Published online before print March 20, 2008, doi: 10.1161/CIRCRESAHA.107.161729
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© 2008 American Heart Association, Inc.
Cellular Biology |
Hypoxia-Inducible Factor-2
Transactivates Abcg2 and Promotes Cytoprotection in Cardiac Side Population Cells
From the Departments of Internal Medicine (C.M.M., A.F., C.H., H.S., A.C., J.A.G., M.G.G., D.J.G.), Pathology (T.G.), and Molecular Biology (D.J.G.) and Donald W. Reynolds Clinical Cardiovascular Center (D.J.G.), University of Texas Southwestern Medical Center, Dallas; Lillehei Heart Institute (C.M.M., A.F., M.G.G., D.J.G.), University of Minnesota, Minneapolis; and Oklahoma Medical Research Foundation (L.I.S.), Oklahoma City.
Correspondence to Daniel J. Garry, MD, PhD, Lillehei Heart Institute, University of Minneapolis, 420 Delaware St SE, MMC 508, Minneapolis, MN 55455. E-mail garry{at}umn.edu
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Abstract |
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Stem and progenitor cell populations occupy a specialized niche and are consequently exposed to hypoxic as well as oxidative stresses. We have previously established that the multidrug resistance protein Abcg2 is the molecular determinant of the side population (SP) progenitor cell population. We observed that the cardiac SP cells increase in number more than 3-fold within 3 days of injury. Transcriptome analysis of the SP cells isolated from the injured adult murine heart reveals increased expression of cytoprotective transcripts. Overexpression of Abcg2 results in an increased ability to consume hydrogen peroxide and is associated with increased levels of
-glutathione reductase protein expression. Importantly, overexpression of Abcg2 also conferred a cell survival benefit following exposure to hydrogen peroxide. To further examine the molecular regulation of the Abcg2 gene, we demonstrated that hypoxia-inducible factor (HIF)-2 binds an evolutionary conserved HIF-2 response element in the murine Abcg2 promoter. Transcriptional assays reveal a dose-dependent activation of Abcg2 expression by HIF-2. These results support the hypothesis that Abcg2 is a direct downstream target of HIF-2 which functions with other factors to initiate a cytoprotective program for this progenitor SP cell population that resides in the adult heart.
Key Words: Abcg2 • cardiac SP • HIF-2 • oxidative stress
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Side population (SP) cells are isolated from embryonic and adult tissues using Hoechst 33342 and dual-wavelength fluorescence-activated cell sorting (FACS) analysis.1–4 This strategy defines a rare population of progenitor cells that can adopt alternative fates in permissive environments.1–3 We have verified that the ability of SP cells to efflux Hoechst 33342 dye is dependent on the expression of Abcg2, which is a member of the family of ATP-binding cassette (ABC) transporters.3,5,6 Although the functional role of the ABC transporters remains ill-defined,7 we established that Abcg2 was able to confer the SP phenotype in a striated muscle cell line.3
Stem and progenitor cell populations, including SP cells, are exposed to environmental stress by virtue of their physical location. Although oxidative stress attributable to unchecked levels of free radical–derived reactive oxygen species (ROS) can damage DNA, proteins, and lipids,8 oxidative stress caused by modestly increased ROS can activate specific signal transduction pathways, leading to either senescence or apoptosis.9 Previous transcriptome analyses of embryonic, hematopoietic, and neural stem cells revealed a common signature of gene expression in these stem cell populations. This profile includes transcripts that function as cytoprotective factors to provide resistance against environmental stress.10–12 Recent studies that examined circulating, blood-derived endothelial progenitor cells reveal enrichment for the expression of genes encoding for antioxidative factors that reduce sensitivity toward ROS-induced cell death.13 Regulation of cytoprotective factors during injury states would be beneficial for survival and expansion of stem and progenitor cell populations.
