• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Am J Respir Cell Mol Biol. Author manuscript; available in PMC Jun 1, 2007.
Published in final edited form as:
PMCID: PMC1868666
NIHMSID: NIHMS17928

Regulation of Bcl-xL expression in lung vascular smooth muscle

Abstract

Pulmonary hypertension is characterized by thickened pulmonary arterial walls due to increased number of pulmonary artery smooth muscle cells (PASMC). Apoptosis of PASMC may play important roles in regulating the PASMC number and may be useful for reducing pulmonary vascular thickening. The present study examined the regulation of an anti-apoptotic protein Bcl-xL. Bcl-xL expression was found to be increased in the pulmonary artery of chronic hypoxia treated rats with pulmonary vascular remodeling. Adenovirus-mediated gene transfer of Bcl-xL indeed showed that this protein has anti-apoptotic activities in PASMC. Treatment of remodeled pulmonary artery with sodium nitroprusside (SNP) reduced Bcl-xL expression by targeting the bcl-xL promoter. The bcl-xL promoter contains two GATA elements, and SNP decreases the GATA-4 DNA binding activity. Overexpression of GATA-4 attenuated the SNP-mediated suppression of Bcl-xL expression, providing direct evidence for the role of GATA-4 in Bcl-xL gene transcription. We identified that SNP targets the 250 proximal region of the gata4 promoter and suppresses its gene transcription. Thus, inducers of pulmonary hypertension enhance anti-apoptotic Bcl-xL gene transcription, which can be suppressed by targeting the gata4 gene transcription.

Keywords: Apoptosis, Genes, Pulmonary hypertension, Smooth muscle

Introduction

Pulmonary hypertension is characterized by the elevation of pulmonary vascular resistance, which interferes with the ejection of blood by the right ventricle and ultimately causes heart failure. It is often developed secondary to various cardiovascular and pulmonary diseases such as left ventricular failure, congenital heart defects, chronic obstructive pulmonary disease, sleep apnea syndrome and post-thrombotic diseases. Pulmonary arterial hypertension can also occur as a genetic disorder. Pulmonary hypertension is associated with increased vasoconstriction in the pulmonary circulation and vascular remodeling in part due to thickening of pulmonary vascular wall because of increased number of smooth muscle cells (SMC). While therapeutic agents are available which target the vasoconstrictive aspect of this condition, preventing or treating pulmonary vascular remodeling is also needed. Recently, apoptosis-based therapeutic strategies to reduce pulmonary vascular thickening gained attention and have been successful in experimental animals (1-5). Thus, further understanding of the regulation of apoptosis in pulmonary vascular smooth muscle should promote developing therapeutic strategies to treat pulmonary hypertension.

The Bcl-2 family consists of anti-apoptotic members including Bcl-2, Bcl-xL, Bcl-w, and Ced9, while Bax, Bid, Bad, Bak and Bcl-xS belong to the pro-apoptotic group. Bcl-x plays a dual role in apoptotic regulation, using the different splicing protein products; one being anti-apoptotic Bcl-xL and another being a shorter form, pro-apoptotic Bcl-xS (6). Bcl-xL often represents the major isoform expressed in various tissues. Bcl-xS is a dominant negative repressor of Bcl-2 and Bcl-xL (7,8). Overexpression of Bcl-xS has been shown to enhance apoptosis of cancer cells (9-13).

A series of observations suggest the role of Bcl-xL in the regulation of apoptosis in smooth muscle cells (SMC) from systemic circulation during various disease conditions. In a rabbit carotid artery balloon injury model, Bcl-xL mRNA and protein levels are upregulated in the atheromatous lesion, and the anti-sense Bcl-xL increases intimal smooth muscle apoptosis and reduces the intimal lesion size and thickness (14). Similarly, in mouse coronary artery with arteriopathy after the cardiac allograft, antisense Bcl-xL increased intimal cell apoptosis and suppressed arterial neointimal formation (15). Angiotensin II increases medial and neointimal apoptosis and downregulates Bcl-xL expression (16). Regulation of Bcl-xL in pulmonary vascular smooth muscle has not been defined. Recently, neonatal rats with pulmonary hypertension were found to have increased expression of Bcl-xL in pulmonary arterial walls (17). Bcl-xL has also been shown to be upregulated as a cytoprotective response in hyperoxic acute lung injury (18).

We here report that mediators of pulmonary vascular remodeling increase the expression of Bcl-xL in pulmonary artery. Bcl-xL levels can effectively be reduced by apoptotic agents such as sodium nitroprusside (SNP). SNP-mediated downregulation of Bcl-xL is dependent on the inhibition of GATA-4 gene expression in remodeled pulmonary artery. We cloned the gata4 promoter and identified the site of SNP actions.

Methods

Culture of pulmonary artery SMC

Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2-6 passages were maintained in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 0.5% fungisone at 5% CO2 and 37°C. Cells were treated with sodium nitroprusside (SNP; Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, Dan 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 experiments, cells were infected with 50 pfu/cell of adenovirus expressing wild-type GATA-4 (a gift from Dr. J. D. Molkentin, Univ. of Cincinnati). Adenovirus expressing Bcl-xL was constructed 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 cells to hypoxia, cells were placed in a cell culture incubator that is controlled to maintain 5% O2, 5% CO2 and 37°C.

Chronic hypoxia treatment of rats

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

Histological measurements

For histological analysis, tissues were immersed in buffered 4% paraformaldehyde with 10% sucrose at 4 °C for 24 h, and were embedded in Microtome Tissue Tek II. Frozen tissues were cut to 7-μm-thick slices and mounted on glass slides. Tissue sections were stained with hematoxylin and eosin for microscopic evaluation at 200x magnification. Wall thickness values were determined by the IP Lab Software (Scanalytics Inc, VA).

