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Hypoxia-inducible factor 1 (HIF-1), and Arnt-containing, heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes
In response to hypoxia, the hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of a
network of genes encoding erythropoietin, vascular endothelial growth factor, and several glycolytic
enzymes. HIF-1 consists of a heterodimer of two basic helix-loop-helix PAS (Per/Arnt/Sim) proteins,
HIF-1alpha (Drosophila homolog: Similar) and Arnt. HIF-1alpha and Arnt mRNAs are constitutively expressed and are not altered
upon exposure of HeLa or HepG2 cells to hypoxia, suggesting that the activity of the HIF-1alpha-Arnt
complex may be regulated by some as yet unknown posttranscriptional mechanism. In support of this
model, it has been demonstrated that Arnt protein levels are not increased under conditions that induce
an hypoxic response in HeLa and HepG2 cells. However, under identical conditions, HIF-1alpha
protein levels are rapidly and dramatically up-regulated, as assessed by immunoblot analysis. In
addition, HIF-1alpha acquires a new conformational state upon dimerization with Arnt, rendering
HIF-1alpha more resistant to proteolytic digestion in vitro. Dimerization as such is not sufficient to
elicit the conformational change in HIF-1alpha, since truncated forms of Arnt that are capable of
dimerizing with HIF-1alpha do not induce this effect. The high affinity DNA binding form of
the HIF-1alpha-Arnt complex is only generated by forms of Arnt capable of eliciting the allosteric
change in conformation. In conclusion, the combination of enhanced protein levels and allosteric
change by dimerization defines a novel mechanism for modulation of transcription factor activity (Kallio, 1997).
Hypoxia-inducible factor-1 (HIF-1), a DNA-binding complex implicated in the regulation of gene
expression by oxygen, has been shown to consist of a heterodimer of two basic helix-loop-helix PAS proteins, HIF-1alpha, and HIF-1beta. One partner, HIF-1beta, is the aryl hydrocarbon receptor nuclear translocator (Arnt), an essential component of the xenobiotic response. In the present work, Arnt-deficient mutant cells, originally derived from the mouse hepatoma line Hepa1c1c7, have been used to analyze the role of
Arnt/HIF-1beta in oxygen-regulated gene expression. Two stimuli were examined: hypoxia itself and
desferrioxamine, an iron-chelating agent that also activates HIF-1. Induction of the DNA binding and
transcriptional activity of HIF-1 is absent in the mutant cells, indicating an essential role for
Arnt/HIF-1beta. Analysis of deleted Arnt/HIF-1beta genes indicates that the basic,
helix-loop-helix, and PAS domains, but not the amino or carboxyl termini, are necessary for function
in the response to hypoxia. Comparison of gene expression in wild type and mutant cells demonstrates
the critical importance of Arnt/HIF-1beta in the hypoxic induction of a wide variety of genes.
Nevertheless, for some genes a reduced response to hypoxia and desferrioxamine persists in these
mutant cells, clearly distinguishing Arnt/HIF-1beta-dependent and Arnt/HIF-1beta-independent
mechanisms of gene activation by both these stimuli (Wood, 1996).
Hypoxia-inducible factor 1 (HIF-1) is a DNA-binding heterodimeric protein complex originally
described in the transcriptional activation of the erythropoietin gene by hypoxia. This protein complex is
composed of two subunits, HIF-1alpha and HIF-1beta (synonymous with aryl hydrocarbon receptor nuclear translocator, Arnt). In this study, Arnt-deficient cells, derived from the mouse hepatoma cell line
Hepa1c1c7, were used to further characterize HIF-1 complex formation and its relationship with gene activation by hypoxia and desferrioxamine (Df). Gel shift assays reveal that Arnt is absolutely required for
the formation of the HIF-1 DNA-binding complex. Results from RNase protection assays and
Northern blots show that the lack of functional HIF-1 complex completely abrogates the response to
hypoxia of three genes, vascular endothelial growth factor (VEGF), the glycolytic enzymes aldolase A (ALDA),
and phosphoglycerate kinase 1 (PGK-1), each of which is known to be upregulated by low oxygen tension.
Desferrioxamine induction of VEGF and PGK-1 genes is reduced in the Arnt-deficient cells, but
unlike the response to hypoxia, the induction is not completely suppressed. These results suggest that Df is able to
activate gene transcription through HIF-1-independent mechanisms. Exposure to either hypoxia or Df does not
induce any changes in HIF-1alpha and -1beta mRNA levels, suggesting that posttranscriptional
mechanisms are involved in HIF-1 complex activation (Salceda, 1996).
