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A New Notch in the HIF Belt: How Hypoxia Impacts Differentiation

2005; Elsevier BV; Volume: 9; Issue: 5 Linguagem: Inglês

10.1016/j.devcel.2005.10.001

1878-1551

Pilar Cejudo–Martín, Randall S. Johnson,

Epigenetics and DNA Methylation

In the current issue of Developmental Cell, work by Gustafsson and coworkers demonstrates that hypoxia synergizes with Notch to inhibit differentiation of myogenic and neural precursor cells (Gustafsson et al., 2005Gustafsson M.V. Zheng X. Pereira T. Gradin K. Jin S. Lundkvist J. Ruas J.L. Poellinger L. Lendahl U. Bondesson M. Dev. Cell. 2005; 9 (this issue): 617-628Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar). This effect requires a newly described interaction between the transcriptionally active form of HIF-1α and the intracellular domain of Notch. In the current issue of Developmental Cell, work by Gustafsson and coworkers demonstrates that hypoxia synergizes with Notch to inhibit differentiation of myogenic and neural precursor cells (Gustafsson et al., 2005Gustafsson M.V. Zheng X. Pereira T. Gradin K. Jin S. Lundkvist J. Ruas J.L. Poellinger L. Lendahl U. Bondesson M. Dev. Cell. 2005; 9 (this issue): 617-628Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar). This effect requires a newly described interaction between the transcriptionally active form of HIF-1α and the intracellular domain of Notch. What role does oxygen, and its delivery, play during embryogenesis? That there is a connection has been apparent for many years. Studies of mammalian embryogenesis in the 1970s showed that successful development of the neural fold is highly sensitive to oxygenation levels; it was demonstrated that when mammalian embryos were cultured, the degree of culture oxygenation must be kept to very specific levels (Morriss and New, 1979Morriss G.M. New D.A. J. Embryol. Exp. Morphol. 1979; 54: 17-35PubMed Google Scholar). Interestingly, these studies also showed that switching from a hypoxic to normoxic and ultimately a hyperoxic state is essential for the successful formation and closure of the neural fold in mammalian embryos, at least ex utero. This embryonic oxygenation shift has never been well characterized at a molecular level. However, an interesting intersection occurred in more recent studies, in which it has been shown that the HIF-1 transcription factor, which regulates most hypoxic response, is also required for embryogenesis, and indeed, when mutated it gives rise to animals that have defects in neural fold formation and closure (Iyer et al., 1998Iyer N.V. Kotch L.E. Agani F. Leung S.W. Laughner E. Wenger R.H. Gassmann M. Gearhart J.D. Lawler A.M. Yu A.Y. Semenza G.L. Genes Dev. 1998; 12: 149-162Crossref PubMed Scopus (1932) Google Scholar, Maltepe et al., 1997Maltepe E. Schmidt J.V. Baunoch D. Bradfield C.A. Simon M.C. Nature. 1997; 386: 403-407Crossref PubMed Scopus (607) Google Scholar, Ryan et al., 1998Ryan H.E. Lo J. Johnson R.S. EMBO J. 1998; 17: 3005-3015Crossref PubMed Scopus (1285) Google Scholar). The exposure to low levels of oxygen triggers a HIF-1 response in virtually every vertebrate cell type studied, including embryonic stem cells (Ryan et al., 1998Ryan H.E. Lo J. Johnson R.S. EMBO J. 1998; 17: 3005-3015Crossref PubMed Scopus (1285) Google Scholar). HIF-1 belongs to the basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) family of transcription factors and is composed of two subunits: HIF-1β (also know as ARNT), which is constitutively expressed, and HIF-1α, which is tightly regulated by oxygen tension. At normoxia, HIF-1α protein is hydroxylated at two proline residues (P402 and P564) by prolyl hydroxylase domain proteins (PHD), making it recognizable by the protein von Hippel-Lindau (pVHL) complex. The interaction between pVHL and HIF-1α drives ubiquitin-mediated degradation by the 26S proteasome. Under physiologically moderate oxygenation levels, HIF-1α