Regulation of Bcl-xL Expression in Lung VascularSmooth MuscleYuichiro J. Suzuki, Hiroko Nagase, Chi Ming Wong, Shilpashree Vinod Kumar, Vivek Jain, Ah-Mee Park, andRegina M. Day

Department of Pharmacology, Georgetown University Medical Center, and Division of Pulmonary and Critical Care Medicine, Department ofMedicine, George Washington University Medical Center, Washington, DC; and Department of Pharmacology, Uniformed ServicesUniversity of the Health Sciences, Bethesda, Maryland

Pulmonary hypertension is characterized by thickened pulmonaryarterial walls due to increased number of pulmonary artery smoothmuscle cells (PASMC). Apoptosis of PASMC may play an importantrole in regulating the PASMC number and may be useful for reducingpulmonary vascular thickening. The present study examined the reg-ulation of an anti-apoptotic protein Bcl-xL. Bcl-xL expression wasfound to be increased in the pulmonary artery of chronic hypoxia–treated rats with pulmonary vascular remodeling. Adenovirus-medi-ated gene transfer of Bcl-xL indeed showed that this protein has anti-apoptotic activities in PASMC. Treatment of remodeled pulmonaryartery with sodium nitroprusside (SNP) reduced Bcl-xL expressionby targeting the bcl-xL promoter. The bcl-xL promoter contains twoGATA elements, and SNP decreases the GATA-4 DNA-binding activ-ity. Overexpression of GATA-4 attenuated the SNP-mediated sup-pression of Bcl-xL expression, providing direct evidence for the roleof GATA-4 in Bcl-xL gene transcription. We established that SNPtargets the 250 proximal region of the gata4 promoter and sup-presses its gene transcription. Thus, inducers of pulmonary hyper-tension enhance anti-apoptotic Bcl-xL gene transcription, whichcan be suppressed by targeting gata4 gene transcription.

Keywords: apoptosis; genes; pulmonary hypertension; smooth muscle

Pulmonary hypertension is characterized by the elevation ofpulmonary vascular resistance, which interferes with the ejectionof blood by the right ventricle and ultimately causes heart failure.It is often developed secondary to various cardiovascular andpulmonary diseases such as left ventricular failure, congenitalheart defects, chronic obstructive pulmonary disease, sleep ap-nea syndrome, and post-thrombotic diseases. Pulmonary arterialhypertension can also occur as a genetic disorder. Pulmonaryhypertension is associated with increased vasoconstriction in thepulmonary circulation and vascular remodeling in part due tothickening of pulmonary vascular wall because of increased num-ber of smooth muscle cells (SMC). Although therapeutic agentsare available that target the vasoconstrictive aspect of this condi-

(Received in original form September 23, 2006 and in final form January 3, 2007 )

This work was supported in part by NIH grants HL67340 (to Y.J.S.), HL72844 (toY.J.S.), and HL73929 (to R.M.D.), and by grants from the American Heart AssociationNew England Affiliate (to Y.J.S.) and American Lung Association/MassachusettsThoracic Society (to Y.J.S.). R.M.D. is a recipient of the Career Development Awardfrom the American Heart Association National Center. This work was pursued inpart by a collaboration through the DC Area Consortium for Integrative Cardio-Pulmonary Biology. The opinions and assertions contained herein are the privateopinions of the authors and are not to be construed as official or reflecting theviews of the Uniformed Services University of the Health Sciences or the Depart-ment of Defense or the Government of the United States.

Correspondence and requests for reprints should be addressed to Dr. Yuichiro J.Suzuki, Department of Pharmacology, Georgetown University Medical Center,NW403 Medical-Dental Building, 3900 Reservoir Road NW, Washington, DC20057. E-mail: ys82@georgetown.edu

Am J Respir Cell Mol Biol Vol 36. pp 678–687, 2007Originally Published in Press as DOI: 10.1165/rcmb.2006-0359OC on February 1, 2007Internet address: www.atsjournals.org

CLINICAL RELEVANCE

Recently, apoptosis-based therapeutic strategies to reducepulmonary vascular thickening have gained attention. Un-derstanding apoptotic regulation in pulmonary vascularsmooth muscle should promote such strategies to treat pul-monary hypertension.

tion, preventing or treating pulmonary vascular remodeling isalso needed. Recently, apoptosis-based therapeutic strategies toreduce pulmonary vascular thickening gained attention and havebeen successful in experimental animals (1–5). Thus, furtherunderstanding of the regulation of apoptosis in pulmonary vascu-lar smooth muscle should promote developing therapeutic strat-egies to treat pulmonary hypertension.

The Bcl-2 family consists of anti-apoptotic members includingBcl-2, Bcl-xL, Bcl-w, and Ced9, while Bax, Bid, Bad, Bak, andBcl-xS belong to the pro-apoptotic group. Bcl-x plays a dualrole in apoptotic regulation, using the different splicing proteinproducts; one being anti-apoptotic Bcl-xL and another being ashorter form, pro-apoptotic Bcl-xS (6). Bcl-xL often representsthe major isoform expressed in various tissues. Bcl-xS is a domi-nant-negative repressor of Bcl-2 and Bcl-xL (7, 8). Overexpres-sion of Bcl-xS has been shown to enhance apoptosis of cancercells (9–13).

A series of observations suggest the role of Bcl-xL in theregulation of apoptosis in SMC from systemic circulation duringvarious disease conditions. In a rabbit carotid artery ballooninjury model, Bcl-xL mRNA and protein levels are up-regulatedin the atheromatous lesion, and the anti-sense Bcl-xL increasesintimal smooth muscle apoptosis and reduces the intimal lesionsize and thickness (14). Similarly, in mouse coronary artery witharteriopathy after the cardiac allograft, antisense Bcl-xL in-creased intimal cell apoptosis and suppressed arterial neointimalformation (15). Angiotensin II increases medial and neointimalapoptosis and down-regulates Bcl-xL expression (16). Regulationof Bcl-xL in pulmonary vascular smooth muscle has not beendefined. Recently, neonatal rats with pulmonary hypertensionwere found to have increased expression of Bcl-xL in pulmonaryarterial walls (17). Bcl-xL has also been shown to be up-regulatedas a cytoprotective response in hyperoxic acute lung injury (18).