For immunohistochemistry, tissue sections were washed with PBS and incubated in 3% H2O2 in methanol for 10 min at room temperature to block endogenous peroxidases. After washing in PBS, tissues were incubated in a 3% BSA solution for 1 h to block non-specific binding. Tissues were then incubated overnight at 4 °C with PBS containing 3% BSA and 1:1000 dilution of the primary antibody, rabbit anti-Bcl-xL or anti-desmin (Santa Cruz Biotechnology, Santa Cruz, CA). Tissue sections were rinsed, incubated with biotinylated goat anti-rabbit secondary antiserum, and immunoreaction was visualized by incubating with 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 were grown in 12-well plates for 24 h followed by treatment with or without testing reagents for 24 h. DePsipher Kit (Trevigen Inc., Gaithersburg, MD) was used to detect changes in mitochondrial membrane potential with a cationic dye 5,5′6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide. In accordance with the manufacture's instruction, cells were incubated for 20 min in 1x reaction buffer, stabilizing solution and DePsipher solution at 37°C. The cells were examined under a fluorescence microscope (Olympus) with a red/green dual filter cube.

Comet Assay

The neutral comet assay was used to measure double stranded DNA breaks as an indication of apoptosis. Cells were treated with apoptotic stimuli, washed in PBS, embedded in 1% agarose and placed on a comet slide (Trevigen, Gaithersburg, MD). 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 electrophoresed for 20 min at 1 V/cm in 1x TBE, fixed in ethanol, followed by staining with Sybr Green and visualized with a fluorescence microscope at 478 nm excitation and 507 nm emission wavelengths. Between 100 and 150 comets were scored per experiment and apoptotic cells were assigned based on their tail moments (20).

Western blot

To prepare lysates, the cells were washed in PBS and solubilized with 50 mM Hepes solution (pH 7.4) containing 1% (v/v) Triton X-100, 4 mM EDTA, 1 mM sodium fluoride, 0.1 mM sodium orthovanadate, 1 mM tetrasodium pyrophosphate, 2 mM PMSF, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Intact tissues were homogenized in this solution with Polytron. Equal protein amounts were electrophoresed through a reducing SDS polyacrylamide gel and electroblotted onto a membrane. The membrane was blocked and incubated with the polyclonal IgG for Bcl-xL or ERK (Santa Cruz Biotechnology). Protein levels were detected with HRP-linked secondary antibodies and ECL (Amersham Life Science, Arlington Heights, IL).

Electrophoretic mobility shift assays (EMSA)

Nuclear extracts were prepared as previously described (21). For EMSA, the binding reactions were performed for 20 min in 5 mM Tris-HCl (pH 7.5), 37.5 mM KCl, 4% (w/v) Ficoll 400, 0.2 mM EDTA, 0.5 mM DTT, 1 μg poly(dI-dC).poly(dI-dC), 0.25 ng (>20,000cpm) 32P-labeled double stranded oligonucleotide, and 2 μg protein of nuclear extract. Electrophoresis of samples through a native 6% polyacrylamide gel will be followed by autoradiography. The double stranded EMSA probes used in the present study include an oligonucleotide with two GATA consensus elements 5′-CAC TTG ATA ACA GAA AGT GAT AA CT CT-3′, the proximal 250 bp region of the gata4 promoter, and an oligonucleotide containing the sequence from positions −95 to −55 of the gata4 promoter. Supershift experiments were performed by incubating nuclear extracts with 2 μg of antibodies for Egr1, Sp1, USF1 and USF2 (Santa Cruz Biotechnology).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA (1 μg) extracted using TRIZOL (Invitrogen, Carlsbad, CA) was reverse-transcribed by oligo(dT) priming and MMLV reverse transcriptase (Applied Biosystems, Foster City, CA). The resultant cDNA was amplified using Taq DNA polymerase (Invitrogen) and resolved on a 1.5% agarose gel containing ethidium bromide. Two sets of PCR primers for human GATA-4 were designed and used in this study to confirm the expression of gata4 mRNA. The primer pair 5′-CTG TGC CAA CTG CCA GAC C-3′ and 5′-CTG CTG TGC CCG TAG TGA G-3′ give expected PCR product size of 306 bp, and the pair 5′-CAA CTC CAG CAA CGC CAC C-3′ and 5′-AAT CCA ACA CCC GCT TCC C-3′ produces 441 bp. Levels of rat mRNA were monitored using PCR primers with the following sequences: for gata4 mRNA, 5′-CAG GCA GAA AGC AAG GAC TA-3′ and 5′-CAT AGC CAG GCT TTG GTA CAT-3′; for bcl-xL mRNA, 5′-AGG ATA CAG CTG GAG TCA G-3′ and 5′-TCT CCT TGT CTA CGC TTT CC-3′. Denaturing was performed at 94C for 45 sec. Annealing processes were for 45 sec 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. Results were obtained at various cycles in order 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 in a 12-well plate. 1 μg DNA/well was transfected using the Fugene 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) in serum-free, antibiotic-free RPMI. Co-transfection of the Renilla reporter (0.1 μg/well) was performed to normalize for transfection differences between wells. Cells were transfected for 6 h, and then medium was replaced with RPMI containing 0.1% FBS with antibiotics. Cells were treated 1 h later.

