HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth.
Journal: 2006/March - Genes and Development
ISSN: 0890-9369
Abstract:
The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic "knock-in" strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1alpha with HIF-2alpha results in expanded expression of HIF-2alpha-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2alpha, but not HIF-1alpha, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2alpha KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2alpha severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2alpha-specific target gene and indicate that HIF-2alpha can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2alpha's tumor promoting activity.
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Genes Dev 20(5): 557-570

HIF-2α regulates Oct-4: effects of hypoxiaon stem cell function, embryonic development, and tumor growth

Department of Cell and Developmental Biology,
Abramson Family Cancer Research Institute,
Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA;
Center for Animal Transgenesis and Germ Cell Research, New Bolton Center, University of Pennsylvania, Philadelphia, Pennsylvania 19348, USA
Present address: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Corresponding author.

E-MAIL ude.nnepu.dem.liam@2etselec; FAX (215) 746-5511.

Received 2005 Dec 8; Accepted 2006 Jan 11.

Abstract

The division, differentiation, and function of stem cells and multipotent progenitors are influenced by complex signals in the microenvironment, including oxygen availability. Using a genetic “knock-in” strategy, we demonstrate that targeted replacement of the oxygen-regulated transcription factor HIF-1α with HIF-2α results in expanded expression of HIF-2α-specific target genes including Oct-4, a transcription factor essential for maintaining stem cell pluripotency. We show that HIF-2α, but not HIF-1α, binds to the Oct-4 promoter and induces Oct-4 expression and transcriptional activity, thereby contributing to impaired development in homozygous Hif-2α KI/KI embryos, defective hematopoietic stem cell differentiation in embryoid bodies, and large embryonic stem cell (ES)-derived tumors characterized by altered cellular differentiation. Furthermore, loss of HIF-2α severely reduces the number of embryonic primordial germ cells, which require Oct-4 expression for survival and/or maintenance. These results identify Oct-4 as a HIF-2α-specific target gene and indicate that HIF-2α can regulate stem cell function and/or differentiation through activation of Oct-4, which in turn contributes to HIF-2α’s tumor promoting activity.

Keywords: HIF, hypoxia, HIF-2α, Oct-4, VEGF, TGF-α, stem cells, cancer
Abstract

Stem cells are characterized by the ability to self-renew and maintain pluripotency. The POU transcription factor Oct-4 (also known as Oct-3/4 and Pou5F1) is essential for maintaining an undifferentiated cell fate in embryonic stem (ES) cells, the embryonic epiblast, and primordial germ cells (PGCs) (Scholer et al. 1990b; Nichols et al. 1998). Oct-4 also plays a critical role in regulating the differentiation of ES cells and maintaining the pluripotent nature of the blastocyst inner cell mass (ICM). In vitro experiments have demonstrated the importance of maintaining strict Oct-4 expression levels. For example, twofold changes in Oct-4 expression cause ES cells to lose pluripotency and differentiate into trophectoderm, mesoderm, neuroectoderm, or endoderm lineages (Niwa et al. 2000; Shimozaki et al. 2003). Recently, Oct-4 has been shown to function in a complex with Nanog and Sox2 to activate and repress genes controlling stem cell identity and differentiation (Boyer et al. 2005).

Oct-4 expression is tightly controlled throughout embryogenesis and postnatal life. For example, down-regulation of Oct-4 is required for differentiation of trophectoderm and primitive endoderm lineages. At the egg cylinder stage, Oct-4 is expressed in the epiblast and subsequently down-regulated during gastrulation, although expression is maintained in PGCs. Furthermore, correlative evidence suggests that when Oct-4 expression is dysregulated, cloned embryos do not develop normally beyond post-implantation stages (Boiani et al. 2002). In the adult, Oct-4 is generally believed to be expressed exclusively in germ cells; however, recent evidence suggests Oct-4 is also present in adult stem cell populations, such as multipotent adult progenitor cells (MAPCs) and hematopoietic stem cells (Jiang et al. 2002; Tai et al. 2005). Finally, ectopic Oct-4 expression contributes to tumor growth and also drives reversible epithelial dysplasia in transgenic mice (Gidekel et al. 2003; Cheng et al. 2004; Hochedlinger et al. 2005). Currently, the mechanisms and factors regulating Oct-4 expression have not been fully elucidated.

Stem cells, as well as germ cells and other multipotent progenitors, reside in complex microenvironments, termed niches (Spradling et al. 2001). Stromal cell contacts, extracellular matrix proteins, temperature, and oxygen (O2) levels in the immediate microenvironment can influence stem cell function and differentiation. In particular, low O2 levels (hypoxia) promote the survival of neural crest stem cells, hematopoietic stem cells, and human ES cells (Morrison et al. 2000; Studer et al. 2000; Danet et al. 2003; Ezashi et al. 2005). Hypoxia also regulates the number of ICM cells in bovine blastocysts and enhances hemangioblast and hematopoietic progenitor development. (Adelman et al. 1999; Harvey et al. 2004; Ramirez-Bergeron et al. 2004). It is therefore possible that hypoxia mediates its effects on stem cell function by altering Oct-4 expression.