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that
regulates hypoxia-inducible genes, including the human erythropoietin (EPO) gene. Expression of vascular endothelial growth factor (VEGF) is induced in cells exposed to hypoxia or
ischemia. Neovascularization stimulated by VEGF occurs in several important clinical contexts,
including myocardial ischemia, retinal disease, and tumor growth. Hypoxia-inducible factor 1 (HIF-1) is
a heterodimeric basic helix-loop-helix protein that activates transcription of the human erythropoietin
gene in hypoxic cells. HIF-1 is involved in the activation of VEGF
transcription. VEGF 5'-flanking sequences mediate transcriptional activation of reporter gene
expression in hypoxic Hep3B cells. A 47-bp sequence located 985 to 939 bp 5' to the VEGF
transcription initiation site mediates hypoxia-inducible reporter gene expression directed by a simian
virus 40 promoter element, which is otherwise minimally responsive to hypoxia. When reporters
containing VEGF sequences, in the context of the native VEGF or heterologous simian virus 40
promoter, are cotransfected with expression vectors encoding HIF-1alpha and Arnt, reporter gene transcription is much greater in both
hypoxic and nonhypoxic cells than in cells transfected with the reporter alone. A HIF-1 binding site
is present in the 47-bp hypoxia response element; a 3-bp substitution eliminates the ability
of the element to bind HIF-1 and to activate transcription in response to hypoxia and/or recombinant
HIF-1. Cotransfection of cells with an expression vector encoding a dominant negative form of
HIF-1alpha inhibits the activation of reporter transcription in hypoxic cells in a dose-dependent
manner. VEGF mRNA is not induced by hypoxia in mutant cells that do not express the Arnt subunit. These findings implicate HIF-1 in the activation of VEGF transcription in hypoxic
cells (Forsythe, 1996).
Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia
and dioxin signaling, respectively. Both proteins are conditionally regulated basic helix-loop-helix
(bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region
of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt.
HIF-1 alpha requires Arnt for DNA binding in vitro and functional activity
in vivo. Both the bHLH and PAS motifs of Arnt are critical for dimerization with HIF-1 alpha.
Strikingly, HIF-1 alpha exhibits very high affinity for Arnt in coimmunoprecipitation assays in vitro,
resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent
with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha
inhibits ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor
function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated
signaling pathways are not mutually exclusive, since activation of dioxin receptor function does not
impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs
are expressed constitutively in a large number of human tissues and cell lines, and these steady-state
expression levels are not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally
regulated by a mechanism that is distinct from induced expression levels, the prevalent model of
activation of HIF-1 alpha function. HIF-1 alpha is associated with
the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation
of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism,
possibly by binding to an as yet unknown class of ligands (Gradin, 1996).
bHLH PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-alpha) and the dioxin (aryl hydrocarbon) receptor (DR). The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain. Dimerization among bHLH.PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-alpha and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. Purified bacterially expressed proteins encompassing the N-terminal bHLH and bHLH.PAS regions of Arnt, DR, and HIF-1alpha have been analyzed and the contribution of the PAS domains to DNA binding in vitro was examined. Recovery of functional DNA binding proteins from bacteria was dramatically enhanced by coexpression of the bHLH.PAS regions of DR or HIF-1alpha with the corresponding region of Arnt. Formation of stable protein-DNA complexes by DR/Arnt and HIF-1alpha/Arnt heterodimers with their cognate DNA sequences requires the PAS A domains and exhibits KD values of 0.4 nM and approximately 50 nM, respectively. In contrast, the presence of the PAS domains of Arnt has little effect on DNA binding by Arnt homodimers, and these bind DNA with a KD of 45 nM. In the case of the DR, both high affinity DNA binding and dimer stability are specific to the Arnt native PAS domain, since a chimera in which the PAS A domain was substituted with the equivalent domain of Arnt generates a destabilized protein that binds DNA poorly (Chapman-Smith, 2004).
The structure and interactions of the C-terminal PAS domain of human HIF-2alpha has been studied by NMR spectroscopy. HIF-2alpha PAS-B binds the analogous ARNT domain in vitro, showing that residues involved in this interaction are located on the solvent-exposed side of the HIF-2alpha central beta-sheet. Mutating residues at this surface not only disrupts the interaction between isolated PAS domains in vitro but also interferes with the ability of full-length HIF to respond to hypoxia in living cells. Extending these findings to other PAS domains, this beta-sheet interface is found to be widely used for both intra- and inter-molecular interactions, suggesting a basis of specificity and regulation of many types of PAS-containing signaling proteins (Erbel, 2003).
Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor
composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator
(ARNT/HIF1ß). ARNT function is required
for murine placental development. Cultured trophoblast stem (TS)
cells were used to investigate the molecular basis of this requirement. In
vitro, wild-type TS cell differentiation is largely restricted to
spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS
cells differentiate into chorionic trophoblasts and syncytiotrophoblasts, as
demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the
HIV receptor CXCR4. During this process, a region of the differentiating
Arnt-null TS cells undergo granzyme B-mediated apoptosis,
suggesting a role for this pathway in murine syncytiotrophoblast turnover.
Surprisingly, HIF1alpha and HIF2alpha are induced during TS cell
differentiation in 20% O2; additionally, pVHL levels are modulated
during the same time period. These results suggest that oxygen-independent HIF
functions are crucial to this differentiation process. Since histone deacetylase
(HDAC) activity has been linked to HIF-dependent gene expression,
whether ARNT deficiency affects this epigenetic regulator was investigated.
Interestingly, Arnt-null TS cells have reduced HDAC activity,
increased global histone acetylation, and altered class II HDAC subcellular
localization. In wild-type TS cells, inhibition of HDAC activity recapitulates
the Arnt-null phenotype, suggesting that crosstalk between the HIFs
and the HDACs is required for normal trophoblast differentiation. Thus, the
HIFs play important roles in modulating the developmental plasticity of stem
cells by integrating physiological, transcriptional and epigenetic inputs (Maltepe, 2005).
Characterization of murine ARNT interacting protein (AINT)
Basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) proteins form dimeric transcription factors to mediate diverse biological functions including xenobiotic metabolism, hypoxic response, circadian rhythm and central nervous system midline development. The Ah receptor nuclear translocator protein (ARNT) plays a central role as a common heterodimerization partner. A novel, embryonically expressed, ARNT interacting protein (AINT) is described that may be a member of a larger coiled-coil PAS interacting protein family. The AINT C-terminus mediates interaction with the PAS domain of ARNT in yeast and interacts in vitro with ARNT and ARNT2 specifically. AINT
localizes to the cytoplasm and overexpression leads to non-nuclear localization of ARNT. A dynamic pattern of AINT mRNA expression during embryogenesis and cerebellum ontogeny supports a role for AINT in development (Sadek, 2000).
The AINT C-terminus is homologous to two human proteins, TACC1 and
TACC2. TACC1 (accession no. AF049910) is embryonically expressed and constitutive expression results in transformation and anchorage independent growth of mouse
fibroblasts. The TACC2 (accession no.
AF095791) gene is located on chromosome 10 and has yet
to be described further. Three additional TACC-related proteins have been described. The coiled-coil region of Xenopus maskin is most closely related to AINT and
TACC3 and interacts with CPEB and eIF-4E to restrict
polyadenylation-induced translation during oocyte matura-
tion. Drosophila TACC (D-TACC) has been shown to interact with and stabilize centrosomal microtubules in embryo extracts via the C-terminal
region (Gergely, 2000). Finally, AZU-1, a variant of
TACC2, is implicated in tumor suppression and tissue
morphogenesis of epithelial cells. A C. elegans sequence cloned from chromosome
III (accession no. Q9XWJ0) also appears to encode the
conserved coiled-coil domain of TACC proteins although
functionally this has not been characterized. To compare
this growing protein family and determine important
conserved sequences in the coiled-coil domains,
the C-terminal ends of each member were aligned and
a phylogenetic tree was constucted based on this conserved
region. The existing TACC proteins can be aligned into two distinct clades, one consisting of TACC1 and TACC2 proteins and a second containing TACC3
proteins, to which AINT belongs. With the sudden growth
in TACC family members it will be interesting to see if
future members will resolve the first clade into two separate
groups for TACC1 and TACC2 proteins (Sadek, 2000).
AINT is not a bHLH-PAS protein. Many recent studies in
the field of bHLH-PAS proteins have served to identify novel
family members and most mechanisms described thus far are
examples of heterodimerization of two bHLH-PAS proteins
via the PAS domain. However, it has become increasingly
apparent that PAS proteins also interact heterotypically with
non-PAS proteins such as AINT. Examples of this type of
interaction may be seen at different stages of the signal transduction pathway. Early in the transduction pathway the AhR interacts with HSP90 and AIP/ARA9/XAP in its non-liganded state, perhaps to maintain a conformation that recognizes ligand. The Drosophila PAS protein Period (Per) forms heterodimers with the
circadian clock protein Timeless (Tim), which does not
contain a PAS domain. This heterodimerization is important
for the regulation of circadian rhythms and is mediated by the
PAS domain of Per. In the later stages of signal transduction,
HIF-1alpha actively recruits CBP to the transcriptional complex. In addition, SRC-1, SRC-2, and SRC-3 have not been widely studied in the context of PAS interactions and, instead, have been shown to bridge interactions
between nuclear receptors and basal transcription machinery
via conserved LXXLL motifs termed nuclear receptor boxes. From
these many examples, it might not be unusual to have
isolated a non-bHLH-PAS protein in the screening for
ARNT interacting proteins. Of the heterotypic interactions mentioned above, the
Per-Tim interaction may be most relevant in the context
of AINT. In the same way that Tim interacts with the PAS
domain of Per, evidence is presented that AINT requires an intact PAS domain of ARNT for interaction. Because the PAS domain provides the dimerization surface between many proteins, this could be a clue to the function of AINT in either enhancing or disrupting homotypic interactions between ARNT and other PAS proteins (Sadek, 2000).