Luciferase assays were performed using the Dual Luciferase Assay Kit (Promega). Transfected cells were washed in PBS and lysed. Cellular debris was removed by centrifugation at 14,000g for 30 sec. Cell lysates were added to Luciferase Assay Reagent II and the firefly luciferase activities were read in a Model TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). An equal volume of Stop and Glow was added, and the Renilla reading was taken. The ratio of firefly luciferase to Renilla luciferase was observed for each well of transfection. The luciferase construct controlled by the 0.6 kb proximal promoter region, pGL2-0.6R (22) was a gift from Dr. Nunez (Univ. of Michigan).

5′ Rapid Amplification of cDNA Ends (5′ RACE)

Total RNA was isolated from the C57BL/6 mouse heart by TRIzol (Invitrogen). Antisense primer (5′-CAG CAT CAA AGC AGA AAC-3′) located within exon 2 was used for first-strand synthesis. Subsequent amplification was performed using the 5′ RACE System for Amplification of cDNA Ends (Invitrogen). In brief, first strand cDNA was tailed with recombinant TdT and linker (dC) oligonucleotide. 5′ RACE was performed by incubating with an aliquot of RACE primer located upstream of anti-sense primer (5′-AGG CTC TGG TTT GCT CAG GAA AAA-3′) and with Abridged Anchor Primer (AAP) using Platinum Taq High-Fidelity DNA polymerase (Invitrogen). Subsequently, nested PCR was performed with a nested primer designed upstream of RACE primer (5′-CCA AAT TGG ATT TGC GGT TGC T-3′) and Abridged Universal Amplification Primer (AUAP). The nested primer was used to sequence the PCR product to determine the transcriptional start site.

Cloning of gata4 gene promoter

Fragments containing proximal 1,000 bp, 500 bp, and 250 bp regions of the gata4 gene promoter were cloned by PCR cloning using mouse genomic DNA obtained from Promega (Madison, WI). Primers for each PCR fragments were: 5′-TGA CAT GGT ACC AAA AGT TTA GCC CAA AGC GCG A-3′ (1000 bp Forward), 5′-TGA CAT GGT ACC AAG GGC CAG TTC AGG TTT TAG TG-3′ (500 bp Forward), 5′-TGA CAT GGT ACC AAG GAC GTC GGG CTG CAC TGA-3′ (250 bp Forward), 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 encodes a Hind III site. PCR was performed with Platinum Taq DNA Polymerase High Fidelity (Invitrogen) with the primers, NTPs, MgSO4, and buffer. PCR reactions were performed for 40 cycles using a 30 sec denaturation at 95°C, 1 min annealing at 65°C for 1000 bp and 500 bp fragments and at 68°C for the 250 bp fragment, and 6 min extension at 72°C. This reaction resulted in one band each of approximately 1000, 500, and 250 bp on agarose gel. The product was purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Both the vector and purified PCR fragments were digested overnight at 37°C with Kpn I and Hind III (New England Biolabs, Beverly, MA); digested fragments were purified by QIAquick PCR Purification Kit (Qiagen) and ligated into the pGL3 luciferase reporter vector (Promega) by T4 DNA Ligase (New England Biolabs, Beverly, MA). Vectors positive for inserts were screened by digestion and subjected to bidirectional sequencing.

Statistical analysis

Means ± S.E. were calculated. Significant differences between all groups were computed by one-way analysis of variance using an F statistics. Statistically significant differences between two groups were determined by the Student's t-test.

Results

Effects of mediators of pulmonary vascular remodeling on Bcl-xL gene expression

To determine if Bcl-xL expression is regulated in remodeled pulmonary artery smooth muscle in vivo, rats were subjected to 2, 7 or 14 days of chronic hypoxia (10% O2) to induce pulmonary hypertension. In these rats, we noted significant increase in the mass of right ventricle/(left ventricle + septum) as early as 7 days of chronic hypoxia, indicating increased pulmonary vascular resistance and resultant right ventricular hypertrophy (Fig. 1A). Pulmonary vascular thickening was evident by 14 days of chronic hypoxia (Fig. 1B). In these remodeled pulmonary arteries, increased Bcl-xL expression was observed by Western blot (Fig. 1C). Histological analyses also revealed an increased expression of Bcl-xL protein in the lung of rats treated with 14 days of chronic hypoxia (Fig. 1D), showing enhanced Bcl-xL expression in pulmonary arteries as well as surrounding lung structures. These results demonstrate in vivo that pulmonary vascular remodeling is associated with increased expression of anti-apoptotic Bcl-xL.

Fig. 1
Effects of the mediators of pulmonary hypertension on Bcl-xL expression. (A) Rats were subjected to chronic hypoxia at 10% O2. Right ventricle (RV)/[left ventricle (LV) + septum (S)] values were measured as indications of the occurrence of pulmonary hypertension ...

Vasoactive agents such as 5-HT and ET-1 also induce pulmonary artery SMC proliferation and are thought to play important roles in promoting pulmonary vascular remodeling (23,24). We found that these agents can also increase Bcl-xL gene expression. The bcl-x promoter is controlled by complex mechanisms, which utilize alternative promoters regulating anti-apoptotic Bcl-xL, pro-apoptotic Bcl-xS and other isoforms (25). The luciferase construct, pGL2-0.6R (22), contains a 0.6 kb fragment within the P1/P2 region of the bcl-x promoter (25) with regulatory sites for specifically expressing anti-apoptotic isoform Bcl-xL. As shown in Fig. 1E, treatment of cells with 5-HT or ET-1, but not hypoxia, promoted luciferase reporter gene expression controlled by the 0.6 kb proximal region of the bcl-x promoter. Western blot also shows the Bcl-xL protein expression being enhanced by 5-HT or ET-1 (Fig. 1F). These results demonstrate that 5-HT and ET-1, which have been shown to elicit signal transduction for cell proliferation, also promote gene transcription of anti-apoptotic Bcl-xL in pulmonary artery SMC.