Hypoxia occurs in a number of physiological and pathophysiological settings, particularly when rapid tissue growth exceeds blood supply. For example, embryogenesis occurs in a physiologic “hypoxic” environment (3%–5% O2). The primary transcriptional regulators of both cellular and systemic hypoxic adaptation in mammals are hypoxia-inducible factors (HIFs). HIFs are heterodimers consisting of a regulated subunit (HIFα) and a constitutive subunit (HIFβ, also known as ARNT [aryl hydrocarbon nuclear translocator]) (Wenger and Gassmann 1997). HIFs regulate the expression of at least 180 genes involved in metabolism, cell survival, erythropoiesis, and vascular remodeling (Semenza 2000) by binding to cis-acting hypoxia response elements (HREs) located in the enhancers and/or promoters of these genes (Pugh et al. 1991).

The first HIFα subunit identified was HIF-1α, which is expressed ubiquitously in human and mouse tissues and considered to be the primary regulator of hypoxic responses. The more recent identification of HIF-2α (also known as endothelial PAS domain protein 1 [EPAS 1], HIF-1-like factor [HLF], and HIF-1-related factor [HRF]) has raised important questions about the relative activity of these factors in mediating hypoxic adaptation. HIF-1α and HIF-2α share a high degree of sequence identity, underscored by their shared ability to heterodimerize with ARNT and bind HREs to activate transcription in various in vitro reporter assays (Wiesener et al. 1998). Whereas HIF-1α is broadly expressed, HIF-2α transcripts are restricted to particular cell types, including vascular endothelial cells, neural crest cell derivatives, lung type II pneumocytes, liver parenchyma, and interstitial cells in the kidney (Ema et al. 1997; Flamme et al. 1997; Tian et al. 1997; Compernolle et al. 2002; Wiesener et al. 2003). Gene targeting experiments in mice have shown that loss of HIF-1α or HIF-2α results in strikingly different phenotypes, suggesting that each protein performs unique physiological functions. It is possible that HIF-1α and HIF-2α proteins are functionally equivalent but perform different physiological roles due to their distinct expression patterns; alternatively, HIF-1α and HIF-2α may regulate overlapping but nonidentical target genes. In studies designed to identify unique HIF-1α and HIF-2α target genes, we and others showed that HIF-1α and HIF-2α activate a number of common genes; however, HIF-1α exclusively induces the hypoxic transcription of glycolytic genes such as phosphoglycerate kinase 1 (Pgk1) and aldolase A (Alda) (Hu et al. 2003; Wang et al. 2005). Expression profiling of a renal clear cell (RCC) carcinoma line expressing HIF-2α, but not HIF-1α, identified a number of putative HIF-2α target genes, including Vascular endothelial growth factor (Vegf) and Transforming growth factor α (Tgf-α) (Gunaratnam et al. 2003; Hu et al. 2003; Wykoff et al. 2004).

To dissect the unique and overlapping roles of HIF-1α and HIF-2α more rigorously, we generated “knock-in” ES cells in which a cDNA encoding HIF-2α was targeted to the Hif-1α locus, generating a Hif-1αHif-2αKI allele, hereafter referred to as Hif-2α KI (Covello et al. 2005). This allele results in expanded HIF-2α expression under the regulatory control of the Hif-1α locus and replaces HIF-1α with HIF-2α, thereby allowing a direct comparison of HIF-1α and HIF-2α function. Previous experiments revealed that teratomas derived from homozygous Hif-2α KI/KI ES cells were significantly larger and more vascularized than wild-type tumors, and exhibited increased expression of HIF-2α target genes, including Tgf-α and Vegf (Covello et al. 2005). Interestingly, Hif-2α KI/KI tumors displayed a large proportion of undifferentiated cells in addition to a distinctive spectrum of differentiated mesodermal and neural cells.

In this report, we investigate the effects of expanded HIF-2α expression on embryonic development by generating Hif-2α KI mice. Surprisingly, homozygous Hif-2α KI/KI embryos were recovered at markedly reduced numbers at embryonic day 6.5–7.5 (E6.5–E7.5), exhibited developmental patterning defects, and displayed increased expression of Vegf, Tgf-α, and Oct-4. Increased Oct-4 mRNA levels in Hif-2α KI/KI embryoid bodies (EBs) correlated with enhanced Oct-4 protein levels and transcriptional activity, and was associated with defects in generating hematopoietic progenitors. Down-regulation of Oct-4 activity in Hif-2α KI/KI cells partly rescued the hematopoietic phenotypes; moreover, Oct-4 inhibition altered the cellular differentiation and diminished the growth of Hif-2α KI/KI teratomas. Finally, Hif-2α embryos displayed strikingly reduced numbers of PGCs, which require Oct-4 expression for survival and/or maintenance. Together, our data identify HIF-2α as an upstream regulator of Oct-4 expression, and suggest a specific mechanism whereby HIF-2α can affect stem cell function and promote tumor growth.

Acknowledgments

We thank Anja Runge, Mercy Gohil, and Kim Tremblay for technical assistance. Thanks to Hans Scholer for providing the Oct-4 reporter plasmids. Special thanks to Marisa Bartolomei, Dan Kessler, and members of the Simon laboratory for thoughtful discussions and reading of the manuscript. This research was supported by National Institutes of Health Grant HL66130 (M.C.S. and B.K.), the American Heart Association (K.L.C.) and the Abramson Family Cancer Research Institute. M.C.S. is an investigator of the Howard Hughes Medical Institute.

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Supplemental material is available at http://www.genesdev.org.

Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1399906

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