Members of the hypoxia-inducible factor (HIF) family are activated by multiple environmental stimuli. HIF-1, a master regulator for hypoxia-inducible gene expression, regulates gene expression to promote energy production as well as oxygen delivery in response to hypoxia.14–16 HIF-2, also known as endothelial PAS domain protein 1 (EPAS1), has many similarities with HIF-1.17–19 However, several molecular, biochemical, and physiological studies have established that HIF-1 and HIF-2 are not redundant but have distinct functional roles.17–19 HIF-2 transcriptional activity is induced in specific tissues (vascular endothelial cells, neural crest cell derivatives, cardiac myocytes, and stem cell populations)17 and is important in ROS homeostasis, apoptosis, and lung and hematopoietic development.18,19 Furthermore, studies have demonstrated that HIF-2 plays the major role in oxidative stress defense mechanisms specifically in the regulation of antioxidant enzymes. Recent studies have also demonstrated that HIF-2 and not HIF-1 is a direct upstream regulator of Oct-4, a transcription factor required for pluripotency of embryonic stem cells. This latter regulatory role for HIF-2 provides a mechanism for HIF-2–dependent regulation of stem cell function.17
In the present study, we define the response of the cardiac SP cells following injury. We undertake a transcriptome analysis to define the common transcriptional signature of the SP cell populations isolated from embryonic and adult lineages. We further define the transcriptional regulation of Abcg2 by HIF-2. Collectively, these studies enhance our understanding of the cardiac SP cell population and provide insight regarding the functional role as well as regulation of Abcg2.
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Materials and Methods |
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FACS Analysis and Transcriptome Analyses
Cells were cultured, harvested, and analyzed as described in the online data supplement, available at http://circres.ahajournals.org. Oligonucleotide array hybridizations were carried out according to the Affymetrix protocol, as previously described.20,21
Quantitative RT-PCR Analyses
cDNA synthesis and quantitative (q)RT-PCR reactions were performed as previously described.20 All primer pairs sequences are listed in the online data supplement.
Myocardial Cryoinjury
A transmural cryoinjury was induced as described in the online data supplement. At specified days following injury, the mice were euthanized and prepared for FACS, molecular, or immunohistochemical analyses and compared with uninjured littermates. All mice were maintained in a pathogen free facility according to the animal care guidelines at the UT Southwestern Medical Center.
Immunohistochemistry
Adult hearts were fixed, embedded in paraffin, and sectioned as previously described.22 Antibodies were used as follows: polyclonal rabbit anti-ABCG2 serum (kindly provided by Susan Bates [National Institute of Health, Bethesda, Md; 1:800 dilution])7 anti–-sarcomeric actinin serum (1:150 dilution; Sigma, St. Louis, Mo).
Cell Culture and Overexpression of Abcg2
C2C12 and mouse embryo fibroblast (MEF) cells were cultured as previously described.23 Cells were transfected with EGFP-N1 plasmid (Clontech) or the pG2-IRES-EGFP bicistronic construct. The cells were analyzed as described in the online data supplement.
Electrophoretic Mobility-Shift Assay and Chromatin Immunoprecipatation Assay
C2C12 cells were transfected with hemagglutinin (HA)-tagged HIF-2. After 24 hours, nuclear extracts were prepared and used for electrophoretic mobility-shift assay (EMSA) as previously described.23 Chromatin immunoprecipatation assays for evaluating Abcg2 promoter binding of HIF-2 were performed as described in the online data supplement.
Reporter Gene Assays
Luciferase assays were performed as previously described.23 C2C12 myoblast cells were transfected with control (pGLT-Luc) or Abcg2-Luc constructs with or without increased amounts of HA-tagged HIF-1 or HIF-2 overexpression plasmids, as described in online data supplement.