also undergoes a hydroxylation on Asn 803 by Factor Inhibiting HIF 1 (FIH-1). This modification inhibits HIF-1 transcriptional activity, as it prevents binding to the coactivator p300/CBP. When oxygen levels decrease, the mechanisms of degradation/inactivation of HIF-1α are inhibited, so it accumulates and translocates into the nucleus, where it binds to HIF-1β and cofactors and activates transcription. Among the defined developmental roles of hypoxic response in low oxygen are stimulation of the proliferation of CNS precursor cells (Studer et al., 2000Studer L. Csete M. Lee S.H. Kabbani N. Walikonis J. Wold B. McKay R. J. Neurosci. 2000; 20: 7377-7383Crossref PubMed Google Scholar) and neural crest stem cells (Morrison et al., 2000Morrison S.J. Csete M. Groves A.K. Melega W. Wold B. Anderson D.J. J. Neurosci. 2000; 20: 7370-7376Crossref PubMed Google Scholar), promotion of survival of the chondrocyte growth plate (Schipani et al., 2001Schipani E. Ryan H.E. Didrickson S. Kobayashi T. Knight M. Johnson R.S. Genes Dev. 2001; 15: 2865-2876PubMed Google Scholar), and inhibition of adipocyte differentiation (Yun et al., 2002Yun Z. Maecker H.L. Johnson R.S. Giaccia A.J. Dev. Cell. 2002; 2: 331-341Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar), but the molecular processes underlying these effects are divergent and in some cases not understood at all. In this regard, the work by the Poellinger and Lendahl groups is groundbreaking, in that it reports a novel link between hypoxia and Notch that at least in part explains how these two pathways synergize to inhibit differentiation of precursor cells during early stages of embryogenesis (Gustafsson et al., 2005Gustafsson M.V. Zheng X. Pereira T. Gradin K. Jin S. Lundkvist J. Ruas J.L. Poellinger L. Lendahl U. Bondesson M. Dev. Cell. 2005; 9 (this issue): 617-628Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar). Notch signaling is a highly evolutionarily conserved pathway that regulates many aspects of cellular differentiation in multicellular organisms. It mediates cell-cell signaling between adjacent cells expressing Notch receptors and Notch ligands. Notch receptors are single-pass transmembrane proteins with several tandem epidermal growth factor-like and LIN12/Notch repeats in the extracellular domain, a RAM domain and ankyrin repeats in the intracellular domain (ICD), as well as nuclear localization sequences. Notch activation takes place after binding to ligand. Notch ligands are also single-pass transmembrane proteins and belong to the DSL family (Delta-Serrate-Lag2). After binding to ligand, the Notch receptor undergoes two proteolytic cleavages: The first is metalloprotease-dependent and takes place in the extracellular domain (S2), and the second is endomembranous and mediated by a γ-secretase complex (S3). This releases the intracellular domain of Notch (ICD), which translocates to the nucleus and associates with the CSL transcription factor and coactivators such as CBP/p300 and mastermind-like proteins, and regulates the expression of target genes such as Hes and Hey. The Poellinger and Lendahl groups found that hypoxic inhibition of differentiation of myogenic cells, satellite cells, and neural stem cells was reverted after incubation with the γ-secretase inhibitor L-685,458; in turn demonstrating that the cleavage of the Notch receptor was necessary for this inhibition (Gustafsson et al., 2005Gustafsson M.V. Zheng X. Pereira T. Gradin K. Jin S. Lundkvist J. Ruas J.L. Poellinger L. Lendahl U. Bondesson M. Dev. Cell. 2005; 9 (this issue): 617-628Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar). Supporting this, hypoxia stimulated the expression of Notch downstream genes Hes-1 and Hey-2. Analysis of the expression of promoter constructs in the presence of an exogenous form of the Notch ICD revealed that it synergizes with hypoxia in the activation of these constructs. Intriguingly, it also synergized with HIF-1α