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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 an important role 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 established 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 gata4 gene transcription.
Recently, apoptosis-based therapeutic strategies to reduce pulmonary vascular thickening have gained attention. Understanding apoptotic regulation in pulmonary vascular smooth muscle should promote such strategies to treat pulmonary hypertension.
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). Although therapeutic agents are available that 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 SMC from systemic circulation during various disease conditions. In a rabbit carotid artery balloon injury model, Bcl-xL mRNA and protein levels are up-regulated 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 down-regulates 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 up-regulated 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 down-regulation 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.
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).
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, and 0.5% fungisone at 5% CO2 and 37°C. Cells were treated with SNP (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 experiments, cells were infected with 50 plaque-forming units (pfu)/cell of adenovirus expressing wild-type GATA-4 (a gift from Dr. J. D. Molkentin, Univ. of Cincinnati, Cincinnati, OH). 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.
Male Sprague Dawley rats (275–300 g) 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 d. Normoxia controls were subjected to ambient 21% O2 for 14 d. Animals were fed normal rat chow during treatment and were used in accordance with institutional guidelines.
For histologic 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 (H&E) for microscopic evaluation at ×200 magnification. Wall thickness values were determined by the IP Lab Software (Scanalytics Inc., Fairfax, 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 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). 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).
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 manufacturer's instruction, cells were incubated for 20 min in 1× reaction buffer, stabilizing solution, and DePsipher solution at 37°C. The cells were examined under a fluorescence microscope (Olympus, Center Valley, PA) with a red/green dual filter cube.
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). 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 1× Tris-Borate-EDTA (TBE), fixed in ethanol, stained 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).
To prepare lysates, the cells were washed in PBS and solubilized with 50 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 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 immunoglobulin (Ig)G for Bcl-xL or extracellular signal–regulated kinase (ERK) (Santa Cruz Biotechnology). Protein levels were detected with horseradish peroxidase–linked secondary antibodies and enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL).
Nuclear extracts were prepared as previously described (21). For electrophoretic mobility shift assays (EMSA), the binding reactions were performed 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 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).
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 94°C for 45 s. Annealing processes were 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. Results were obtained at various cycles to obtain information at a linear range (15, 20, 25, and 30 cycles).
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, Madison, WI). Transfected cells were washed in PBS and lysed. Cellular debris was removed by centrifugation at 14,000 × g for 30 s. 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 (University of Michigan, Ann Arbor, MI).
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′ rapid amplification of cDNA ends (5′ RACE) System (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.
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. Primers for PCR fragments were: 5′-TGA CAT GGT ACC AAA AGT TTA GCC CAA AGC GCG A-3′ (1,000 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-s denaturation at 95°C, 1 min annealing at 65°C for 1,000-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 ~ 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.
Means ± SE were calculated. Significant differences between all groups were computed by one-way ANOVA using an F statistic. Statistically significant differences between two groups were determined by the Student's t test.
To determine if Bcl-xL expression is regulated in remodeled pulmonary artery smooth muscle in vivo, rats were subjected to 2, 7, or 14 d 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 d of chronic hypoxia, indicating increased pulmonary vascular resistance and resultant right ventricular hypertrophy (Figure 1A). Pulmonary vascular thickening was evident by 14 d of chronic hypoxia (Figure 1B). In these remodeled pulmonary arteries, increased Bcl-xL expression was observed by Western blot (Figure 1C). Histologic analyses also revealed an increased expression of Bcl-xL protein in the lungs of rats treated with 14 d of chronic hypoxia (Figure 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.
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 use 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 the anti-apoptotic isoform Bcl-xL. As shown in Figure 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 (Figure 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 (Figure 2A). SNP also caused the disruption of mitochondrial membrane potential, an early event of apoptosis, as indicated in green color in Figure 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 (Figure 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.
The ability of NO donors to induce apoptosis of pulmonary artery SMC (26–28) might play important roles in their beneficial effects 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 in the remodeled pulmonary artery. Rats were subjected to chronic hypoxia for 2 wk 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 (Figure 3A). Similarly, a treatment of BPASMC with 100 μM SNP for 24 h resulted in decreased expression of Bcl-xL protein (Figure 3B).
To determine whether the down-regulated 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 Figure 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 (Figure 3D). These results suggest that SNP inhibits Bcl-xL gene expression by modulating the activity of the 0.6-kb promoter region.
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 (Figure 4A). Densitometry analysis demonstrated that a treatment of BPASMC with 300 μM SNP for 20 h resulted in 90% reduction in GATA activity. SNP also inhibited the GATA-binding activity in HPASMC, which also contain GATA-4 (data not shown).
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 (Figure 4B). These results provide direct evidence for the role of GATA in SNP-induced down-regulation of Bcl-xL.
We next investigated the mechanisms of GATA down-regulation induced by SNP in pulmonary artery SMC. In HPASMC, the expression of gata4 mRNA was confirmed by RT-PCR experiments using PCR primers derived from the known human mRNA sequence. Further, down-regulation 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 (Figure 4C). In organ culture of remodeled rat pulmonary artery, SNP was also found to effectively reduce gata4 mRNA expression (Figure 4D).
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′-RACE analysis (Figure 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 (Figure 5B).
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 (Figure 5C), suggesting that the 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 (Figure 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 Figure 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 (Figure 6B), suggesting the binding of these transcription factors to the proximal 250-bp region of the gata4 promoter.
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 (Figure 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 (Figure 6D), indicating the importance of USF1/USF2 in the basal expression of gata4. Further, overexpression of wild-type Egr1 inhibited the gata4 promoter activity (Figure 6E), providing evidence that the increased Egr1 binding might lead to suppressed gata4 gene transcription.
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 up-regulation 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 down-regulate 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 isoform 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 down-regulates the GATA-4 expression. Adenovirus-mediated gene transfer of GATA-4 attenuated SNP-induced down-regulation 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 that 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 with 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 and coworkers (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 and colleagues (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 and coworkers reported that GATA-4 is the primary transcription factor for the two GATA elements in first noncoding exon of bcl-x gene in cardiac myocytes (33).
Recent work from our laboratory has 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 down-regulate gata4 gene transcription. These results suggest that Bcl-xL expression is enhanced by mediators of pulmonary hypertension and is down-regulated by agents that 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.
The authors 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.
This work was supported in part by NIH grants HL67340 (to Y.J.S.), HL72844 (to Y.J.S.), and HL73929 (to R.M.D.), and by grants from the American Heart Association New England Affiliate (to Y.J.S.) and American Lung Association/Massachusetts Thoracic Society (to Y.J.S.). R.M.D. 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.
Originally Published in Press as DOI: 10.1165/rcmb.2006-0359OC on February 1, 2007
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.