FM19G11, a New Hypoxia-inducible Factor (HIF) Modulator, Affects Stem Cell Differentiation Status

Hypoxia-inducible factors (HIF) are heterodimeric (alpha/beta) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-alpha subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-alpha for degradation. In the absence of oxygen, HIF-alpha hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-alpha protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2alpha protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-alpha in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-alpha was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O2 as well as at 1% O2. Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.

The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Although several stromal cells and molecular pathways have been identified, the microenvironment of the stem cell niche remains largely unclear. Recent evidence suggests that stem cells are localized in areas with low oxygen. We have hypothesized that hypoxia maintains the undifferentiated phenotype of stem/precursor cells. In this report, we demonstrate that hypoxia reversibly arrests preadipocytes in an undifferentiated state. Consistent with this observation, hypoxia maintains the expression of pref-1, a key stem/precursor cell gene that negatively regulates adipogenic differentiation. We further demonstrate that the hypoxia-inducible factor-1 (HIF-1) constitutes an important mechanism for the inhibition of adipogenic differentiation by hypoxia. Our findings suggest that hypoxia in the stem cell niche is critical for the maintenance of the undifferentiated stem or precursor cell phenotype.

The multipotentiality and self-renewal ability of stem cells are controlled by intrinsic genetic pathways that are subject to regulation by extrinsic signals emanating from the stem cell niche. The stem cell niche provides a microenvironment composed of cellular structures or extracellular matrix in which stem cells are maintained as undifferentiated (1Li L. Xie T. Annu. Rev. Cell Dev. Biol. 2005; 21: 605-631Crossref PubMed Scopus (942) Google Scholar, 2Lin H. Nat. Rev. Genet. 2002; 3: 931-940Crossref PubMed Scopus (305) Google Scholar, 3Scadden D.T. Nature. 2006; 441: 1075-1079Crossref PubMed Scopus (1508) Google Scholar, 4Spradling A. Drummond-Barbosa D. Kai T. Nature. 2001; 414: 98-104Crossref PubMed Scopus (1215) Google Scholar, 5Watt F.M. Hogan B.L. Science. 2000; 287: 1427-1430Crossref PubMed Scopus (1474) Google Scholar, 6Fuchs E. Tumbar T. Guasch G. Cell. 2004; 116: 769-778Abstract Full Text Full Text PDF PubMed Scopus (1481) Google Scholar). 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Two HSC niches have been proposed in murine BM, an osteoblastic niche and a vascular niche, in which osteoblasts and vascular endothelial cells have been demonstrated as major components, respectively (34Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. Ross J. Haug J. Johnson T. Feng J.Q. Harris S. Wiedemann L.M. Mishina Y. Li L. Nature. 2003; 425: 836-841Crossref PubMed Scopus (2385) Google Scholar, 35Calvi L.M. Adams G.B. Weibrecht K.W. Weber J.M. Olson D.P. Knight M.C. Martin R.P. Schipani E. Divieti P. Bringhurst F.R. Milner L.A. Kronenberg H.M. Scadden D.T. Nature. 2003; 425: 841-846Crossref PubMed Scopus (2799) Google Scholar, 36Arai F. Hirao A. Ohmura M. Sato H. Matsuoka S. Takubo K. Ito K. Koh G.Y. Suda T. Cell. 2004; 118: 149-161Abstract Full Text Full Text PDF PubMed Scopus (1540) Google Scholar, 37Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. 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Immunity. 