Original article
Synergistic effects of the GATA-4-mediated miR-144/451 cluster in protection against simulated ischemia/reperfusion-induced cardiomyocyte death

https://doi.org/10.1016/j.yjmcc.2010.08.007Get rights and content

Abstract

Among the identified microRNAs (miRs) thus far, ~ 50% of mammalian miRs are clustered in the genome and transcribed as polycistronic primary transcripts. However, whether clustered miRs mediate non-redundant and cooperative functions remains poorly understood. In this study, we first identified activation of the promoter of miR-144/451 by GATA-4, a critical transcription factor in the heart. Next, we observed that ectopic expression of miR-144 and -451 individually augmented cardiomyocyte survival, which was further improved by overexpression of miR-144/451, compared to control cells in response to simulated ischemia/reperfusion. In contrast, knockdown of endogenous miR-144 and -451 revealed opposite effects. Using luciferase reporter assay and western blot analysis, we also validated that both miR-144 and miR-451 target CUG triplet repeat-binding protein 2 (CUGBP2), a ubiquitously expressed RNA-binding protein, known to interact with COX-2 3′UTR and inhibit its translation. Accordingly, protein levels of CUGBP2 were greatly reduced and COX-2 activity was markedly increased in miR-144-, miR-451-, and miR-144/451-overexpressing cardiomyocytes, compared to GFP cells. Furthermore, inhibition of COX-2 activity by either NS-398 or DUP-697 partially offset protective effects of the miR-144/451 cluster. Together, these data indicate that both partners of the miR-144/451 cluster confer protection against simulated I/R-induced cardiomyocyte death via targeting CUGBP2–COX-2 pathway, at least in part. Thus, both miR-144 and miR-451 may represent new therapeutic agents for the treatment of ischemic heart disease.

Research Highlights

►The miR-144/451 cluster is directly regulated by cardiac transcription factor GATA-4. ►Increased levels of miR-144 and miR-451 protect adult cardiomyocytes against simulated ischemia/reperfusion-induced cell death, and overexpression of miR-144/451 reveals additive effects on cardioprotection. ►Both miR-144 and miR-451 target CUGBP2, an RNA-binding protein, which interacts with COX-2 3′UTR and inhibits its mRNA translation. ►COX-2 is upregulated in miR-144-, miR-451-, and miR-144/451-overexpressing cardiomyocytes. ►Inhibition of COX-2 partially abrogates miR-144/451-mediated cardioprotection.

Introduction

Accumulating evidence has established cardiomyocyte death as a critical component in the pathogenesis of ischemia/reperfusion injury and heart failure [1], [2]. Thus, inhibition or prevention of cardiac cell death is a desirable endpoint. Given the intricacy of cell death signaling cascade, multiple levels of intervention will likely be required to modulate this process. Recent discovered microRNAs (miRs), targeting hundreds of cellular proteins, may widely influence the signaling networks associated with cell death/survival [3], [4]. For example, miR-21 has been confirmed to target programmed cell death 4 (PDCD4), phosphatase and tensin homology deleted from chromosome 10 (PTEN), sprouty1/2 (SPRY1/2), and superoxide dismutase 2 (SOD2), which activate Akt, ERK-MAPK, and other survival signaling pathways in cardiomyocytes [5], [6], [7], [8]. Therefore, it should be an attractive therapeutic strategy to identify a new miR that suppresses aberrant cell loss and promotes cell survival following myocardial injury.