Western Blot Analysis
Protein extracts from Abcg2-overexpressing SP cells and respective control cell populations were prepared, and Western blot analysis was preformed as previously described.24,25 Blots were probed with a polyclonal rabbit anti–glutathione reductase serum (BD PharMingen; 1:2000) and the polyclonal rabbit -tubulin serum (Sigma-Aldrich; 1:2000 dilution).24,26
H2O2 Consumption Assays
Following the addition of 250 µmol/L H2O2 to the sample of interest, 100 µL was removed at specific time points and added to 2.0 mL of 25 mmol/L K2HPO4, 0.1%Triton X-100 (pH 7.25) with 500 µmol/L hydroxyphenyllactic acid (Sigma) and 2.0 U/mL horseradish peroxidase (added shortly before use).27
Glutathione/Oxidized Glutathione Measurements
Abcg2-overexpressing SP cells and respective control cell populations were pelleted, and glutathione (GSH) and oxidized glutathione (GSSG) were extracted with 75 µL of 5% meta-phosphoric acid. Following centrifugation, GSH and GSSG present in the supernatant were resolved by reverse-phase high-performance liquid chromatography and quantified by electrochemical detection as previously described.28
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Results |
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We have previously reported that Abcg2 is expressed in cardiac SP cells that are resident in the developing and adult murine heart.3 The ability of SP cells to efflux Hoechst 33342 dye is dependent on the expression of Abcg2.3,4 Using FACS, cardiac SP cells were isolated from the unperturbed adult murine heart and at 3, 7, and 14 days after cryoinjury (Figure 1). We observed that the cardiac SP cells increased 3.3-fold by day 3 following injury. The increase was sustained through day 7 (after cryoinjury), which revealed a 3.1-fold increase compared with baseline (ie, the unperturbed adult heart) but then returned to near baseline levels by day 14 following injury. Analysis of the CD45+ (a pan-hematopoietic marker) cell population of the cardiac SP cells using FACS analysis, revealed that the percentage of CD45+ cells was not significantly changed at any of the time points (Figure 1C). This suggests that the increase in the number of cardiac SP cells is largely from proliferation of the resident cardiac SP cell pool. Future studies will be necessary to further verify the source(s) of the cardiac SP cells following injury. The increase in the number of cardiac SP cells following injury was further confirmed using immunohistochemical techniques. Abcg2-expressing cells were observed rarely (within the interstitium) in the unperturbed heart and at 1 and 14 days following cryoinjury of the adult heart (Figure 1E and 1H). At 3 days following injury, there is a significant increase in the cardiac Abcg2-positive cells at low magnification (Figure 1F) and high magnification (Figure 1G). Statistical analysis of the sections revealed an
10-fold increase in the Abcg2-positive cells at day 3 (after cryoinjury) compared with days 1 and 14 (following injury) (29.7±4.2 versus 3.2±1.5 and 2.5±1.9 respectively; n=6; * P<0.001; Figure 1D). These results support the conclusion that Abcg2-expressing cardiac SP cells increase in number following myocardial injury.
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To define the molecular signature of the cardiac SP cells following injury, we isolated cardiac SP cells at 3 and 7 days following injury and analyzed the respective transcriptome using Affymetrix array technology. The results were then compared with cardiac SP cells harvested from the uninjured adult murine heart. The analysis of the transcriptome profiling experiments revealed a common molecular program of significantly expressed transcripts that were induced at both days 3 and 7 following myocardial injury. As shown in the Venn diagram, 333 transcripts were significantly induced in cardiac SP cells isolated on days 3 and 7 following injury (Figure IA in the online data supplement). The fold induction of representative transcripts that were commonly increased in cardiac SP cells isolated at both time periods following myocardial injury is presented in supplemental Figure IB. Interestingly, not only were there higher numbers of Abcg2-expressing SP cells, but the increased Abcg2 transcript expression was observed in individual SP cells. The transcriptome results were confirmed using qRT-PCR for several candidate genes on separately harvested samples of cardiac SP cells isolated from adult hearts 3 and 7 days following injury (supplemental Figure IC).
To define a common SP cell signature, SP cells were harvested from murine embryonic stem cells, adult mouse bone marrow, skeletal muscle, or cardiac tissue (Figure 2A through 2H), and the molecular signature was defined using Affymetrix array technology. The array data from the SP cells isolated from these lineages were compared with their respective main population (MP) and significantly upregulated transcripts are illustrated in the Venn diagrams in Figure 2I. Each SP cell population displayed a distinct pattern of transcript enrichment, although a common SP cell program was also defined. The fold changes of representative significantly enriched transcripts are shown in supplemental Figure IIA. The transcriptome results were confirmed using qRT-PCR for several candidate genes on separately harvested samples (supplemental Figure IIB). These results support the conclusion that the common SP molecular program includes cell cycle regulatory genes (ie, p21), signaling pathways (Tgfb), and cytoprotective factors (Jun, Smad7, Myc, Ndrg1, and Txnl1).
