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Transcription factor gata4 regulates cardiac BCL2 gene expression in vitro and in vivo

Satoru Kobayashi^*, Troy Lackey^*, Yuan Huang^*, Egbert Bisping, William T. Pu, Linda M. Boxer and Qiangrong Liang^*,1

^* Cardiovascular Research Institute, University of South Dakota School of Medicine, South Dakota Health Research Foundation, Sioux Falls, South Dakota, USA;
Department of Cardiology, Children’s Hospital Boston, Boston, Massachusetts, USA; and
Department of Medicine, Stanford University School of Medicine, Stanford, California, USA

¹Correspondence: Cardiovascular Research Institute, University of South Dakota School of Medicine, South Dakota Health Research Foundation, Sioux Falls, SD 57105, USA. E-mail: qliang{at}usd.edu

SPECIFIC AIM

The transcription factor GATA-4 regulates diverse cardiac-specific genes and confers cardiomyocytes a survival signal against anticancer drug doxorubicin (DOX) -induced cardiotoxicity. The mechanism whereby GATA4 protects cardiomyocytes from DOX-induced apoptosis is not established. This study will determine whether the prototypical antiapoptotic factor Bcl2 is a direct target gene of GATA4 that may mediate the prosurvival function of GATA4 in cardiomyocytes in vitro and in vivo.

PRINCIPAL FINDINGS

1. Overexpression of GATA4 is sufficient to prevent DOX-induced Bcl2 depletion in cardiomyocytes in vitro and in vivo through up-regulating Bcl2 gene expression
DOX induced-apoptosis in neonatal rat ventricular cardiomyocytes (NRVC) is associated with GATA4 depletion, while overexpression of GATA4 attenuates DOX-induced apoptosis in NRVC, suggesting that GATA4 is a critical survival factor that can protect against cell death. We screened a panel of proteins involved in regulating apoptosis in an attempt to identify potential downstream effectors for GATA4. As shown in Fig. 1 , DOX reduced the protein levels of GATA4, Bcl2, and Bclx in NRVC in a time-dependent manner (Fig. 1A ), which was prevented by adenovirus-mediated gene transfer of GATA4 (Fig. 1B ). GATA4 also moderately increased the baseline protein levels of Bcl2 and BclxL (30 and 28%, respectively. n=4, P<0.05; Fig. 1B, C ). The mRNA levels of GATA4, Bcl2, and BclxL as determined by semiquantitative RT-PCR correlated well with the changes in protein levels (Fig. 1B , right panel). These results suggest that overexpression of GATA4 is sufficient to up-regulate Bcl2 and Bclx genes in NRVC, and both Bcl2 and Bclx are potential downstream target genes of GATA4 that may contribute to the prosurvival effects of GATA4 in NRVC. However, in GATA4-overexpressing transgenic mice, only Bcl2 but not BclxL protein and mRNA levels were up-regulated in the heart (2.7-fold). Similarly, only Bcl2 but not BclxL levels were reduced by DOX treatment in wild-type (WT) hearts, and the reduction in Bcl2 levels were prevented in GATA4 transgenic hearts (Fig. 2 A–C). These results suggest that Bcl2 is a preferred target gene for GATA4 in vivo although both Bcl2 and Bclx genes are regulated by GATA4 in NRVC.

View larger version (52K): [in this window] [in a new window]   Figure 1. Up-regulation of Bcl2 gene expression in NRVC by GATA4. A) DOX induces a time-dependent depletion of GATA4 protein levels, which correlates with reduced Bcl2 and BclxL levels. NRVC were treated with DOX (1 µM), and cells were harvested at indicated hours (h) after the treatment. Western blot analysis was performed using 50 µg of whole cell protein extracts. B) Overexpression of GATA4 prevents DOX-induced decrease in Bcl2 and BclxL protein levels (determined by Western blot analysis in left panel) and mRNA levels (determined by RT-PCR and shown in ethidium bromide-stained agarose gel in right panel). NRVC were infected with Adßgal or AdGATA4 for 24 h at a MOI of 100, and treated with 1 µM of DOX for 18 h. C) Western blots were quantified by densitometry and results are presented as mean ± SE (n=4). Values were initially normalized with GAPDH, and compared with Adßgal-infected samples in the absence of DOX. WB Ab, Western blot antibody; *P < 0.05 as compared with Adßgal.

View larger version (46K): [in this window] [in a new window]   Figure 2. GATA4 up-regulates Bcl2 gene expression in transgenic hearts. GATA4 TG mice and WT control mice were treated with a single dose of DOX (20 mg/kg), and the protein extracts and total RNA were prepared from the hearts 48 h after the treatment. A) Western blot showing the protein levels of GATA4, Bcl2, BclxL, and Bax in GATA4 TG and WT mouse hearts. B) Protein levels of GATA4, Bcl2, BclxL, and Bax in the hearts were determined by densitometry. Data are mean ± SE (n=4). *P < 0.05 vs. WT mice without DOX treatment. C) Ethidium bromide stained agarose gel showing the transcript levels of GATA4, Bcl2, BclxL, and Bax in GATA4 TG and WT hearts as determined by semiquantitative RT-PCR. The experiment was repeated multiple times with similar results.

2. GATA4 is required for Bcl2 gene expression in NRVC
An adenovirus encoding a short hairpin RNA targeted at GATA4 (AdG4i) was used to specifically knock down GATA4 in NRVC. The results showed that AdG4i at 60 MOI reduced GATA4 protein levels by 84.7%, and Bcl2 protein levels by 48%, while an adenovirus encoding a scrambled short hairpin RNA did not affect GATA4 and Bcl2 levels in NRVC. The reduction in protein levels correlated with the transcript levels of GATA4 and Bcl2 as determined by semiquantitative RT-PCR. Surprisingly, neither protein levels nor mRNA levels of Bclx gene were affected by AdG4i. These results indicate that GATA4 is required for Bcl2 but not for Bclx constitutive gene expression regardless of the ability of GATA4 to up-regulate both genes in NRVC.