To confirm the anti-apoptotic functions of Bcl-xL in pulmonary artery SMC, adenovirus expressing human Bcl-xL was constructed and the effects of Bcl-xL overexpression on cell death were studied. Enhanced expression of Bcl-xL via adenovirus-mediated gene transfer in BPASMC effectively attenuated the ability of SNP to induce cell death (Fig. 2A). SNP also caused the disruption of mitochondrial membrane potential, an early event of apoptosis, as indicated in green color in Fig. 2B. These effects of SNP on mitochondrial membrane potential were almost completely prevented by the overexpression of Bcl-xL, demonstrating a functional role of Bcl-xL in regulating pulmonary artery SMC apoptosis. Further, the apoptosis of pulmonary artery SMC induced by agents such as SNAP and DNR was attenuated by 5-HT or ET-1 (Fig. 2C), which activates Bcl-xL. Collectively, these results demonstrate that pulmonary vascular remodeling is associated with increased expression of Bcl-xL which can indeed serve as an anti-apoptotic factor in pulmonary artery SMC. Thus, suppressing this anti-apoptotic protein might be a way to induce apoptosis for regressing pulmonary vascular thickening.

Fig. 2
Effects of Bcl-xL on pulmonary vascular SMC apoptosis. (A) BPASMC were infected with adenovirus expressing Bcl-xL (AdBcl-xL) for 48 h, then treated with SNP (300 μM) for 17 h. Cells were washed with PBS, trypsinized, incubated with Trypan Blue ...

SNP downregulates the expression of anti-apoptotic protein Bcl-xL

The ability of NO donors to induce apoptosis of pulmonary artery SMC (26-28) might play important roles in their beneficial effects observed in pulmonary hypertension patients (29-32). Thus, we tested the hypothesis that an NO donor, SNP, might reduce the expression of Bcl-xL expression, particularly in the remodeled pulmonary artery. Rats were subjected to chronic hypoxia for 2 wks to induce pulmonary vascular thickening and the organ culture of isolated pulmonary vessels was treated with SNP. RT-PCR analysis of RNA obtained from these preparations revealed the expression of bcl-xL mRNA that was decreased by the SNP treatment (Fig. 3A). Similarly, a treatment of BPASMC with 100 μM SNP for 24 h resulted in decreased expression of Bcl-xL protein (Fig. 3B).

Fig. 3
Effects of SNP on Bcl-xL expression. (A) Rats were subjected to chronic hypoxia with 10% O2 in an OxyCycler Oxygen Profiler for 2 weeks to elicit pulmonary vascular remodeling. Remodeled pulmonary arteries were surgically isolated, cut into ~2 ...

To determine whether the downregulated Bcl-xL protein expression is due to suppressed gene transcription, effects of SNP on the bcl-xL promoter activity were studied. Using the luciferase reporter construct with the 0.6 kb bcl-x promoter, we found that SNP inhibited the transcriptional activity. As shown in Fig. 3C, transfection of BPASMC with the bcl-x promoter-controlled reporter vector increased firefly luciferase activity that was apparent by 24 h and peaked at 48 h. Treatment with SNP suppressed this activity. Cells were co-transfected with thymidine kinase promoter-controlled Renilla luciferase reporter construct to assess the specificity of SNP actions to the bcl-x promoter. The ratio of the bcl-x promoter activity to thymidine kinase promoter activity was decreased by 70% with 150 μM SNP and 90% with 300 μM SNP (Fig. 3D). These results suggest that SNP inhibits Bcl-xL gene expression by modulating the activity of the 0.6 kb promoter region.

SNP downregulates GATA DNA binding activity

The 0.6 kb region of the bcl-x promoter contains two GATA consensus motifs (22), and a recent study in cardiac muscle cells identified that these two sites are regulated by GATA-4 (33). We have previously reported that BPASMC express GATA binding factors including GATA-4. Further, mediators of pulmonary hypertension and inducers of pulmonary artery SMC growth such as 5-HT and ET-1 activate GATA-4 DNA binding (21). Treatment of BPASMC with SNP caused dose-dependent suppression of GATA DNA binding activity (Fig. 4A). Densitometry analysis demonstrated that a treatment of BPASMC with 300 μM SNP for 20 h resulted in a 90% reduction in GATA activity. SNP also inhibited the GATA-binding activity in HPASMC, which also contain GATA-4 (data not shown).

Fig. 4
Effects of SNP on GATA-4. (A) BPASMC were treated with SNP for 20 h. Nuclear extracts were prepared and the GATA DNA-binding activity was monitored by EMSA. The bar graph represents means ± S.E. of the intensity of GATA activity from cells untreated ...

To determine whether GATA-4 is involved in the regulation of Bcl-xL expression, effects of ectopic expression of GATA-4 were tested. Adenovirus-mediated gene transfer of wild-type GATA-4 increased the GATA-4 protein expression and GATA DNA binding activity in BPASMC. SNP had no effects on ectopically induced GATA activity (data not shown). We found that the overexpression of GATA-4 via adenovirus-mediated gene transfer attenuated SNP-induced suppression of Bcl-xL (Fig. 4B). These results provide direct evidence for the role of GATA in SNP-induced downregulation of Bcl-xL.