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The transcripts that were commonly expressed in SP cell populations isolated from embryonic and adult lineages were largely associated with the cellular response to oxidative stress. Although these transcripts were commonly expressed in the SP cell populations, the transcriptome analysis could not distinguish, whether expression of these factors was a direct effect of Abcg2 itself or whether stem/progenitor cells were expressing a common program that included Abcg2 expression. To further examine whether the common transcript expression could be a direct effect of Abcg2, we overexpressed Abcg2 in C2C12 cells and isolated the Abcg2-expressing SP cells as well as MP cells (Figure 3A). Native C2C12 cells lack Abcg2 expression and therefore lack SP cells (Figure 3A). Following forced expression of Abcg2 in C2C12 myoblasts, a significant increase in the number of SP cells was observed and the ability of these cells to efflux Hoechst dye was completely blocked with fumitremorgin (FTC), a specific Abcg2 inhibitor (Figure 3A).
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To determine whether Abcg2 expression affects the transcriptome, the molecular signature of the Abcg2-expressing C2C12 SP cells was compared with the native C2C12 MP cells. The fold changes of representative significantly enriched transcripts in the C2C12 SP cells are shown in supplemental Figure IIIA. Many of the same transcripts (that function as cytoprotective factors/pathways) that were significantly expressed in the common SP cell molecular program in ES, bone marrow, skeletal muscle, and cardiac SP cells were also enriched in the Abcg2-expressing C2C12 SP cells. Transcripts such as Atf3, Ndr1, Gsta4, and Ddit3 were significantly upregulated in the Abcg2-expressing C2C12 SP cells compared with the native C2C12 MP cells. This induction of gene expression was further confirmed using qRT-PCR analysis (supplemental Figure IIIB).
The Abcg2-expressing C2C12 SP cell transcriptome data indicate that Abcg2 expression results in the upregulation of the oxidative stress pathway. These results indicate that either Abcg2 expression was itself cytoprotective or that the expression (or overexpression) of Abcg2 results in an oxidative stress that activates oxidative stress signaling pathways. We note that no difference in cell death was observed between the experimental and control samples, indicating that Abcg2 expression does not result in unchecked oxidative stress (data not shown). Although there was not significant oxidative stress to cause cell death in the Acbg2-overexpressing cells, we hypothesized that Abcg2 was inducing a low level of oxidative stress that was "priming" or preconditioning the cells to be more resistant to oxidative stress.29,30 To examine this hypothesis, we measured the ratio of reduced to oxidized glutathione (GSH/GSSG) in Abcg2-expressing C2C12 SP cells compared with C2C12 cells, which lack Abcg2. We observed that there was a lower ratio of reduced to oxidized (GSH/GSSG) glutathione in the Abcg2-expressing C2C12 cells (11.1±0.8) compared with wild-type C2C12 cells (16.4±4.7; n=6; *P<0.05; Figure 3B). To determine whether Abcg2 expression itself affects the cellular survival in other cell types, we overexpressed Abcg2 in mouse embryonic fibroblast (MEF) cells given their well-described use in oxidative stress experiments (Figure 3C).31,32 Abcg2-overexpressing MEFs and wild-type MEFs were then exposed to hydrogen peroxide. The Abcg2-overexpressing MEF SP cells displayed a survival benefit (64±4.5% cell death) compared with MEF MP cells (74±2.5% cell death n=5; *P<0.05; Figure 3D) after exposure to oxidative stress. This finding confirmed that the overexpression of Abcg2 was not generating an oxidative stress that was deleterious to the cells, but rather Abcg2 overexpression resulted in a survival benefit.
To further define the biochemical mechanism of the induction of oxidative stress genes, hydrogen peroxide consumption assays were performed in Abcg2-expressing C2C12 SP cells. We observed that the Abcg2-expressing C2C12 SP cells are able to consume hydrogen peroxide at a higher rate compared with C2C12 cells, which lack Abcg2, consistent with the notion that Abcg2 induces cytoprotective pathways (Figure 3E). Given that glutathione reductase is responsible for recycling GSSG to GSH, we hypothesized that there would be an induction of this enzyme in the Abcg2-expressing C2C12 SP cells.29 This was confirmed using Western blot analysis, which revealed higher levels of -glutathione reductase (Gsr) expression in the Abcg2-overexpressing C2C12 SP cells compared with the C2C12 MP cells (ratio of Gsr to -tubulin loading control, 0.547±0.1 in C2C12 MP versus 0.81±0.1 in Abcg2-overexpressing C2C12 SP cells; n=3; *P<0.05; Figure 3F and 3G). Collectively, these findings further confirmed that the upregulation of cytoprotective factors involved in the oxidative stress response is directly related to overexpression of Abcg2.