3. GATA4 activates Bcl2 gene transcription partially through directly binding to the –266 GATA motif on the Bcl2 promoter
We transfected HEK cells with the Bcl2-1281 promoter-luciferase construct and a GATA4 expression plasmid. The result showed that GATA4 dose dependently activated the Bcl2-1281 promoter, which correlated with the expression levels of Bcl2 and GATA4 proteins. The 1281 bp Bcl2 promoter region contains two putative GATA binding sites at positions –266 and –1025. The –266 GATA site is conserved between human, mouse, rat, bovine, dog, and cat. Transfection of HEK cells with GATA4 plasmid and deletion and mutation constructs of Bcl2 promoter-luciferase reporter showed that the –1025 GATA site did not contribute to GATA4-induced Bcl2 promoter activity. However, GATA4-induced promoter activity was moderately but significantly attenuated by removing the –266 GATA motif from Bcl2-293 reporter or by mutating the –266 GATA site to CCTA on the Bcl2-293 reporter, indicating that GATA4 is able to activate Bcl2 promoter and this effect is mediated, at least in part, through the –266 GATA binding site. To determine if GATA4 can directly interact with the –266 GATA site in vitro and in vivo, we performed electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. Both GST-GATA4 and nuclear GATA4 from AdGATA4-infected NRVC were able to bind to the ³²P-labeled oligonucleotide probe containing the –266 GATA site but not the –1025 GATA site. ChIP assay showed that GATA4 antibodies specifically pulled down DNA fragments containing the –266 GATA motif, but not DNA fragments containing the –1025 GATA site. These results confirmed that GATA4 can directly bind to –266 GATA but not the –1025 GATA site in vitro and in vivo. Overall, these data suggest that the direct interaction of GATA4 with the –266 GATA site is likely responsible for the activation of Bcl2 promoter and subsequent up-regulation of Bcl2 gene expression by GATA4.

CONCLUSIONS AND SIGNIFICANCE

GATA4 is a cardiac enriched transcription factor that is essential for various physiological and adaptive responses of cardiomyocytes. Recent studies have shown that GATA4 was able to protect cardiomyocytes from DOX-induced apoptosis, indicating that GATA4 also conveys a survival signal for cardiomyocytes. However, the downstream mechanism of GATA4 prosurvival effect has not been elucidated. Using both gain- and loss-of-function approaches, our current study provided strong evidence to suggest that Bcl2, the prototype of antiapoptotic factors, is a direct target gene for GATA4 that may mediate GATA4 myocardial protection against DOX-induced cardiotoxicity both in vitro and in vivo. To the best of our knowledge, GATA4 is the first transcription factor that has demonstrated the ability to up-regulate Bcl2 gene at both mRNA and protein levels in cardiomyocytes in vitro and in vivo. GATA4 is also necessary for the baseline Bcl2 gene expression in cardiomyocytes as shown by siRNA-mediated GATA4 knockdown although the necessity of GATA4 in vivo remains to be determined in animals with cardiac-specific disruption of GATA4 gene.

The present study demonstrates that the transcriptional activation of Bcl2 gene by GATA4 in HEK cells and cardiomyocytes is mediated through the P2 promoter in contrast to the predominant use of the P1 promoter in other cell types including normal B cells. This is probably because GATA binding motifs are found only on the P2 promoter. We show that GATA4 directly bind to the conserved –266 GATA consensus motif on the P2 promoter as demonstrated by EMSA in vitro and ChIP assay in vivo. The deletion or mutation of the –266 GATA site resulted in a moderate decrease in the P2 promoter activity, suggesting that the direct interaction of GATA4 with the –266 GATA site is at least partially responsible for the activation of Bcl2 promoter and subsequent up-regulation of Bcl2 gene expression by GATA4.

Our ongoing research is attempting to establish a novel molecular paradigm in which GATA4 protects the heart from DOX toxicity via the up-regulation of Bcl2 gene expression. Our unpublished data indicate that DOX-induced GATA4 depletion is independent of DOX-induced generation of reactive oxygen species (ROS). The latter is a well-established mechanism for DOX cardiotoxicity. Thus, DOX-induced GATA4 depletion is likely a distinct mechanism that acts in parallel with ROS leading to DOX cardiotoxicity, which might explain the limited effect of some anti-oxidants to prevent DOX cardiotoxicity in clinical trials. We predict that therapeutic regimen that blocks GATA4 depletion and/or enhances GATA4-Bcl2 signaling will presumably be able to attenuate DOX cardiotoxicity (Fig. 3 ). The GATA4-Bcl2 survival pathway may also turn out to be a common mechanism for myocardial protection in various heart diseases such as those induced by ischemia-reperfusion injury. Bcl2 may not be the only downstream mediator of GATA4 survival effect. It is possible that GATA4 may regulate other uncharacterized factors that may then work in concert with Bcl2 to confer myocardial protection.

View larger version (24K): [in this window] [in a new window]   Figure 3. Proposed model for DOX cardiotoxicity. DOX-induced depletion of GATA4 and Bcl2 is a distinct mechanism that acts in parallel with ROS generation leading to cardiomyocyte death. Therapeutic strategy that enhances GATA4-Bcl2 signaling will presumably be able to attenuate DOX cardiotoxicity.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5426fje;

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