We next investigated the mechanisms of GATA downregulation induced by SNP in pulmonary artery SMC. In HPASMC, the expression of gata4 mRNA was confirmed by RTPCR experiments using PCR primers derived from the known human mRNA sequence. Further, downregulation of the GATA activity by SNP appears to be due to decreased expression of GATA-4, as the mRNA expression of gata4 was suppressed to 50% of control by 1 h and to <10% by 4 h in response to treatment with 100 μM SNP (Fig. 4C). In organ culture of remodeled rat pulmonary artery, SNP was also found to effectively reduce gata4 mRNA expression (Fig. 4D).

SNP suppresses gata4 gene transcription

To determine whether the inhibitory effects of SNP on GATA-4 expression is regulated at the level of gata4 gene transcription, we first identified the transcriptional start site and then cloned the 1,000 bp mouse gata4 promoter into the pGL3 luciferase vector. This promoter region is directly upstream from the transcriptional start site that is 4.1 kb upstream from the translational start site as determined by the 5′-Rapid Amplification of cDNA Ends (RACE) analysis (Fig. 5A), and shares 90% homology with the promoter of the rat gata4 gene. Transfection of this luciferase construct in BPASMC resulted in expression of firefly luciferase within 24 h after transfection. The activity of this gata4 promoter fragment was suppressed by treating cells with SNP (Fig. 5B).

Fig. 5
Effects of SNP on the gata4 gene promoter activity. (A) A scheme depicting the mouse gata4 gene structure with the major transcriptional start site of mouse gata4 gene identified by 5′RACE to occur 4.1 kb upstream of the translational start site. ...

To examine the site of gata4 promoter, which might be responsible for SNP-induced inhibition of gata4 promoter-dependent gene transcription, the 1,000 bp region was further truncated to 500 and 250 bp fragments. We found that truncation of the 1,000 bp region did not significantly alter the basal transcriptional activity (Fig. 5C), suggesting that 250 bp proximal region contains important regulatory elements for the basal expression of GATA-4 in pulmonary artery SMC. We found that SNP similarly affected gene transcription controlled by the 1,000, 500 and 250 bp regions (Fig. 5D), suggesting that the SNP-target site might reside within the proximal 250 bp region of the gata4 promoter. Thus, SNP appears to affect the proximal 250 bp region, suppress GATA-4 expression, and in turn inhibit gene transcription of Bcl-xL.

To identify transcription factors, which regulate the proximal 250 bp region of the gata4 promoter, supershift experiments were performed using antibodies against putative binding factors. As shown in Fig. 6A, the proximal 250 bp region of the gata4 promoter contains an early growth response 1 (Egr1)/specificity protein 1 (Sp1) overlapping site as well as binding sites for upstream stimulating factors (USF). Results showed that antibodies against Egr1, USF1 and USF2 supershifted the band (Fig. 6B), suggesting the binding of these transcription factors to the proximal 250 bp region of the gata4 promoter.

Fig. 6
Effects of SNP on transcription factors which bind to the proximal 250 bp gata4 promoter. (A) The sequence of the 250 bp gata4 promoter proximal to the transcriptional start site. Putative binding sites for transcription factors are indicated. (B) Nuclear ...

To further examine the role of these factors in the regulation of gata4 gene transcription, a shorter 40 bp EMSA probe, which contains the sequence from −55 to −95 within the gata4 promoter, was constructed. We found a band that is supershifted by antibodies against Egr1, USF1 and USF2 in EMSA experiments using BPASMC nuclear extracts and this shorter 40 bp EMSA probe. We found that this band was increased in response to treating BPASMC with SNP (Fig. 6C). These results led us to hypothesize that one or more factors binding to this region of the gata4 promoter might serve as negative regulators of gata4 gene transcription. We found that the mutation of the USF binding sites inhibited gata4 promoter activity (Fig. 6D), indicating the importance of USF1/USF2 in the basal expression of gata4. Further, overexpression of wild-type Egr1 inhibited the gata4 promoter activity (Fig. 6E), providing evidence that the increased Egr1 binding might lead to suppressed gata4 gene transcription.

Discussion

In the present study, we found that the expression of anti-apoptotic Bcl-xL is increased by the mediators of pulmonary hypertension. In the in vivo rat model of chronic hypoxia, pulmonary vascular remodeling was found to be associated with increased protein expression of Bcl-xL. In cultured pulmonary artery SMC, 5-HT and ET-1 both increased gene transcription of Bcl-xL. Adenovirus-mediated gene transfer of Bcl-xL demonstrated that this protein indeed serves as an anti-apoptotic factor in pulmonary artery SMC. Thus, this upregulation of Bcl-xL might be related to increased pulmonary vascular thickness in pulmonary hypertension.

Regression of thickened pulmonary vasculature by inducing apoptosis of SMC may serve as an effective way to treat patients with pulmonary hypertension (1-5). Thus, it is helpful to understand mechanisms of apoptotic regulation in pulmonary artery SMC. Some of the apoptotic agents might induce apoptosis of pulmonary vascular SMC by suppressing the actions of anti-apoptotic Bcl-xL. Indeed, we found that SNP can downregulate Bcl-xL gene expression. SNP inhibits the transcriptional activity of the 0.6 kb proximal region of the bcl-x gene promoter that is responsible for the expression of anti-apoptotic isofrom Bcl-xL. This region of the bcl-x promoter contains two GATA binding sites. We previously reported that GATA-4 is expressed in pulmonary artery SMC (21), and the present study showed that SNP downregulates the GATA-4 expression. Adenovirus-mediated gene transfer of GATA-4 attenuated SNP-induced downregulation of Bcl-xL, providing direct evidence for the GATA-4 regulation of Bcl-xL gene transcription. We also identified an SNP-responsive site in the 250 bp upstream from the transcriptional start site of the gata4 gene. Although further investigations are needed to determine the exact mechanism of SNP actions, the present study provided evidence that the factors which regulate the −55 to −95 region might play a role. We propose that Egr1 binding might suppress the gata4 gene transcription by affecting neighboring factors such as USF1 and USF2.