Recent studies support the notion that HIF-2 (EPAS1) may serve as a master regulator of oxidative stress response pathways.17–19 Because we have demonstrated, Abcg2 expression is associated with induction of oxidative stress pathways, we hypothesized that HIF-2 could be an upstream regulator of Abcg2. Database analysis of the 3-kb upstream fragment of the Abcg2 gene revealed the presence of an evolutionarily conserved hypoxia-response element (HRE) (Figure 4 A). The HRE was tested for its ability to interact specifically with Abcg2 in vitro using an EMSA. As outlined in Figure 4B and 4C, using the EMSA, we demonstrated that HIF-2 binds to this site (ie, HRE) because it forms a HIF-2 -DNA complex (lane 2), which is competed in the presence of excess cold competitor (lanes 3 and 4) but not the mutant (lanes 5 and 6) cold probe, and the DNA-protein complex is supershifted by anti-HA serum. As a further control, heat denaturation of the antibody before adding to the reaction fails to supershift the complex (lanes 7 and 8). Using chromatin immunoprecipitation assays, we confirmed that HIF-2 binds to the Abcg2 promoter in vivo. The database analysis revealed that the HRE was flanked by three HIF accessory sequences within the upstream fragment of the Abcg2 gene, as outlined in Figure 4D. Chromatin solutions, prepared from C2C12 cells transfected with either HA-tagged HIF-2 (+) or HA-tagged control (–) vector, were used to immunoprecipitate the HIF-2–DNA complex by anti-HA serum and control IgG serum and analyzed by PCR amplification. Using primers designed to amplify the HRE, we were able to amplify product from the DNA bound to protein precipitated with the anti-HA serum only from the HIF-2–overexpressing cells but not from control antibodies which further establishes the specificity of HIF-2 binding to the Abcg2 promoter (Figure 4D).
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We then undertook transcriptional activation assays to confirm that the binding of HIF-2 to the Abcg2 promoter was biologically significant. Initially, we analyzed whether the Abcg2 transcript was upregulated following HIF-2 overexpression. RNA was isolated from C2C12 myoblasts transfected either with HA-tagged HIF-2 (+) or control (–) vectors and corresponding cDNA was used for qRT-PCR analysis. In response to HIF-2 overexpression, we observed a 2.7-fold induction of Abcg2 transcript (Figure 4E), whereas no significant induction was seen of Car9, a known downstream target of HIF-1 (supplemental Figure IV). This activation was further confirmed using transcriptional assays. We fused a 3-kb Abcg2 promoter fragment to the luciferase reporter. We transfected C2C12 myoblasts with the Abcg2 promoter–reporter construct and increasing HIF-2 amounts (Figure 4F). We observed a 6.1-fold increase of Abcg2 transcription in response to HIF-2. Moreover, HIF-2 in a dose-dependent fashion transcriptionally activated the Abcg2 gene compared with the control (ie, empty vector). However, when we transfected C2C12 myoblasts with the Abcg2 promoter–reporter construct and maximum doses of HIF-1, we observed only a 2-fold increase of Abcg2 transcription (supplemental Figure VA). These results further establish that HIF-2 binds and transactivates Abcg2 gene expression to promote cytoprotection in the cardiac SP cell population and that this response is specific for HIF-2.
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Discussion |
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Many adult tissues harbor a resident stem cell or progenitor cell population that participates in the maintenance and regeneration of the respective tissues in response to an injury. Accumulating evidence supports the notion that the adult heart is capable of limited regeneration in response to an injury. SP cells have been shown to function as stem/progenitor cells in a number of lineages.1–3 In the present study, we have begun to decipher the regulatory program of the cardiac SP cell population by making 3 principal findings that enhance our mechanistic understanding of this cell population. First, we demonstrated that the Abcg2-expressing cardiac SP cell population increases in number following a focal myocardial injury. Using numerous technologies, we observed that the cardiac SP cells following myocardial injury increased in number compared with the cardiac SP cells resident in the uninjured adult heart. These techniques do not definitively distinguish whether the increase in cardiac SP cells is attributable to recruitment from extracardiac tissues (ie, bone marrow) or proliferation of the resident cardiac SP cell population. Future studies will be necessary to further define the source of the increased SP cell population following injury. The transcriptome analysis also revealed that not only was there an increase in the numbers of cardiac SP cells in response to injury, but there was an induction of Abcg2 gene expression in cardiac SP cells isolated from the injured heart. The induction of Abcg2 in this cell population isolated from the injured heart supported the notion that Abcg2 may have an important functional role in this cell population.