We here report that mediators of pulmonary vascular remod-eling increase the expression of Bcl-xL in pulmonary artery. Bcl-xL levels can effectively be reduced by apoptotic agents such assodium nitroprusside (SNP). SNP-mediated down-regulation ofBcl-xL is dependent on the inhibition of GATA-4 gene expres-sion in remodeled pulmonary artery. We cloned the gata4 pro-moter and identified the site of SNP actions.

Suzuki, Nagase, Wong, et al.: Regulation of Bcl-xL in Smooth Muscle 679

MATERIALS AND METHODS

All animal studies were approved by the Georgetown University Insti-tutional Animal Care and Use Committee, and were conducted inaccordance with the NRC Guide to the Care and Use of LaboratoryAnimals (National Academy Press, Washington DC, 1996).

Culture of Pulmonary Artery SMC

Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmo-nary arteries and human pulmonary artery SMC (HPASMC) (CellApplications, San Diego, CA) at 2–6 passages were maintained in RPMI1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin,and 0.5% fungisone at 5% CO2 and 37�C. Cells were treated withSNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine(SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma),serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1;Sigma) in media supplemented only with antibiotics. In some experi-ments, cells were infected with 50 plaque-forming units (pfu)/cell ofadenovirus expressing wild-type GATA-4 (a gift from Dr. J. D. Molkentin,Univ. of Cincinnati, Cincinnati, OH). Adenovirus expressing Bcl-xL wasconstructed from pORF-hBclXL vector (InvivoGen, San Diego, CA)using Adeno-X Expression System 2 (Clontech, Mountain View, CA).Control adenovirus did not express any proteins. For subjecting cellsto hypoxia, cells were placed in a cell culture incubator that is controlledto maintain 5% O2, 5% CO2, and 37�C.

Chronic Hypoxia Treatment of Rats

Male Sprague Dawley rats (275–300 g) were placed in an OxyCyclerOxygen Profile Controller (BioSpherix, Redfield, NY) that was set tomaintain 10% O2. Animals were subjected to chronic hypoxia for 2, 7,and 14 d. Normoxia controls were subjected to ambient 21% O2 for14 d. Animals were fed normal rat chow during treatment and wereused in accordance with institutional guidelines.

Histologic Measurements

For histologic analysis, tissues were immersed in buffered 4% paraform-aldehyde with 10% sucrose at 4�C for 24 h, and were embedded inMicrotome Tissue Tek II. Frozen tissues were cut to 7-�m-thick slicesand mounted on glass slides. Tissue sections were stained with hematox-ylin and eosin (H&E) for microscopic evaluation at �200 magnification.Wall thickness values were determined by the IP Lab Software (Scana-lytics Inc., Fairfax, VA).

For immunohistochemistry, tissue sections were washed with PBSand incubated in 3% H2O2 in methanol for 10 min at room temperatureto block endogenous peroxidases. After washing in PBS, tissues wereincubated in a 3% BSA solution for 1 h to block nonspecific binding.Tissues were then incubated overnight at 4�C with PBS containing 3%BSA and 1:1,000 dilution of the primary antibody, rabbit anti–Bcl-xL

or anti-desmin (Santa Cruz Biotechnology, Santa Cruz, CA). Tissuesections were rinsed, incubated with biotinylated goat anti-rabbit sec-ondary antiserum, and immunoreaction was visualized by incubatingwith 3,3�-diaminobenzidine containing H2O2 using DAB Substrate Kit(Vector Laboratories, Burlingame, CA).

Measurements of Mitochondrial Membrane Potential

To measure mitochondrial membrane potential disruption, cells weregrown in 12-well plates for 24 h followed by treatment with or withouttesting reagents for 24 h. DePsipher Kit (Trevigen Inc., Gaithersburg,MD) was used to detect changes in mitochondrial membrane potentialwith a cationic dye 5,5�6,6�-tetrachloro-1,1�,3,3�-tetraethylbenzimidazo-lylcarbocyanine iodide. In accordance with the manufacturer’s instruc-tion, cells were incubated for 20 min in 1� reaction buffer, stabilizingsolution, and DePsipher solution at 37�C. The cells were examinedunder a fluorescence microscope (Olympus, Center Valley, PA) witha red/green dual filter cube.

Comet Assay

The neutral comet assay was used to measure double-stranded DNAbreaks as an indication of apoptosis. Cells were treated with apoptoticstimuli, washed in PBS, embedded in 1% agarose, and placed on acomet slide (Trevigen). Cells were then placed in a lysis solution (2.5

M NaCl, 1% Na-lauryl sarcosinate, 100 mM EDTA, 10 mM Tris base,0.01% Triton X-100) for 30 min. The nuclei were subsequently electro-phoresed for 20 min at 1 V/cm in 1� Tris-Borate-EDTA (TBE), fixedin ethanol, stained with Sybr Green, and visualized with a fluorescencemicroscope at 478 nm excitation and 507 nm emission wavelengths.Between 100 and 150 comets were scored per experiment, and apoptoticcells were assigned based on their tail moments (20).

Western Blot

To prepare lysates, the cells were washed in PBS and solubilized with50 mM Hepes solution (pH 7.4) containing 1% (vol/vol) Triton X-100,4 mM EDTA, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate,1 mM tetrasodium pyrophosphate, 2 mM PMSF, 10 �g/ml leupeptin,and 10 �g/ml aprotinin. Intact tissues were homogenized in this solutionwith Polytron. Equal protein amounts were electrophoresed through areducing SDS polyacrylamide gel and electroblotted onto a membrane.The membrane was blocked and incubated with the polyclonal immuno-globulin (Ig)G for Bcl-xL or extracellular signal–regulated kinase (ERK)(Santa Cruz Biotechnology). Protein levels were detected with horse-radish peroxidase–linked secondary antibodies and enhanced chemilu-minescence (Amersham Life Science, Arlington Heights, IL).

