and Lagna labs for helpful suggestions and critical discussions. This work was supported by grants from the National Institutes of Health (HL086572 [G.L.], HD042149, and HL082854), the American Heart Association (0940095N), and the LeDucq Foundation (Transatlantic Networks of Excellence program [A.H.]). REFERENCES 1. Alexandrova, E. M., and G. H. Thomsen. 2006. Smurf1 regulates neural patterning and folding in Xenopus embryos by antagonizing the BMP/Smad1 pathway. Dev. Biol. 299:398–410. 2. Andrae, J., R. Gallini, and C. Betsholtz. 2008. Role of platelet-derived growth factors in physiology and medicine. Genes Dev. 22:1276–1312. 3. Aramburu, J., et al. 1999. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science 285:2129–2133. 4. Bonnet, S., et al. 2007. The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted. Proc. Natl. Acad. Sci. U. S. A. 104:11418–11423. 5. Chan, M. C., et al. 2010. Molecular basis for antagonism between PDGF and the TGF-␤ family of signalling pathways by control of miR-24 expression. EMBO J. 29:559–573. 6. Chan, M. C., et al. 2007. A novel regulatory mechanism of the bone morphogenetic protein (BMP) signaling pathway involving the carboxyl-terminal tail domain of BMP type II receptor. Mol. Cell. Biol. 27:5776–5789. 7. Chu, X. P., et al. 2002. Proton-gated channels in PC12 cells. J. Neurophysiol. 87:2555–2561. 8. Dai, X. Q., et al. 2007. Inhibition of TRPP3 channel by amiloride and analogs. Mol. Pharmacol. 72:1576–1585. 9. D’Angelo, G., E. Duplan, N. Boyer, P. Vigne, and C. Frelin. 2003. Hypoxia up-regulates prolyl hydroxylase activity: a feedback mechanism that limits HIF-1 responses during reoxygenation. J. Biol. Chem. 278:38183–38187. 10. D’Angelo, G., E. Duplan, P. Vigne, and C. Frelin. 2003. Cyclosporin A prevents the hypoxic adaptation by activating hypoxia-inducible factor-1alpha Pro-564 hydroxylation. J. Biol. Chem. 278:15406–15411. 11. Davis, B. N., A. C. Hilyard, G. Lagna, and A. Hata. 2008. SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454:56–61. 12. Drummond, H. A., N. L. Jernigan, and S. C. Grifoni. 2008. Sensing tension: epithelial sodium channel/acid-sensing ion channel proteins in cardiovascular homeostasis. Hypertension 51:1265–1271. 13. Feske, S., J. Giltnane, R. Dolmetsch, L. M. Staudt, and A. Rao. 2001. Gene regulation mediated by calcium signals in T lymphocytes. Nat. Immunol. 2:316–324. 14. Grifoni, S. C., N. L. Jernigan, G. Hamilton, and H. A. Drummond. 2008. ASIC proteins regulate smooth muscle cell migration. Microvasc. Res. 75: 202–210. 15. Grinstein, S., D. Rotin, and M. J. Mason. 1989. Na⫹/H⫹ exchange and growth factor-induced cytosolic pH changes. Role in cellular proliferation. Biochim. Biophys. Acta 988:73–97. 16. Guo, J., et al. 2004. Expression of genes in the TGF-␤ signaling pathway is significantly deregulated in smooth muscle cells from aorta of aryl hydrocarbon receptor knockout mice. Toxicol. Appl. Pharmacol. 194:79–89. 17. Jernigan, N. L., M. L. Paffett, B. R. Walker, and T. C. Resta. 2009. ASIC1 contributes to pulmonary vascular smooth muscle store-operated Ca(2⫹) entry. Am. J. Physiol. Lung Cell. Mol. Physiol. 297:L271–L285. 18. Koulmann, N., et al. 2006. Cyclosporin A inhibits hypoxia-induced pulmonary hypertension and right ventricle hypertrophy. Am. J. Respir. Crit. Care Med. 174:699–705. 19. Kranzhofer, R., et al. 1993. Suppression of neointimal thickening and smooth muscle cell proliferation after arterial injury in the rat by inhibitors of Na(⫹)-H⫹ exchange. Circ. Res. 73:264–268. 20. Lagna, G., et al. 2007. Control of phenotypic plasticity of smooth muscle cells by BMP signaling through the myocardin-related transcription factors. J. Biol. Chem. 282:37244–37255. 21. Macian, F. 2005. NFAT proteins: key regulators of T-cell development and function. Nat. Rev. Immunol. 5:472–484. 22. Misiakos, E. P., et al. 2001. Expression of PDGF-A, TGF-␤ and VCAM-1 during the developmental stages of experimental atherosclerosis. Eur. Surg. Res. 33:264–269. 23. Mitsuka, M., M. Nagae, and B. C. Berk. 1993. Na(⫹)-H⫹ exchange inhibitors decrease neointimal formation after rat carotid injury. Effects on smooth muscle cell migration and proliferation. Circ. Res. 73:269–275. 24. Monzen, K., R. Nagai, and I. Komuro. 2002. A role for bone morphogenetic protein signaling in cardiomyocyte differentiation. Trends Cardiovasc. Med. 12:263–269. 25. Moser, M., and C. Patterson. 2005. Bone morphogenetic proteins and vascular differentiation: BMPing up vasculogenesis. Thromb. Haemost. 94:713– 718.

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