The growth of pulmonary artery SMC is an important component of the pathogenesis of pulmonary hypertension, and depends on the balance between proliferation and death of cells. Anti-proliferative and pro-apoptotic events may regress thickened arterial walls (34,35). Interestingly, vasoconstrictors of pulmonary arteries such as 5-HT and ET-1 induce proliferation as well as anti-apoptotic responses, whereas vasodilators, such as nitric oxide, suppress cell growth and induce the apoptosis of pulmonary artery SMC (36). Lung tissues from patients with primary pulmonary hypertension have fewer apoptotic cells compared to normal lungs (37) and have increased levels of anti-apoptotic genes such as Bcl-2 (38). Apoptotic cells are detected in rat main pulmonary arteries during reversal of remodeling produced by chronic hypoxia (39). McMurtry et al. (3) demonstrated that the induction of SMC apoptosis prevents and reverses pulmonary hypertension. Recently, gene therapy with inhalation of an adenovirus expressing phosphorylation-deficient mutant of survivin was found to induce pulmonary artery SMC apoptosis and reverse pulmonary hypertension in rats (4). Therefore, further identifications of molecular mechanisms of apoptotic and anti-apoptotic signaling in pulmonary artery SMC should be useful to develop therapeutic strategies to treat pulmonary hypertension.

Proteins derived from the bcl-x gene play important roles in the regulation of apoptosis. There are five Bcl-x isoforms identified so far, including Bcl-xL, Bcl-xS, Bcl-xβ, Bcl-xγ and Bcl-xΔTM (25). These protein isoforms are translated from different mRNAs that are transcribed under the regulation of five bcl-x gene promoters named P1, P2, P3, P4 and P5 (25). Proximal promoter regions P1 and P2, which are located within 802 bp upstream from the ATG site, are responsible for the regulation of anti-apoptotic Bcl-xL (25). There are two GATA elements located in the P1/P2 region (22). The GATA family of transcription factors include six genes, with a highly conserved zinc-finger DNA binding domain, that interacts with the consensus (A/T)GATA(A/G) sequence. Gregory et al. (40) demonstrated that GATA-1 induces expression of erythroid cell Bcl-xL. In cardiac muscle cells, GATA-4 was found to play an important role in the regulation of apoptosis (41) and Bcl-xL expression induced by survival factors such as hepatocyte growth factor (42). Aries et al. reported that GATA-4 is the primary transcription factor for the two GATA elements in first non-coding exon of bcl-x gene in cardiac myocytes (33).

Recent work from our laboratory have demonstrated that pulmonary artery SMC express GATA-4 which appears to mediate the growth of these cells (21), thus this transcription factor may be involved in the development of pulmonary hypertension. Inducers of pulmonary artery SMC growth and mediators of pulmonary hypertension, such as 5-HT and ET-1, activate GATA-4 via the MEK-ERK pathway (21). This signal transduction pathway activates genes associated with pulmonary vascular disease such as S100A4/Mts1 (43). 5-HT and ET-1 can also elicit cell survival signaling, as this study demonstrated that these agents protect pulmonary artery SMC against apoptosis, perhaps through regulation by GATA-4 and Bcl-xL. In the present study, an anti-proliferative and apoptotic agent, SNP, was found to inhibit Bcl-xL expression and downregulate gata4 gene transcription. These results suggest that Bcl-xL expression is enhanced by mediators of pulmonary hypertension and is downregulated by agents, which suppress pulmonary vascular remodeling, at least in part via the GATA-4 dependent mechanism. Further work is needed to establish the roles of Bcl-xL and GATA-4 in the regulation of pulmonary vascular thickening, and to determine whether these factors might serve as therapeutic targets to prevent and/or treat pulmonary vascular disease.

Acknowledgements

This work was supported in part by NIH grants HL67340 (to YJS), HL72844 (to YJS), HL73929 (to RMD), American Heart Association New England Affiliate (to YJS), and American Lung Association/Massachusetts Thoracic Society (to YJS). RMD is a recipient of the Career Development Award from the American Heart Association National Center. This work was pursued in part by a collaboration through the DC Area Consortium for Integrative Cardio-Pulmonary Biology. The opinions and assertions contained herein are the private opinions of the authors and are not to be construed as official or reflecting the views of the Uniformed Services University of the Health Sciences or the Department of Defense or the Government of the United States. All animal studies were approved by the Georgetown University Institutional Animal Care and Use Committee, and were conducted in accordance with the NRC Guide to the Care and Use of Laboratory Animals (National Academy Press, Washington DC, 1996). We thank Chia Chi Tan, Sarah Fitch, Jason Tilan, Tufani SenGupta, Young Lee, Drazenka Nemcic-Moerl, Kai Nie and Karen Pitlyk for excellent technical assistance, Dr. Aiguo Ma for help in comet assay at the onset of this project, Dr. Takayuki Ikeda for providing PCR primers, and Dr. Michael Segel for help with transfection of BPASMC.