Electrophoretic Mobility Shift Assays

Nuclear extracts were prepared as previously described (21). For elec-trophoretic mobility shift assays (EMSA), the binding reactions wereperformed for 20 min in 5 mM Tris-HCl (pH 7.5), 37.5 mM KCl,4% (wt/vol) Ficoll 400, 0.2 mM EDTA, 0.5 mM DTT, 1 �g poly(dI-dC)·poly(dI-dC), 0.25 ng (� 20,000cpm) 32P-labeled double-strandedoligonucleotide, and 2 �g protein of nuclear extract. Electrophoresisof samples through a native 6% polyacrylamide gel will be followedby autoradiography. The double-stranded EMSA probes used in thepresent study include an oligonucleotide with two GATA consensuselements 5�-CAC TTG ATA ACA GAA AGT GAT AA CT CT-3�, theproximal 250-bp region of the gata4 promoter, and an oligonucleotidecontaining the sequence from positions –95 to –55 of the gata4 promoter.Supershift experiments were performed by incubating nuclear extractswith 2 �g of antibodies for Egr1, Sp1, USF1, and USF2 (Santa CruzBiotechnology).

RT-PCR

Total RNA (1 �g) extracted using TRIZOL (Invitrogen, Carlsbad,CA) was reverse-transcribed by oligo(dT) priming and MMLV reversetranscriptase (Applied Biosystems, Foster City, CA). The resultantcDNA was amplified using Taq DNA polymerase (Invitrogen) andresolved on a 1.5% agarose gel containing ethidium bromide. Two setsof PCR primers for human GATA-4 were designed and used in thisstudy to confirm the expression of gata4 mRNA. The primer pair 5�-CTG TGC CAA CTG CCA GAC C-3� and 5�-CTG CTG TGC CCGTAG TGA G-3� give expected PCR product size of 306 bp, and thepair 5�-CAA CTC CAG CAA CGC CAC C-3� and 5�-AAT CCAACA CCC GCT TCC C-3�produces 441 bp. Levels of rat mRNA weremonitored using PCR primers with the following sequences: for gata4mRNA, 5�-CAG GCA GAA AGC AAG GAC TA-3� and 5�-CATAGC CAG GCT TTG GTA CAT-3�; for bcl-xL mRNA, 5�-AGG ATACAG CTG GAG TCA G-3� and 5�-TCT CCT TGT CTA CGC TTTCC-3�. Denaturing was performed at 94�C for 45 s. Annealing processeswere for 45 s at 58�C (for human gata4), 60�C (for rat gata4), and 61�C(for rat bcl-xL). Polymerase reactions were for 2 min at 72�C. Resultswere obtained at various cycles to obtain information at a linear range(15, 20, 25, and 30 cycles).

Transfection and Luciferase Assays

The day before transfection, cells were plated at 1.4 � 105 cells/well ina 12-well plate. 1 �g DNA/well was transfected using the Fugene 6transfection reagent (Roche Diagnostics, Indianapolis, IN) in serum-free, antibiotic-free RPMI. Co-transfection of the Renilla reporter (0.1�g/well) was performed to normalize for transfection differences be-tween wells. Cells were transfected for 6 h, and then medium wasreplaced with RPMI containing 0.1% FBS with antibiotics. Cells weretreated 1 h later.

Luciferase assays were performed using the Dual Luciferase AssayKit (Promega, Madison, WI). Transfected cells were washed in PBS

680 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 36 2007

and lysed. Cellular debris was removed by centrifugation at 14,000 �g for 30 s. Cell lysates were added to Luciferase Assay Reagent IIand the firefly luciferase activities were read in a Model TD-20/20luminometer (Turner Designs, Sunnyvale, CA). An equal volume ofStop and Glow was added, and the Renilla reading was taken. Theratio of firefly luciferase to Renilla luciferase was observed for eachwell of transfection. The luciferase construct controlled by the 0.6-kbproximal promoter region, pGL2–0.6R (22), was a gift from Dr. Nunez(University of Michigan, Ann Arbor, MI).

5� Rapid Amplification of cDNA EndsTotal RNA was isolated from the C57BL/6 mouse heart by TRIzol(Invitrogen). Antisense primer (5�-CAG CAT CAA AGC AGA AAC-

Figure 1. Effects of themediators of pulmonaryhypertension on Bcl-xL

expression. (A) Rats weresubjected to chronic hyp-oxia at 10% O2. Rightventricle (RV)/[left ventri-cle (LV) � septum (S)]values were measured asindications of the occur-rence of pulmonary hy-pertension and resultantright ventricular hyper-trophy. (B ) H&E stainshows thickened pul-monary artery smoothmuscle in rats subjectedto chronic hypoxia. (C )Pulmonary arteries wereisolated from rats sub-jected to chronic hypoxiaand homogenized. Tis-sue homogenates (40 �gprotein) were subjectedto Western blot to moni-tor Bcl-xL expression. ERKprotein levels are shownas loading controls. Thebar graph representsmeans � SE of the in-tensity of the Bcl-xL

band. (D) Immunohisto-chemical staining showsincreased Bcl-xL proteinexpression in the lung(including pulmonaryartery) of rats subjectedto chronic hypoxia. Thefigure also shows the ex-pression of desmin (asmooth muscle marker)and negative controlwithout the use of pri-mary antibodies. (E )BPASMC transfectedwith firefly luciferasegene controlled by the0.6-kb bcl-x promoterwere treated with 5-HT

(1 �M), ET-1 (30 nM), or hypoxia (5% O2) for 20 h. Cell lysates were prepared and luciferase activities were measured. (F ) BPASMC were treatedwith 5-HT or ET-1, cell lysates were prepared, and Bcl-xL protein expression was monitored by Western blot. Bar graph represents means � SE ofintensity of Bcl-xL protein levels in untreated and ET-1–treated cells. Representative results of the effects of 5-HT on Bcl-xL protein expression arealso shown. Asterisk denotes values significantly different from controls at P 0.05.