2006; 25: 977-988Abstract Full Text Full Text PDF PubMed Scopus (1693) Google Scholar). Drosophila ovary and testis provide attractive models for stem cell niche studies. In Drosophila ovary, cap cells, a specific type of somatic cell located at the tip of the germarium in the ovary, function as the niche for GSCs. An E-cadherin/β-catenin-formed cell-cell adhesion junction mediates the physical interaction between GSCs and their niche cells (23Song X. Xie T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14813-14818Crossref PubMed Scopus (184) Google Scholar, 46Song X. Zhu C.H. Doan C. Xie T. Science. 2002; 296: 1855-1857Crossref PubMed Scopus (389) Google Scholar), ensuring stem cell control by niche signals. Normally, asymmetric division of stem cells results in two daughter cells with different fates: one daughter cell attaches to the niche and is maintained as a stem cell (self-renewal), whereas the other daughter cell leaves the niche and develops into a cystoblast (1Li L. 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Interestingly, the newly formed cysts (prior to the eight-cell cyst stage, while still encapsulated by the escort cell) can revert to the stem cell state under certain circumstances, suggesting that they may still retain (albeit limited) stem cell properties (47Kai T. Spradling A. Nature. 2004; 428: 564-569Crossref PubMed Scopus (267) Google Scholar). A similar niche is also found in Drosophila testis and is composed of hub cells located at the end of the testis (48Le Bras S. Van Doren M. Dev. Biol. 2006; 294: 92-103Crossref PubMed Scopus (96) Google Scholar). Male GSCs surrounding the hub cells are interspersed with CPs (which are counterparts of ESCs in the ovary). Both GSCs and CPs are attached to the hub cells through an adhesion junction. Similar to ESCs in the ovary, proliferation and differentiation of CPs always accompany proliferation and differentiation of GSCs. CPs in the testis produce cyst cells, which encapsulate the gonialblasts (the immediate daughters of male GSCs) during spermatogonium formation (Fig. 1b). The newly formed gonialblasts can also revert to stem cells under certain circumstances, resembling their counterpart cells (CBs and cysts) in the ovary (49Brawley C. Matunis E. Science. 2004; 304: 1331-1334Crossref PubMed Scopus (326) Google Scholar). The role of cap and hub cells in GSC regulation has been well studied, but the contribution of ESCs and their offspring in the ovary and CPs and their progeny in the testis to GSC self-renewal, proliferation, and differentiation is largely unknown. However, it is known that a dialogue between the two types of cells is required for the coordination during organogenesis (30King F.J. Szakmary A. Cox D.N. Lin H. Mol. Cell. 2001; 7: 497-508Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Cap and hub cells provide an attachment point for anchoring GSCs to the niche; they also produce signals that inhibit differentiation but promote self-renewal of stem cells (17Tulina N. Matunis E. Science. 2001; 294: 2546-2549Crossref PubMed Scopus (506) Google Scholar). The stem cell niches in mammals are very complicated because of the complexity of cellular components of mammalian tissues, even though the mechanism for niche regulation is conserved from invertebrate to vertebrate. In mammals, BM tissue is composed of more than eight different hematopoietic cell lineages supported by a network of mesenchymal stromal cells and vascular endothelial cells. The two HSC niches, osteoblastic (34Zhang J. Niu C. Ye L. Huang H. He X. Tong W.G. Ross J. Haug J. Johnson T. Feng J.Q. Harris S. Wiedemann L.M. Mishina Y. Li L. Nature. 2003; 425: 836-841Crossref PubMed Scopus (2385) Google Scholar, 35Calvi L.M. Adams G.B. Weibrecht K.W. Weber J.M. Olson D.P. Knight M.C. Martin R.P. Schipani E. 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NOTCH-mediated exvivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia.