Abbreviations

5-HT
serotonin
BPASMC
bovine pulmonary artery smooth muscle cells
DNR
daunorubicin
Egr1
early growth response 1
EMSA
electrophoretic mobility shift assays
ET-1
endothelin-1
HPASMC
human pulmonary artery smooth muscle cells
RT-PCR
reverse transcription-polymerase chain reaction
SMC
smooth muscle cells
SNAP
S-nitroso-N-acetylpenicillamine
SNP
sodium nitroprusside
Sp1
specificity protein 1
USF
upstream stimulating factor

References

1. Nishimura T, Vaszar LT, Faul JL, Zhao G, Berry GJ, Shi L, Qiu D, Benson G, Pearl RG, Kao PN. Simvastatin rescues rats from fatal pulmonary hypertension by inducing apoptosis of neointimal smooth muscle cells. Circulation. 2003;108:1640–1645. [PubMed]
2. Brevnova EE, Platoshyn O, Zhang S, Yuan JX. Overexpression of human KCNA5 increases IK V and enhances apoptosis. Am J Physiol. 2004;287:C715–C722. [PubMed]
3. McMurtry MS, Bonnet S, Wu X, Dyck JR, Haromy A, Hashimoto K, Michelakis ED. Dichloroacetate prevents and reverses pulmonary hypertension by inducing pulmonary artery smooth muscle cell apoptosis. Circ Res. 2004;95:830–840. [PubMed]
4. McMurtry MS, Archer SL, Altieri DC, Bonnet S, Haromy A, Harry G, Bonnet S, Puttagunta L, Michelakis ED. Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension. J Clin Invest. 2005;115:1479–1491. [PMC free article] [PubMed]
5. Merklinger SL, Jones PL, Martinez EC, Rabinovitch M. Epidermal growth factor receptor blockade mediates smooth muscle cell apoptosis and improves survival in rats with pulmonary hypertension. Circulation. 2005;112:423–431. [PubMed]
6. Sorenson CM. Bcl-2 family members and disease. Biochim Biophys Acta. 2004;1644:169–177. [PubMed]
7. Minn AJ, Boise LH, Thompson CB. Bcl-xS antagonizes the protective effects of Bcl-xL. J Biol Chem. 1996;271:6306–6312. [PubMed]
8. Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nunez G, Thompson CB. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993;74:597–608. [PubMed]
9. Sumantran VN, Ealovega MW, Nunez G, Clarke MF, Wicha MS. Overexpression of Bcl-XS sensitizes MCF-7 cells to chemotherapy-induced apoptosis. Cancer Res. 1995;55:2507–2510. [PubMed]
10. Sumantran VN, Lee DS, Woods Ignatoski KM, Ethier SP, Wicha MS. A bcl-xS adenovirus selectively induces apoptosis in transformed cells compared to normal mammary cells. Neoplasia. 2000;2:251–260. [PMC free article] [PubMed]
11. Ealovega MW, McGinnis PK, Sumantran VN, Clarke MF, Wicha MS. bcl-xS gene therapy induces apoptosis of human mammary tumors in nude mice. Cancer Res. 1996;56:1965–1969. [PubMed]
12. Dole MG, Clarke MF, Holman P, Benedict M, Lu J, Jasty R, Eipers P, Thompson CB, Rode C, Bloch C, Nunez G, Castle VP. Bcl-xS enhances adenoviral vector-induced apoptosis in neuroblastoma cells. Cancer Res. 1996;56:5734–5740. [PubMed]
13. Clarke MF, Apel IJ, Benedict MA, Eipers PG, Sumantran V, Gonzalez-Garcia M, Doedens M, Fukunaga N, Davidson B, Dick JE, Minn AJ, Boise LH, Thompson CB, Wicha M, Nunez G. A recombinant bcl-xS adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells. Proc Natl Acad Sci USA. 1995;92:11024–11028. [PMC free article] [PubMed]
14. Pollman MJ, Hall JL, Mann MJ, Zhang L, Gibbons GH. Inhibition of neointimal cell bcl-x expression induces apoptosis and regression of vascular disease. Nature Med. 1998;4:222–227. [PubMed]
15. Suzuki J, Isobe M, Morishita R, Nishikawa T, Amano J, Kaneda Y. Antisense Bcl-x oligonucleotide induces apoptosis and prevents arterial neointimal formation in murine cardiac allografts. Cardiovasc Res. 2000;45:783–787. [PubMed]
16. Suzuki J, Iwai M, Nakagami H, Wu L, Chen R, Sugaya T, Hamada M, Hiwada K, Horiuchi M. Role of angiotensin II-regulated apoptosis through distinct AT1 and AT2 receptors in neointimal formation. Circulation. 2002;106:847–853. [PubMed]
17. Jankov RP, Kantores C, Belcastro R, Yi M, Tanswell AK. Endothelin-1 inhibits apoptosis of pulmonary arterial smooth muscle in the neonatal rat. Pediatr Res. 2006;60:245–251. [PubMed]
18. He CH, Waxman AB, Lee CG, Link H, Rabach ME, Ma B, Chen Q, Zhu Z, Zhong M, Nakayama K, Nakayama KI, Homer R, Elias JA. Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury. J Clin Invest. 2005;115:1039–1048. [PMC free article] [PubMed]
19. Lee SL, Wang WW, Fanburg BL. Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture. Circ Res. 1991;68:1362–1368. [PubMed]
20. Kitta K, Day RM, Ikeda T, Suzuki YJ. Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis. Free Radic Biol Med. 2001;31:902–910. [PubMed]
21. Suzuki YJ, Day RM, Tan CC, Sandven TH, Liang Q, Molkentin JD, Fanburg BL. Activation of GATA-4 by serotonin in pulmonary artery smooth muscle cells. J Biol Chem. 2003;278:17525–17531. [PubMed]
22. Grillot DA, Gonzalez-Garcia M, Ekhterae D, Duan L, Inohara N, Ohta S, Seldin MF, Nunez G. Genomic organization, promoter region analysis, and chromosome localization of the mouse bcl-x gene. J Immunol. 1997;158:4750–4757. [PubMed]