3�) located within exon 2 was used for first-strand synthesis. Subsequentamplification was performed using the 5� rapid amplification of cDNAends (5� RACE) System (Invitrogen). In brief, first-strand cDNA wastailed with recombinant TdT and linker (dC) oligonucleotide. 5� RACEwas performed by incubating with an aliquot of RACE primer locatedupstream of anti-sense primer (5�-AGG CTC TGG TTT GCT CAGGAA AAA-3�) and with Abridged Anchor Primer (AAP) using Plati-num Taq High-Fidelity DNA polymerase (Invitrogen). Subsequently,nested PCR was performed with a nested primer designed upstreamof RACE primer (5�-CCA AAT TGG ATT TGC GGT TGC T-3�)and Abridged Universal Amplification Primer (AUAP). The nestedprimer was used to sequence the PCR product to determine the tran-scriptional start site.

Suzuki, Nagase, Wong, et al.: Regulation of Bcl-xL in Smooth Muscle 681

Cloning of gata4 Gene Promoter

Fragments containing proximal 1,000-bp, 500-bp, and 250-bp regionsof the gata4 gene promoter were cloned by PCR cloning using mousegenomic DNA obtained from Promega. Primers for PCR fragmentswere: 5�-TGA CAT GGT ACC AAA AGT TTA GCC CAA AGCGCG A-3� (1,000 bp forward), 5�-TGA CAT GGT ACC AAG GGCCAG TTC AGG TTT TAG TG-3� (500 bp forward), 5�-TGA CATGGT ACC AAG GAC GTC GGG CTG CAC TGA-3� (250 bp for-ward), and 5�-CGG AAA GCT TCT CCG GCT TGT CCC CTG CTC-3� (reverse). The primers encode restriction digest sites (underlined)for cloning into the pGL3 basic luciferase reporter vector (Promega);forward primer encodes a Kpn I site and the reverse primer encodesa Hind III site. PCR was performed with Platinum Taq DNA Polymer-ase High Fidelity (Invitrogen) with the primers, NTPs, MgSO4, andbuffer. PCR reactions were performed for 40 cycles using a 30-s denatur-ation at 95�C, 1 min annealing at 65�C for 1,000-bp and 500-bp frag-ments, and at 68�C for the 250-bp fragment, and 6 min extension at72�C. This reaction resulted in one band each of � 1000, 500, and 250bp on agarose gel. The product was purified using the QIAquick GelExtraction Kit (Qiagen, Valencia, CA). Both the vector and purifiedPCR fragments were digested overnight at 37�C with Kpn I and HindIII (New England Biolabs, Beverly, MA); digested fragments werepurified by QIAquick PCR Purification Kit (Qiagen) and ligated intothe pGL3 luciferase reporter vector (Promega) by T4 DNA Ligase(New England Biolabs, Beverly, MA). Vectors positive for inserts werescreened by digestion and subjected to bidirectional sequencing.

Statistical Analysis

Means � SE were calculated. Significant differences between all groupswere computed by one-way ANOVA using an F statistic. Statistically

Figure 2. Effects of Bcl-xL on pulmonary vas-cular SMC apoptosis. (A) BPASMC were in-fected with adenovirus expressing Bcl-xL

(AdBcl-xL) for 48 h, then treated with SNP(300 �M) for 17 h. Cells were washed withPBS, trypsinized, incubated with Trypan Blue,and the number of viable cells was countedon a hematocytometer. The values representmeans � SE. Letters (a and b ) denote that thevalues with the same letter are significantlydifferent from each other at P 0.05. Toppanel shows the expression level of Bcl-xL withor without adenovirus (adv)-mediated over-expression. (B ) BPASMC were infected withAdBcl-xL or control adenovirus (AdCont) for48 h, then treated with SNP (100 �M) for17 h. Mitochondrial membrane potentialswere measured by DePsipher Kit. In healthymitochondria, the DePsipher dye aggregatesand forms red fluorescence. When the mem-brane potential is disrupted during the earlystage of apoptosis, the dye cannot cross themitochondrial membrane and is visualized asa monomeric form with green fluorescencein the cytosol. Fluorescence signals were ob-tained using a red/green dual filter, and un-der these conditions, green fluorescence wasnot observed in cells without staining withthe dye. Green apoptotic cells are indicatedby the arrows. (C ) BPASMC were pretreatedwith 5-HT or ET-1 for 2 h, then treated withSNAP (100 �M) or DNR (2 �M) for 24 h.Percent of apoptotic cells were monitoredby neutral comet assay. Values representmeans � SE. The values denoted by the sameletter (a, b, or c ) are significantly differentfrom each other at P 0.05.

significant differences between two groups were determined by theStudent’s t test.

RESULTS

Effects of Mediators of Pulmonary Vascular Remodeling onBcl-xL Gene Expression

To determine if Bcl-xL expression is regulated in remodeledpulmonary artery smooth muscle in vivo, rats were subjected to2, 7, or 14 d of chronic hypoxia (10% O2) to induce pulmonaryhypertension. In these rats, we noted significant increase in themass of right ventricle/(left ventricle � septum) as early as 7d of chronic hypoxia, indicating increased pulmonary vascularresistance and resultant right ventricular hypertrophy (Figure1A). Pulmonary vascular thickening was evident by 14 d ofchronic hypoxia (Figure 1B). In these remodeled pulmonaryarteries, increased Bcl-xL expression was observed by Westernblot (Figure 1C). Histologic analyses also revealed an increasedexpression of Bcl-xL protein in the lungs of rats treated with 14d of chronic hypoxia (Figure 1D), showing enhanced Bcl-xL

expression in pulmonary arteries as well as surrounding lungstructures. These results demonstrate in vivo that pulmonaryvascular remodeling is associated with increased expression ofanti-apoptotic Bcl-xL.