Little is known regarding how the Oct1 transcription factor regulates target gene expression. Using murine fibroblasts and two target genes, Polr2a and Ahcy, we show that Oct1 recruits the Jmjd1a/KDM3A lysine demethylase to catalyze the removal of the inhibitory histone H3K9 dimethyl mark and block repression. Using purified murine T cells and the Il2 target locus, and a colon cancer cell line and the Cdx2 target locus, we show that Oct1 recruits the NuRD chromatin-remodeling complex to promote a repressed state, but in a regulated manner can switch to a different capacity and mediate Jmjd1a recruitment to block repression. These findings indicate that Oct1 maintains repression through a mechanism involving NuRD and maintains poised gene expression states through an antirepression mechanism involving Jmjd1a. We propose that, rather than acting as a primary trigger of gene activation or repression, Oct1 is a switchable stabilizer of repressed and inducible states.

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Efficient and specific signaling by mitogen-activated protein kinases (MAPKs) is enhanced by docking sites found on many MAPK substrates and regulators. Here we show that the MAPKs ERK1 and ERK2 form a stable complex (Kd approximately 6 microm) with their substrate the microphthalmia-associated transcription factor (MITF). Complex formation requires a domain of MITF of approximately 100 residues that is nearby, but C-terminal to, the MAPK phosphorylation site at Ser73. MITF derivatives lacking this ERK-binding domain do not bind ERK2 and are phosphorylated less efficiently by ERK2. The ERK-binding domain of MITF bears no obvious resemblance to previously characterized MAPK docking motifs; in particular, it does not contain a consensus D-site. Consistent with this, ERK2-MITF binding does not require the integrity of the CD/sevenmaker region of ERK2. Furthermore, D-site peptides, which are able to potently inhibit ERK2-mediated phosphorylation of the Elk-1 transcription factor (IC50= 3 microm), are relatively poor inhibitors of ERK2-mediated phosphorylation of MITF, exhibiting >15-fold selectivity for inhibition of Elk-1 versus MITF. These observations demonstrate substrate-selective kinase inhibition: the possibility that small molecules that target docking interactions may be used to selectively inhibit the phosphorylation of a subset of the substrates of a kinase.

Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7(F/F) mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stem cells was sustained in association with the accumulation of Notch1 and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stem cells in the brain.

The morphogenesis of the hemochorial placenta is dependent upon the precise expansion and differentiation of trophoblast stem (TS) cells. SATB homeobox 1 (SATB1) and SATB2 are related proteins that have been implicated as regulators of some stem cell populations. SATB1 is highly expressed in TS cells, which prompted an investigation of SATB1 and the related SATB2 as regulators of TS cells. SATB1 and SATB2 were highly expressed in rat TS cells maintained in the stem state and rapidly declined following induction of differentiation. SATB proteins were also present within the rat placenta during early stages of its morphogenesis and disappeared as gestation advanced. Silencing Satb1 or Satb2 expression decreased TS cell self-renewal and increased differentiation, whereas ectopic expression of SATB proteins promoted TS cell expansion and blunted differentiation. Eomes, a key transcriptional regulator of TS cells, was identified as a target for SATB proteins. SATB knockdown decreased Eomes transcript levels and promoter activity, whereas SATB ectopic expression increased Eomes transcript levels and promoter activity. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation analyses demonstrated that SATB proteins physically associate with a regulatory site within the Eomes promoter. We conclude that SATB proteins promote TS cell renewal and inhibit differentiation. These actions are mediated in part by regulating the expression of the TS cell stem-associated transcription factor, EOMES.

Are transplantable stem cells required for adult hematopoiesis?

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.