Previous studies have demonstrated that stem/progenitor cell populations have cytoprotective mechanisms that promote survival in response to stressful stimuli following a severe injury.10,11 Our second principle observation is that Abcg2 promotes a cytoprotective response in SP cell populations by inducing antioxidant stress pathways. We and others have previously demonstrated that Abcg2 functions to efflux Hoechst dye in SP cell populations, although the physiological role for this multidrug resistance protein is unclear.3,4 Using a gene disruption strategy, Abcg2-null mice are viable, and they have a relative absence of SP cells and increased toxicity with exposure to antineoplastic drugs.33 Although it is possible that other members of the ABC transporter superfamily may compensate in the absence of Abcg2, accumulating data support the notion that Abcg2 expression in stem cells may mediate the ability to respond to stressful stimuli. Recent studies have examined the global gene expression or the transcriptome of embryonic, hematopoietic, and neural stem cells and have defined a common signature of gene expression that provides resistance against environmental stress.10–12 These common programs of gene expression (including genes involved in cytoprotection) in these stem cell populations have been proposed to be a "stemness" feature, which may promote stem cell survival and regeneration of injured tissues.10–12 The transcriptome results in the present study are largely in agreement with the previous studies because they too reported an induction of stress responsive genes in SP cells (compared with MP cells). In the present study, we observed that forced expression of Abcg2 resulted in an induction of antioxidant stress pathways that resulted in increased consumption of hydrogen peroxide and increased viability. These results support the conclusion that Abcg2 expression in SP cells has a cytoprotective role and promotes cellular viability following a severe injury.
The third major finding of the present study is that HIF-2 is a potent transcriptional regulator of the Abcg2 gene. HIF-2 is a basic helix–loop–helix/PAS domain transcription factor that is similar in composition to HIF-1 but has distinct functions.19 For example, mice lacking HIF-2 have multiple organ pathologies (ie, heart, skeletal muscle, liver, etc), increased generation of reactive oxygen species, and decreased expression of antioxidant enzymes,.19 In addition, the results of transcriptional assays revealed that HIF-2 was a direct upstream regulator of antioxidant enzymes.19 Collectively, these studies support the hypothesis that HIF-2 is a primary sensor of the oxidative stress response and promotes a cytoprotective response to maintain ROS homeostasis. Importantly, these functional roles for HIF-2 were distinct from HIF-1. Recent studies further support a specific role for HIF-2 in stem and progenitor cell populations. For example, HIF-2 is a specific upstream transcriptional activator of the pluripotency factor, Oct4 in stem cell populations.17 In the present study, we provide molecular biological and biochemical data supporting the role of HIF-2 as an upstream regulator of the Abcg2 gene. These results extend the repertoire of gene expression that is regulated by HIF-2 and provide a mechanistic insight toward the molecular response of progenitor cell populations to stressful stimuli. Moreover, these results further highlight the molecular programs characteristic of stem/progenitor cell populations that promote survival during the postinjury period, including the regulation of Abcg2 gene expression.
In conclusion, these studies demonstrate that the cardiac SP cells are resident in the adult heart and increase in number in response to injury. Furthermore, these studies also unveil a cytoprotective functional role of Abcg2 in response to oxidative stress, which are downstream of HIF-2. These studies further enhance our understanding of the cardiac SP cell population, define a functional role of Abcg2 in the SP cell population, and decipher signal transduction pathways in which stem/progenitor cell populations are protected from oxidative stress.
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Acknowledgments |
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We acknowledge the technical assistance provided by Sean Goetsch and Nan Jiang.
Sources of Funding
Funding was provided by GlaxoSmithKline (to C.M.M.), the American Heart Association (to D.J.G.), and the March of Dimes Associations (to D.J.G.).
Disclosures
None.
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Footnotes |
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Original received August 10, 2007; revision received February 26, 2008; accepted March 7, 2008.
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