Vasoactive agents such as 5-HT and ET-1 also induce pulmo-nary artery SMC proliferation and are thought to play importantroles in promoting pulmonary vascular remodeling (23, 24). Wefound that these agents can also increase Bcl-xL gene expression.

682 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 36 2007

Figure 3. Effects of SNP on Bcl-xL expression. (A ) Rats were subjectedto chronic hypoxia with 10% O2 in an OxyCycler Oxygen Profiler for2 wk to elicit pulmonary vascular remodeling. Remodeled pulmonaryarteries were surgically isolated, cut into � 2-mm segments, and placedin Dulbecco’s modified Eagle’s medium (DMEM). Arterial segmentswere treated with SNP (300 �M) for 20 h. Tissues were homogenizedin Trizol and total RNA was prepared. RT-PCR was performed withprimers for bcl-xL and g3pdh mRNA. The bar graph represents means� SE of the intensity of the bcl-xL band expressed in arbitrary units(a.u.). (B ) BPASMC were treated with SNP for 24 h at various concentra-tions. Cell lysates (10 �g protein) were subjected to Western blot tomonitor levels of Bcl-xL. ERK was used as a loading control. The bargraph represents means � SE of the intensity of Bcl-xL band from cellsuntreated or treated with 100 �M SNP (n 3). (C ) BPASMC weretransfected with the luciferase gene controlled by the 0.6-kb bcl-xL

promoter region without (open circles) or with (solid triangles) SNP.Cell lysates were prepared at various time points after transfection andluciferase activities were measured and expressed in resonance units(R.U.). (D ) Cells were co-transfected with firefly luciferase gene con-trolled by 0.6-kb bcl-xL promoter (pBcl-xL) and Renilla luciferase con-trolled by thymidine kinase promoter (pTK), and treated with variousconcentrations of SNP. Cell lysates were prepared and luciferase activi-ties were measured. The values represent means � SE of the ratio offirefly luciferase activity to Renilla luciferase activity. Asterisk denotes asignificantly different value compared with control at P 0.05.

The bcl-x promoter is controlled by complex mechanisms, whichuse alternative promoters regulating anti-apoptotic Bcl-xL, pro-apoptotic Bcl-xS, and other isoforms (25). The luciferase con-struct, pGL2–0.6R (22), contains a 0.6-kb fragment within theP1/P2 region of the bcl-x promoter (25) with regulatory sitesfor specifically expressing the anti-apoptotic isoform Bcl-xL. Asshown in Figure 1E, treatment of cells with 5-HT or ET-1, butnot hypoxia, promoted luciferase reporter gene expression con-trolled by the 0.6-kb proximal region of the bcl-x promoter.

Western blot also shows the Bcl-xL protein expression beingenhanced by 5-HT or ET-1 (Figure 1F). These results demon-strate that 5-HT and ET-1, which have been shown to elicitsignal transduction for cell proliferation, also promote gene tran-scription of anti-apoptotic Bcl-xL in pulmonary artery SMC.

To confirm the anti-apoptotic functions of Bcl-xL in pulmo-nary artery SMC, adenovirus expressing human Bcl-xL was con-structed and the effects of Bcl-xL overexpression on cell deathwere studied. Enhanced expression of Bcl-xL via adenovirus-mediated gene transfer in BPASMC effectively attenuated theability of SNP to induce cell death (Figure 2A). SNP also causedthe disruption of mitochondrial membrane potential, an earlyevent of apoptosis, as indicated in green color in Figure 2B.These effects of SNP on mitochondrial membrane potential werealmost completely prevented by the overexpression of Bcl-xL,demonstrating a functional role of Bcl-xL in regulating pulmo-nary artery SMC apoptosis. Further, the apoptosis of pulmonaryartery SMC induced by agents such as SNAP and DNR wasattenuated by 5-HT or ET-1 (Figure 2C), which activates Bcl-xL.Collectively, these results demonstrate that pulmonary vascularremodeling is associated with increased expression of Bcl-xL

which can indeed serve as an anti-apoptotic factor in pulmonaryartery SMC. Thus, suppressing this anti-apoptotic protein mightbe a way to induce apoptosis for regressing pulmonary vascularthickening.

SNP Down-Regulates the Expression of Anti-ApoptoticProtein Bcl-xL

The ability of NO donors to induce apoptosis of pulmonaryartery SMC (26–28) might play important roles in their beneficialeffects observed in patients with pulmonary hypertension (29–32). Thus, we tested the hypothesis that an NO donor, SNP,might reduce the expression of Bcl-xL expression, particularly inthe remodeled pulmonary artery. Rats were subjected to chronichypoxia for 2 wk to induce pulmonary vascular thickening andthe organ culture of isolated pulmonary vessels was treated withSNP. RT-PCR analysis of RNA obtained from these prepara-tions revealed the expression of bcl-xL mRNA that was decreasedby the SNP treatment (Figure 3A). Similarly, a treatment ofBPASMC with 100 �M SNP for 24 h resulted in decreasedexpression of Bcl-xL protein (Figure 3B).

To determine whether the down-regulated Bcl-xL protein ex-pression is due to suppressed gene transcription, effects of SNPon the bcl-xL promoter activity were studied. Using the luciferasereporter construct with the 0.6-kb bcl-x promoter, we found thatSNP inhibited the transcriptional activity. As shown in Figure3C, transfection of BPASMC with the bcl-x promoter-controlledreporter vector increased firefly luciferase activity that was ap-parent by 24 h and peaked at 48 h. Treatment with SNP sup-pressed this activity. Cells were co-transfected with thymidinekinase promoter-controlled Renilla luciferase reporter constructto assess the specificity of SNP actions to the bcl-x promoter.The ratio of the bcl-x promoter activity to thymidine kinasepromoter activity was decreased by 70% with 150 �M SNP and90% with 300 �M SNP (Figure 3D). These results suggest thatSNP inhibits Bcl-xL gene expression by modulating the activityof the 0.6-kb promoter region.