hormone response element peroxisome proliferator-activated receptor thyroid hormone receptor estrogen receptor ligand binding domain nuclear receptor corepressor silencing mediator of retinoic acid and thyroid hormone receptor imitation SWI cAMP response element-binding protein CREB-binding protein histone acetyltransferase mitogen-activated protein histone deacetylase steroid receptor coactivator RAR interacting protein glucocorticoid receptor interacting protein T3R receptor associated protein vitamin receptor D interacting protein Members of the nuclear receptor superfamily directly activate or repress target genes by binding to hormone response elements (HREs)1 in promoter or enhancer regions, and by binding to other DNA sequence-specific activators and can inhibit the transcriptional activities of other classes of transcription factors by transrepression. Hormone response elements provide specificity to receptor homodimer heterodimer binding (reviewed in Ref. 2Bourguet W. Germain P. Gronemeyer H. Trends Pharm. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Nuclear receptor functions are directed by specific activation domains, referred to as activation function 1 (AF-1), which resides in the N terminus, and activation function 2 (AF-2), which resides in the C-terminal ligand binding domain (LBD) (reviewed in Ref. 1Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Regulation of gene transcription by nuclear receptors requires the recruitment of proteins characterized as coregulators, with ligand-dependent exchange of corepressors for coactivators serving as the basic mechanism for switching gene repression to activation. In this review, we discuss biochemical and genetic studies suggesting that coregulatory complexes are differentially utilized in both a cell- and promoter-specific fashion to activate or repress gene transcription. These coregulatory components, themselves targets of diverse intracellular signaling pathways, provide a combinatorial code for tissue- and gene-specific responses, utilizing both enzymatic and platform assembly functions to mediate the actions of nuclear receptor genetic programs critical for developmental and homeostatic processes in metazoan organisms. A diverse group of proteins have emerged as potential coactivators for nuclear receptors. Ligand-dependent recruitment of coactivators is dependent on AF-2, which consists of a short conserved helical sequence within the C terminus of the LBD (2Bourguet W. Germain P. Gronemeyer H. Trends Pharm. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Biochemical and expression cloning approaches have been used to identify a large number of factors that interact with nuclear receptors in either a ligand-independent or a ligand-dependent manner and are often components of large multiprotein complexes. Many of these factors are capable of potentiating nuclear receptor activity in transient cotransfection assays. In addition, a distinct set of coactivators is associated with the AF-1 domain. As the number of potential coregulators clearly exceeds the capacity for direct interaction by a single receptor, the most plausible hypothesis is that transcriptional activation by nuclear receptors involves the actions of multiple factors. These factors act in a sequential and/or combinatorial manner to reorganize chromatin templates and to modify and recruit basal factors and RNA polymerase II (3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). 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Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar, 12Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (897) Google Scholar). Mammalian homologues of Drosophila SWI2/SNF2 such as BRG1/hBrm function as components of large multiprotein complexes. Transfection of ATPase-defective alleles of either Brg1 orhBrm into several mammalian cell lines leads to a significant decrease in the ability of several nuclear receptors to activate transcription (3Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 4Wade P.A. Wollfe A.P. Curr. 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Nature. 1996; 384: 641-643Crossref PubMed Scopus (1549) Google Scholar, 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2448) Google Scholar). Rates of gene transcription roughly correlate with the degree of histone acetylation, with hyperacetylated regions of the genome appearing to be more actively transcribed than hypoacetylated regions (reviewed in Ref. 7Pazin M.J. Kadonaga J.T. Cell. 1997; 89: 325-328Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). The specific recruitment of a complex with histone acetyltransferase activity to a promoter may play a critical role in overcoming repressive effects of chromatin structure on transcription (4Wade P.A. Wollfe A.P. Curr. Biol. 1999; 9: R221-R224Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 5Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar, 6Pazin M.J. 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The central conserved domain mediates ligand-dependent interactions with the nuclear receptor LBD, whereas the conserved C-terminal transcriptional activation domains mediate interactions with either CBP/p300 or protein-arginine methyltransferase (16Chen D. Ma H. Hong H. Koh S.S. Huang S.-M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2176Crossref PubMed Scopus (1019) Google Scholar, 17Koh S. Chen D. Lee Y. Stallcup M. J. Biol. Chem. 2001; 276: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Based on the presence of three regulatory domains, members of the p160 family have been suggested to function as coactivators, at least in part, by serving as adapter molecules that recruit CBP and/or p300 complexes to promoter-bound nuclear receptors in a ligand-dependent manner (18Kurokawa R. Kalafus D. Ogliastro M.-H. Kioussi C. Xu L. Torchia J. Rosenfeld M.G. Glass C.K. 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Aging of intestinal stem cells.

Wnt Signaling, Lgr5, and Stem Cells in the Intestine and Skin

Giardia lamblia differentiates into resistant walled cysts for survival outside the host and transmission. During encystation, synthesis of cyst wall proteins is coordinately induced. The E2F family of transcription factors in higher eukaryotes is involved in cell cycle progression and cell differentiation. We asked whether Giardia has E2F-like genes and whether they influence gene expression during Giardia encystation. Blast searches of the Giardia genome database identified one gene (e2f1) encoding a putative E2F protein with two putative DNA-binding domains. We found that the e2f1 gene expression levels increased significantly during encystation. Epitope-tagged E2F1 was found to localize to nuclei. Recombinant E2F1 specifically bound to the thymidine kinase and cwp1-3 gene promoters. E2F1 contains several key residues for DNA binding, and mutation analysis revealed that its binding sequence is similar to those of the known E2F family proteins. The E2F1-binding sequences were positive cis-acting elements of the thymidine kinase and cwp1 promoters. We also found that E2F1 transactivated the thymidine kinase and cwp1 promoters through its binding sequences in vivo. Interestingly, E2F1 overexpression resulted in a significant increase of the levels of CWP1 protein, cwp1-3 gene mRNA, and cyst formation. We also found E2F1 can interact with Myb2, a transcription factor that coordinate up-regulates the cwp1-3 genes during encystation. Our results suggest that E2F family has been conserved during evolution and that E2F1 is an important transcription factor in regulation of the Giardia cwp genes, which are key to Giardia differentiation into cysts.