MiR-144 and miR-451 are closely clustered and evolutionally conserved [9]. They are processed from a single gene locus that is regulated by the essential hematopoietic transcription factor GATA-1 [9]. Furthermore, maturation of miR-144 and miR-451 seems to be posttranscriptionally regulated in a sequence-specific manner [10], [11]. As a result, expression levels of mature miR-144 and miR-451 are different in cells. Previously, miR-144 and miR-451 were identified to have erythroid-specific expression in zebra fish [12]. Knockdown of miR-451 in zebra fish embryos, but not miR-144, significantly impaired erythroid maturation [12]. Nonetheless, the miR-144/451 null mice were born without any structural defects [13]. Further study indicated that these miR-144/451 null erythrocytes were sensitive to oxidative stress [13]. In cancer cells, miR-144 was shown to reduce TRAIL-induced apoptosis by targeting caspase-3 [14], and miR-451 was confirmed to target macrophage migration inhibitory factor (MIF) and the calcium-binding protein 39 (CAB39)/AMPK signaling pathway [15], [16]. Importantly, both miR-144 and miR-451 are predicted to target CUG triplet repeat-binding protein 2 (CUGBP2), an RNA-binding protein which interacts with COX-2 mRNA 3′UTR and inhibits its translation [17]. Collectively, these data suggest that the miR-144/451 cluster may play a critical role in modulation of cell death/survival. Recently, we and others observed that miR-451 (currently, no information available on alteration of miR-144 in the heart) was significantly downregulated in ex vivo ischemic/reperfused animal hearts and human failing hearts [18], [19]. However, miR-451 was upregulated in human infarcted hearts with less than 7 days old, but no changes with more than 4 weeks old [20]. Whether transiently increased miR-451 represents an adaptive response or reduced miR-451 contributes to the process of cardiac disease is not clear. Additionally, it is not known as to whether overexpression of the miR-144/451 cluster in cardiomyocytes augments each other's phenotype.

In the present study, we employed gain-of-function and loss-of-function approaches to determine the functional role of the miR-144/451 cluster in cardiomyocyte death under simulated ischemic conditions. For the first time, we found that GATA-4 directly regulated the miR-144/451 expression in cardiomyocytes. Upregulation of the miR-144/451 cluster was associated with cardioprotection against hypoxic stress via targeting the CUGBP2–COX-2 signaling pathway. We also observed that there was a functional cooperation within the miR-144/451 cluster to protect against simulated ischemia/reperfusion-induced cardiomyocyte death. Thus, results from the present study will potentially advance our understanding of how miR orchestrates the complex genetic networks responsible for cardiovascular homeostasis and disease.

Section snippets

miR mimics/inhibitors and plasmid constructs

miR mimics/inhibitors to miR-144 or miR-451, and miR mimic/inhibitor controls were purchased from the Dharmacon (www.dharmacon.com). The plasmid harboring GATA-4 and the adenoviral vector encoding GATA-4 were provided by Dr. Molkentin (Cincinnati Children's Hospital Medical Center). The miR-144/451 promoter containing one cis-GATA-4 motif, or two cis-GATA-4 motifs was amplified by PCR, and subcloned into the pGL3-basic vector (Promega, Madison, WI) at the KpnI and BglII sites. A CUGBP2-3′UTR

GATA-4 directly regulates the MiR-144/451 cluster expression in cardiomyocytes

The GATA family of zinc finger transcription factors has been implicated in regulating cell growth, differentiation, and survival [25], [26], [27], [28]. Of the six GATA family members, GATA-1/2/3 are predominant in blood and ectodermal derivatives, whereas GATA-4/5/6 are expressed in heart and endodermal derivatives [25]. Recently, an elegant study has shown that the miR-144/451 cluster bearing miR-144 and miR-451 is directly activated by GATA-1 in erythroid cells [9]. Additionally, analysis

Discussion

Among the identified miRs thus far, ~ 50% of mammalian miRs are clustered in the genome and transcribed as polycistronic primary transcripts, whereas ~ 40% are expressed as individual transcripts from intronic locations and ~ 10% from intergenic regions [31]. It is believed that distinct miRs within the same cluster may work in concert to regulate a cellular process [31]. However, two miRs co-expressed from miR-1/133 loci showed the intriguingly opposite effects on apoptosis [32]. Overexpression

Acknowledgments

Funding sources: This study was supported by NIH grants HL-087861 and HL087861-03S1 (G.-C. Fan), HL089824 and HL081859 (Y. Wang), and University of Cincinnati CEG grant no. 1007636 (G.-C. Fan).

Conflict of interest disclosures: None.

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