inductors such as CoCl2, suggesting a role for HIF-1α in this process. The next step was to identify potential molecular mediators acting between hypoxic and Notch activation pathways. Pulse-chase experiments demonstrated that hypoxia increases the half-life of Notch, an effect that requires the presence of HIF-1α. Immunoprecipitation experiments showed that there is a physical interaction between HIF-1α and Notch 1 ICD. To further characterize this, a series of truncated HIF-1α constructs were expressed in P19 cells together with a Notch ICD plasmid, finding that there are two interaction domains in HIF-1α, one located in the N-terminal domain of HIF, spanning residues 1–390, and the second between residues 390 and 531. Interestingly, cotransfection of HIF-1α and Notch 1 ICD resulted in increased expression of the 12XCSL-luc plasmid, both at normoxia and hypoxia, but the truncated forms of HIF-1α failed to do so, demonstrating that only a transcriptionally active form of HIF-1α can increase Notch signaling. Finally, the authors demonstrate with chromatin immunoprecipitation assays that HIF-1α is recruited to the Hes Notch-responsive promoter under activation of both Notch signaling and low oxygen levels; they also show, using HIF-1α mutant cell lines, that Notch transcriptional activation during hypoxia requires HIF-1 function. These findings suggest a new mode of action of HIF-1α under hypoxia that differs from the canonical response, in which it needs to dimerize with HIF-1β in order to activate the transcription of hypoxia-responsive genes. Based on these data, the authors propose a model in which HIF-1α, once stabilized by hypoxia, interacts with the Notch 1 ICD and is an active part of the Notch 1 ICD/CSL transcriptional complex. There, HIF-1α would contribute to stabilize Notch 1 ICD and would enhance the transcriptional activity of the complex through the recruitment of coactivators such as CBP/p300. This model has strong similarities with the mechanism of interactions between Notch and BMP/TGF-β signaling pathways, in which the intracellular mediators SMAD1 and SMAD3 interact with Notch 1 ICD, and there is no need of SMAD binding to DNA to promote a response (Blokzijl et al., 2003Blokzijl A. Dahlqvist C. Reissmann E. Falk A. Moliner A. Lendahl U. Ibanez C.F. J. Cell Biol. 2003; 163: 723-728Crossref PubMed Scopus (269) Google Scholar, Dahlqvist et al., 2003Dahlqvist C. Blokzijl A. Chapman G. Falk A. Dannaeus K. Ibanez C.F. Lendahl U. Development. 2003; 130: 6089-6099Crossref PubMed Scopus (201) Google Scholar). Whether this model of interaction with Notch is followed by other stimuli promoting stem cell dedifferentiation remains to be elucidated. Despite the evidence suggesting a major role for HIF-1α in the interaction between hypoxia and Notch, the involvement of other hypoxia-related molecules cannot be ruled out. Whereas Notch regulation seems to be independent of pVHL, immunoprecipitation experiments showed FIH-1 physically interacts with Notch 1 ICD. Also, FIH-1 expression in P-19 cells decreased 12xCSL-luc activity when coexpressed with HIF-1α, but also in its absence, suggesting a more direct role of FIH-1 in the regulation of Notch signaling; this will certainly be worthy of study in the future. It would also be interesting to study the effect of Notch in the hypoxic response. Even though Notch 1 ICD was not recruited to the promoter of the hypoxia responsive gene PGK-1, the absence of the Notch ligand Serrate-1 in the culture caused a decrease in the amount of HIF-1α bound to the promoter, and incubation with L-685,458 decreased the mRNA expression of the HIF-1 target PGK-1 at normoxia and hypoxia. The interaction between these two essential pathways thus appears to be robust, and will likely spawn a great deal of further effort to understand what was first approached in embryo cultures more than 30 years ago: the role played by oxygen in regulating developmental fate.