SNP Down-Regulates GATA DNA Binding Activity

The 0.6-kb region of the bcl-x promoter contains two GATAconsensus motifs (22), and a recent study in cardiac muscle cellsidentified that these two sites are regulated by GATA-4 (33).We have previously reported that BPASMC express GATAbinding factors including GATA-4. Further, mediators of pulmo-nary hypertension and inducers of pulmonary artery SMC

Suzuki, Nagase, Wong, et al.: Regulation of Bcl-xL in Smooth Muscle 683

Figure 4. Effects of SNP on GATA-4. (A )BPASMC were treated with SNP for 20 h.Nuclear extracts were prepared and theGATA DNA-binding activity was moni-tored by EMSA. The bar graph representsmeans � SE of the intensity of GATA activ-ity from cells untreated or treated withSNP (300 �M) (n 3). Asterisk denotesa value significantly different from thecontrol value at P 0.05. (B ) BPASMCwere infected with control adenovirus(AdCont) or adenovirus expressing wild-type GATA-4 (AdGATA4) for 24 h, thentreated with SNP (100 �M) for 24 h. Celllysates were prepared and levels of Bcl-xL

expression were monitored by Westernblot. The ERK antibody was used as a load-ing control. The values in the bar graphrepresent means � SE. Asterisk denotesvalues significantly different from SNP-treated values at P 0.05. (C ) HPASMCwere treated with SNP (100 �M) for dura-tions indicated. Total RNA was isolatedand mRNA expression levels of gata4 andg3pdh were determined by RT-PCR. Thebar graph shows the ratio of gata4 tog3pdh bands. Similar results were ob-tained in three separate experiments. (D )Rats were subjected to chronic hypoxiawith 10% O2 in an OxyCycler OxygenProfiler for 2 wk to elicit pulmonary vascu-lar remodeling. Remodeled pulmonaryarteries were surgically isolated, cut into� 2-mm segments, and plated in DMEM.Arterial segments were treated with SNP(300 �M) for 20 h. Arterial segments werehomogenized in Trizol and total RNA wasprepared. RT-PCR was performed withprimers for gata4 and g3pdh mRNA. Thebar graph represents means � SE of theintensity of the gata4 band. Asterisk de-notes a value significantly different fromthe control value at P 0.05.

growth such as 5-HT and ET-1 activate GATA-4 DNA binding(21). Treatment of BPASMC with SNP caused dose-dependentsuppression of GATA DNA binding activity (Figure 4A). Densi-tometry analysis demonstrated that a treatment of BPASMCwith 300 �M SNP for 20 h resulted in 90% reduction in GATAactivity. SNP also inhibited the GATA-binding activity inHPASMC, which also contain GATA-4 (data not shown).

To determine whether GATA-4 is involved in the regulationof Bcl-xL expression, effects of ectopic expression of GATA-4were tested. Adenovirus-mediated gene transfer of wild-typeGATA-4 increased the GATA-4 protein expression and GATADNA binding activity in BPASMC. SNP had no effects on ectopi-cally induced GATA activity (data not shown). We found thatthe overexpression of GATA-4 via adenovirus-mediated genetransfer attenuated SNP-induced suppression of Bcl-xL (Figure4B). These results provide direct evidence for the role of GATAin SNP-induced down-regulation of Bcl-xL.

We next investigated the mechanisms of GATA down-regulationinduced by SNP in pulmonary artery SMC. In HPASMC, theexpression of gata4 mRNA was confirmed by RT-PCR experi-ments using PCR primers derived from the known humanmRNA sequence. Further, down-regulation of the GATA activ-

ity by SNP appears to be due to decreased expression ofGATA-4, as the mRNA expression of gata4 was suppressed to50% of control by 1 h and to 10% by 4 h in response totreatment with 100 �M SNP (Figure 4C). In organ culture ofremodeled rat pulmonary artery, SNP was also found to effec-tively reduce gata4 mRNA expression (Figure 4D).

SNP Suppresses gata4 Gene Transcription

To determine whether the inhibitory effects of SNP on GATA-4expression is regulated at the level of gata4 gene transcription,we first identified the transcriptional start site and then clonedthe 1,000-bp mouse gata4 promoter into the pGL3 luciferasevector. This promoter region is directly upstream from the tran-scriptional start site that is 4.1 kb upstream from the translationalstart site as determined by the 5�-RACE analysis (Figure 5A),and shares 90% homology with the promoter of the rat gata4gene. Transfection of this luciferase construct in BPASMC re-sulted in expression of firefly luciferase within 24 h after transfec-tion. The activity of this gata4 promoter fragment was suppressedby treating cells with SNP (Figure 5B).

To examine the site of gata4 promoter, which might be responsi-ble for SNP-induced inhibition of gata4 promoter-dependent gene

684 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 36 2007

transcription, the 1,000-bp region was further truncated to 500-and 250-bp fragments. We found that truncation of the 1,000-bpregion did not significantly alter the basal transcriptional activity(Figure 5C), suggesting that the 250-bp proximal region containsimportant regulatory elements for the basal expression ofGATA-4 in pulmonary artery SMC. We found that SNP similarlyaffected gene transcription controlled by the 1,000-, 500-, and250-bp regions (Figure 5D), suggesting that the SNP-target sitemight reside within the proximal 250-bp region of the gata4promoter. Thus, SNP appears to affect the proximal 250-bpregion, suppress GATA-4 expression, and in turn inhibit genetranscription of Bcl-xL.