23. Eddahibi S, Humbert M, Fadel E, Raffestin B, Darmon M, Capron F, Simonneau G, Dartevelle P, Hamon M, Adnot S. Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension. J Clin Invest. 2001;198:1141–1150. [PMC free article] [PubMed]
24. Davie N, Haleen SJ, Upton PD, Polak JM, Yacoub MH, Morrell NW, Wharton J. ETA and ETB receptors modulate the proliferation of human pulmonary artery smooth muscle cells. Am J Respir Crit Care Med. 2002;165:398–405. [PubMed]
25. Pecci A, Viegas LR, Baranao JL, Beato M. Promoter choice influences alternative splicing and determines the balance of isoforms expressed from the mouse bcl-X gene. J Biol Chem. 2001;276:21062–21069. [PubMed]
26. Smith JD, McLean SD, Nakayama DK. Nitric oxide causes apoptosis in pulmonary vascular smooth muscle cells. J Surg Res. 1998;79:121–127. [PubMed]
27. Krick S, Platoshyn O, Sweeney M, McDaniel SS, Zhang S, Rubin LJ, Yuan JXJ. Nitric oxide induces apoptosis by activating K+ channels in pulmonary vascular smooth muscle cells. Am J Physiol. 2002;282:H184–H193. [PubMed]
28. Wedgwood S, Black SM. Molecular mechanisms of nitric oxide-induced growth arrest and apoptosis in fetal pulmonary arterial smooth muscle cells. Nitric Oxide. 2003;9:201–210. [PubMed]
29. Roberts JD, Polaner DM, Lan P, Zapol WM. Inhaled nitric oxide in persistent pulmonary hypertension of the newborn. Lancet. 1992;340:818–819. [PubMed]
30. Snell GI, Salamonsen RF, Bergin P, Esmore DS, Khan S, Williams TJ. Inhaled nitric oxide used as a bridge to heart-lung transplantation in a patient with end-stage pulmonary hypertension. Am J Respir Crit Care Med. 1995;151:1263–1266. [PubMed]
31. Channick RN, Newhart JW, Johnson FW, Williams PJ, Auger WR, Fedullo PF, Moser KM. Pulse delivery of inhaled nitric oxide to patients with primary pulmonary hypertension. Chest. 1996;109:1545–1549. [PubMed]
32. Perez-Penate G, Julia-Serda G, Pulido-Duque JM, Gorriz-Gomez E, Cabrera-Navarro P. One-year continuous inhaled nitric oxide for primary pulmonary hypertension. Chest. 2001;119:970–973. [PubMed]
33. Aries A, Paradis P, Lefebvre C, Schwartz RJ, Nemer M. Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity. Proc Natl Acad Sci USA. 2004;101:6975–6980. [PMC free article] [PubMed]
34. Cowan KN, Jones PL, Rabinovitch M. Regression of hypertrophied rat pulmonary arteries in organ culture is associated with suppression of proteolytic activity, inhibition of tenascin-C, and smooth muscle cell apoptosis. Circ Res. 1999;84:1223–1233. [PubMed]
35. Pollman MJ, Yamada T, Horiuchi M, Gibbons GH. Vasoactive substances regulate vascular smooth muscle cell apoptosis. Countervailing influences of nitric oxide and angiotensin II. Circ Res. 1996;79:748–756. [PubMed]
36. Hampl V, Herget J. Role of nitric oxide in the pathogenesis of chronic pulmonary hypertension. Physiol Rev. 2000;80:1337–1372. [PubMed]
37. Kasahara Y, Tuder RM, Cool CD, Lynch DA, Flores SC, Voelkel NF. Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am J Respir Crit Care Med. 2001;163:737–744. [PubMed]
38. Geraci MW, Moore M, Gesell T, Yeager ME, Alger L, Golpon H, Gao B, Loyd JE, Tuder RM, Voelkel NF. Gene expression patterns in the lungs of patients with primary pulmonary hypertension: a gene microarray analysis. Circ Res. 2001;88:555–562. [PubMed]
39. Riley DJ, Thakker-Varia S, Wilson FJ, Poiani GJ, Tozzi CA. Role of proteolysis and apoptosis in regression of pulmonary vascular remodeling. Physiol Res. 2000;49:577–585. [PubMed]
40. Gregory T, Yu C, Ma A, Orkin SH, Blobel GA, Weiss MJ. GATA-1 and erythropoietin cooperate to promote erythroid cell survival by regulating bcl-xL expression. Blood. 1999;94:87–96. [PubMed]
41. Kim Y, Ma AG, Kitta K, Fitch SN, Ikeda T, Ihara Y, Simon AR, Evans T, Suzuki YJ. Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol. 2003;63:368–377. [PubMed]
42. Kitta K, Day RM, Kim Y, Torregroza I, Evans T, Suzuki YJ. Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells. J Biol Chem. 2003;278:4705–4712. [PubMed]
43. Lawrie A, Spiekerkoetter E, Martinez EC, Ambartsumian N, Sheward WJ, MacLean MR, Harmar AJ, Schmidt AM, Lukanidin E, Rabinovitch M. Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a gene associated with pulmonary vascular disease. Circ Res. 2005;97:227–235. [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • Gene
    Gene
    Gene links
  • Gene (nucleotide)
    Gene (nucleotide)
    Records in Gene identified from shared sequence links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene
    HomoloGene links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links
  • Taxonomy
    Taxonomy
    Related taxonomy entry
  • Taxonomy Tree
    Taxonomy Tree

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...