In addition to controlling a switch to glycolytic metabolism and induction of erythropoiesis and angiogenesis, hypoxia promotes the undifferentiated cell state in various stem and precursor cell populations. The latter process requires Notch signaling. Hypoxia blocks neuronal and myogenic differentiation in a Notch-dependent manner. Hypoxia activates Notch-responsive promoters and increases expression of Notch direct downstream genes. The Notch intracellular domain (ICD) interacts with HIF-1alpha, a global regulator of oxygen homeostasis, and HIF-1alpha is recruited to Notch-responsive promoters upon Notch activation under hypoxic conditions. Taken together, these data provide molecular insights into how reduced oxygen levels control the cellular differentiation status and demonstrate a role for Notch in this process (Gustafsson, 2005).
The data presented here indicate that Notch ICD and HIF-1α are important at the convergence point between the two signaling mechanisms. The importance of Notch ICD is underlined by the ability of γ-secretase inhibitors, which block the S3 cleavage of the Notch receptor and thus liberation of Notch ICD, to strongly reduce the hypoxic response on Notch downstream genes and promoters. Furthermore, the signaling output from an exogenously introduced Notch 1 ICD was modified by hypoxia, leading to increased activation of 12XCSL-luc and Hes-luc in a Notch 1 ICD-dependent manner. The importance of HIF-1α in this process receives support from the observed direct physical interaction between HIF-1α and Notch 1 ICD, the lack of an hypoxia-induced effect on Notch signaling in fibroblasts devoid of HIF-1α, and that both the amount and activity status of HIF-1α correlate with the level of Notch activation. The latter notion is based on the observations that: (1) transfected HIF-1α elevates the Notch downstream response; (2) a transactivation-inactive form of HIF-1α leaves the Notch response unchanged, and (3) the response is augmented in cells lacking VHL. Finally, HIF-1α is recruited to the Hey-2 promoter in C2C12 cells in a Notch- and hypoxia-dependent manner. This suggests a mechanism involving an effect of direct transcriptional activation of a Notch-responsive promoter by HIF-1α, probably as part of a Notch ICD/CSL transcriptional complex. This model is consistent with the observation that a transcriptionally inactive form of HIF-1α, which was capable of interacting with Notch 1 ICD, does not augment the Notch downstream response. It is therefore reasoned that hypoxia-dependent stabilization of Notch 1CD is not sufficient for activation of the Notch response but may require the recruitment of a form of HIF-1α containing the C-terminal transactivation domain to the Notch ICD/CSL regulatory complex, possibly potentiating the interaction with transcriptional coactivators. This hypothesis is based on the finding that HIF-1α is recruited to promoters of Notch downstream genes and the observation that a mutated form of Notch ICD, unable to interact with CBP/p300, is transcriptionally active at hypoxia. In this context, it will be interesting to learn whether HIF-1β also participates in such a regulatory complex (Gustafsson, 2005).
The link between hypoxia and Notch described here may have ramifications for other aspects of hypoxia, such as tumor development, in which deregulation of both HIF-1α- and Notch-mediated signaling events have been implicated. Since many tumors show elevated expression of HIF-1α, caused by hypoxia inherent to growing tumors and/or genetic loss of VHL, it will be interesting to investigate whether the elevated levels of HIF-1α are paralleled by increased Notch signaling, and whether the ensuing Notch induction contributes to tumor development (Gustafsson, 2005).
In conclusion, the data presented here demonstrate a link between hypoxia and Notch signaling and provide insights into how hypoxia maintains the undifferentiated cell state, by using the Notch signaling mechanism. The data also point to an important role for HIF-1α in this process and to the fact that it can interact with the Notch intracellular domain to link hypoxic information to a Notch response. These data advance the understanding of how Notch crosstalks with other signaling mechanisms and may open up possibilities to control various aspects of the hypoxic response by experimentally manipulating Notch signaling (Gustafsson, 2005).
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