To identify transcription factors, which regulate the proximal250-bp region of the gata4 promoter, supershift experiments

�

Figure 5. Effects of SNP on the gata4 gene promoter activity. (A ) Ascheme depicting the mouse gata4 gene structure with the major tran-scriptional start site of mouse gata4 gene identified by 5�RACE to occur4.1 kb upstream of the translational start site. The 1,000 bp upstreamfrom the identified transcriptional start site (shaded area) that is con-served among various species was cloned into a luciferase reportervector. (B ) BPASMC were co-transfected with the firefly luciferase con-struct controlled by the 1,000-bp proximal region of the gata4 promoter(pGATA4) and Renilla luciferase construct controlled by the thymidinekinase promoter (pTK). Cells were then treated with SNP (300 �M) for24 h, cell lysates were prepared, and luciferase activities were measured.Values represent means � SE of the ratio of pGATA4-luciferase to pTKluciferase activities (n 4). Asterisk denotes values significantly differentfrom the untreated control value at P 0.05. (C ) The region of thegata4 promoter 1,000 bp proximal to the transcriptional start site wastruncated to generate regions of the promoter 500 or 250 bp upstreamfrom the transcriptional start site. BPASMC were transfected with lucifer-ase constructs controlled by these regions of the gata4 promoter. Valuesrepresent means � SE of the ratio of pGATA4-luciferase and pTK lucifer-ase activities (n 6–11). (D) BPASMC were co-transfected with the fireflyluciferase construct controlled by the 1,000, 500 or 250 bp fragment ofthe gata4 promoter and pTK-Renilla luciferase construct. Cells werethen treated with SNP (200 �M) for 24 h, cell lysates were prepared,and luciferase activities were measured. Values represent means � SEof percent of the ratio of pGATA4-luciferase and pTK luciferase activitiesrelative to untreated controls.

were performed using antibodies against putative binding fac-tors. As shown in Figure 6A, the proximal 250-bp region of thegata4 promoter contains an early growth response 1 (Egr1)/specificity protein 1 (Sp1) overlapping site as well as bindingsites for upstream stimulating factors (USF). Results showedthat antibodies against Egr1, USF1, and USF2 supershifted theband (Figure 6B), suggesting the binding of these transcriptionfactors to the proximal 250-bp region of the gata4 promoter.

To further examine the role of these factors in the regulationof gata4 gene transcription, a shorter 40-bp EMSA probe, whichcontains the sequence from �55 to �95 within the gata4 pro-moter, was constructed. We found a band that is supershifted byantibodies against Egr1, USF1, and USF2 in EMSA experimentsusing BPASMC nuclear extracts and this shorter 40-bp EMSAprobe. We found that this band was increased in response totreating BPASMC with SNP (Figure 6C). These results led usto hypothesize that one or more factors binding to this regionof the gata4 promoter might serve as negative regulators of gata4gene transcription. We found that the mutation of the USFbinding sites inhibited gata4 promoter activity (Figure 6D), indi-cating the importance of USF1/USF2 in the basal expression ofgata4. Further, overexpression of wild-type Egr1 inhibited thegata4 promoter activity (Figure 6E), providing evidence that theincreased Egr1 binding might lead to suppressed gata4 genetranscription.

DISCUSSION

In the present study, we found that the expression of anti-apoptotic Bcl-xL is increased by the mediators of pulmonaryhypertension. In the in vivo rat model of chronic hypoxia, pulmo-nary vascular remodeling was found to be associated with in-creased protein expression of Bcl-xL. In cultured pulmonary ar-tery SMC, 5-HT and ET-1 both increased gene transcription ofBcl-xL. Adenovirus-mediated gene transfer of Bcl-xL demon-strated that this protein indeed serves as an anti-apoptotic factorin pulmonary artery SMC. Thus, this up-regulation of Bcl-xL

Suzuki, Nagase, Wong, et al.: Regulation of Bcl-xL in Smooth Muscle 685

Figure 6. Effects of SNP on transcriptionfactors which bind to the proximal 250bp gata4 promoter. (A ) The sequence ofthe 250-bp gata4 promoter proximal tothe transcriptional start site. Putativebinding sites for transcription factors are in-dicated. (B) Nuclear extracts were preparedfrom untreated BPASMC, and the DNA-binding activity toward the 32P-labeled dou-ble-stranded 250 bp gata4 promoter probewas monitored by EMSA. Supershift experi-ments were performed with antibodies(ab) indicated. No ab indicates controlsfor supershift experiments without the in-clusion of any antibodies in nuclear ex-tracts from untreated cells. The letter Aindicates the DNA-protein complex with-out supershift. The letter B indicates thefree probe. The gel at the bottom showsthat the band A can be eliminated by in-creasing amounts of the cold 250-bpgata4 promoter probe. (C ) BPASMC weretreated with SNP (100 or 300 �M) for2 h, and nuclear extracts were prepared.The DNA-binding activity toward the32P-labeled double stranded oligonucleo-tide probe, which contains the sequencefrom position –95 to –55 as indicated inthe box in A, was monitored by EMSA.Supershift experiments were performedwith antibodies (ab) indicated. Solidarrows indicate supershifted bands. Theopen arrow indicates the band of interest.The line graph indicates means � SE ofthe intensity of the band of interest(n 3). Asterisks denote values signifi-cantly different from the untreated con-trol value at P 0.05. (D ) BPASMC weretransfected with the luciferase constructcontrolled by the wild-type (wt) 250-bpproximal region of the gata4 promoterand this promoter with two-nucleotidemutation at –71 and –70 (from CA to TT)within the USF binding site for 48 h. Cell

lysates were then prepared and luciferase activities were measured. Values represent means � SE of the luciferase activity (n 8). Asterisk denotesvalues significantly different from the wt value at P 0.05. (E ) BPASMC were co-transfected with the luciferase construct controlled by the250-bp proximal region of the gata4 promoter and CMV promoter-controlled Egr1 vector for 48 h. Cell lysates were then prepared and luciferaseactivities were measured. Values represent means � SE of the ratio of luciferase activity (n 4). Asterisk denotes values significantly different fromthe control value at P 0.05.

might be related to increased pulmonary vascular thickness inpulmonary hypertension.

Regression of thickened pulmonary vasculature by inducingapoptosis of SMC may serve as an effective way to treat patientswith pulmonary hypertension (1–5). Thus, it is helpful to under-stand mechanisms of apoptotic regulation in pulmonary arterySMC. Some of the apoptotic agents might induce apoptosis ofpulmonary vascular SMC by suppressing the actions of anti-apoptotic Bcl-xL. Indeed, we found that SNP can down-regulateBcl-xL gene expression. SNP inhibits the transcriptional activityof the 0.6-kb proximal region of the bcl-x gene promoter that isresponsible for the expression of anti-apoptotic isoform Bcl-xL.This region of the bcl-x promoter contains two GATA-bindingsites. We previously reported that GATA-4 is expressed inpulmonary artery SMC (21), and the present study showed thatSNP down-regulates the GATA-4 expression. Adenovirus-

mediated gene transfer of GATA-4 attenuated SNP-induceddown-regulation of Bcl-xL, providing direct evidence for theGATA-4 regulation of Bcl-xL gene transcription. We also identi-fied an SNP-responsive site in the 250 bp upstream from thetranscriptional start site of the gata4 gene. Although furtherinvestigations are needed to determine the exact mechanism ofSNP actions, the present study provided evidence that the factorsthat regulate the –55 to –95 region might play a role. We proposethat Egr1 binding might suppress the gata4 gene transcriptionby affecting neighboring factors such as USF1 and USF2.

The growth of pulmonary artery SMC is an important compo-nent of the pathogenesis of pulmonary hypertension, and de-pends on the balance between proliferation and death of cells.Anti-proliferative and pro-apoptotic events may regress thick-ened arterial walls (34, 35). Interestingly, vasoconstrictors ofpulmonary arteries such as 5-HT and ET-1 induce proliferation

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as well as anti-apoptotic responses, whereas vasodilators, suchas nitric oxide, suppress cell growth and induce the apoptosisof pulmonary artery SMC (36). Lung tissues from patients withprimary pulmonary hypertension have fewer apoptotic cellscompared with normal lungs (37) and have increased levels ofanti-apoptotic genes such as Bcl-2 (38). Apoptotic cells are de-tected in rat main pulmonary arteries during reversal of remodel-ing produced by chronic hypoxia (39). McMurtry and coworkers(3) demonstrated that the induction of SMC apoptosis preventsand reverses pulmonary hypertension. Recently, gene therapywith inhalation of an adenovirus expressing phosphorylation-deficient mutant of survivin was found to induce pulmonaryartery SMC apoptosis and reverse pulmonary hypertension inrats (4). Therefore, further identifications of molecular mecha-nisms of apoptotic and anti-apoptotic signaling in pulmonaryartery SMC should be useful to develop therapeutic strategiesto treat pulmonary hypertension.

Proteins derived from the bcl-x gene play important roles inthe regulation of apoptosis. There are five Bcl-x isoforms identi-fied so far, including Bcl-xL, Bcl-xS, Bcl-x�, Bcl-x , and Bcl-x�TM

(25). These protein isoforms are translated from differentmRNAs that are transcribed under the regulation of five bcl-xgene promoters named P1, P2, P3, P4, and P5 (25). Proximalpromoter regions P1 and P2, which are located within 802 bpupstream from the ATG site, are responsible for the regulationof anti-apoptotic Bcl-xL (25). There are two GATA elementslocated in the P1/P2 region (22). The GATA family of transcrip-tion factors include six genes, with a highly conserved zinc-fingerDNA binding domain, that interacts with the consensus (A/T)GATA(A/G) sequence. Gregory and colleagues (40) demon-strated that GATA-1 induces expression of erythroid cell Bcl-xL.In cardiac muscle cells, GATA-4 was found to play an importantrole in the regulation of apoptosis (41) and Bcl-xL expressioninduced by survival factors such as hepatocyte growth factor(42). Aries and coworkers reported that GATA-4 is the primarytranscription factor for the two GATA elements in first noncod-ing exon of bcl-x gene in cardiac myocytes (33).

Recent work from our laboratory has demonstrated thatpulmonary artery SMC express GATA-4, which appears to me-diate the growth of these cells (21); thus, this transcription factormay be involved in the development of pulmonary hypertension.Inducers of pulmonary artery SMC growth and mediators ofpulmonary hypertension, such as 5-HT and ET-1, activateGATA-4 via the MEK–ERK pathway (21). This signal transduc-tion pathway activates genes associated with pulmonary vasculardisease such as S100A4/Mts1 (43). 5-HT and ET-1 can also elicitcell survival signaling, as this study demonstrated that theseagents protect pulmonary artery SMC against apoptosis, perhapsthrough regulation by GATA-4 and Bcl-xL. In the present study,an anti-proliferative and apoptotic agent, SNP, was found toinhibit Bcl-xL expression and down-regulate gata4 gene transcrip-tion. These results suggest that Bcl-xL expression is enhancedby mediators of pulmonary hypertension and is down-regulatedby agents that suppress pulmonary vascular remodeling, at leastin part via the GATA-4–dependent mechanism. Further workis needed to establish the roles of Bcl-xL and GATA-4 in theregulation of pulmonary vascular thickening, and to determinewhether these factors might serve as therapeutic targets to pre-vent and/or treat pulmonary vascular disease.

Conflict of Interest Statement : None of the authors has a financial relationshipwith a commercial entity that has an interest in the subject of this manuscript.

Acknowledgments : The authors thank Chia Chi Tan, Sarah Fitch, Jason Tilan,Tufani SenGupta, Young Lee, Drazenka Nemcic-Moerl, Kai Nie, and Karen Pitlykfor excellent technical assistance; Dr. Aiguo Ma for help in comet assay at theonset of this project; Dr. Takayuki Ikeda for providing PCR primers; and Dr. MichaelSegel for help with transfection of BPASMC.

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