Hydrocarbon Nuclear Translocator Research Articles

The role of nitric oxide (NO) in stability regulation of hypoxia inducible factor-1alpha (HIF-1alpha).

Hypoxia inducible factor-1 (HIF-1) is a master regulator under conditions of decreased oxygen availability. As a hypoxia inducible transcription factor HIF-1 is a heterodimer composed of the helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) proteins HIF-1alpha and the aryl hydrocarbon nuclear translocator (ARNT) also known as HIF-1beta. The HIF-1 transcriptional system senses decreased oxygen availability and transmits this signal into patho-physiological responses such as angiogenesis, erythropoiesis, vasomotor control, an altered energy metabolism, as well as cell survival decisions. Among recent advances are the discoveries that reactive nitrogen species (RNS), oxygen species (ROS), cytokines, and growth factors participate in stability regulation of HIF-1alpha and HIF-1 transactivation during normoxia. Here we summarize current knowledge and existing concepts that help to understand how NO affects protein accumulation of HIF-1alpha. Considering the fundamental role of radicals, especially NO, as signaling molecules makes HIF-1alpha an attractive target under conditions of NO formation that may be attributed to both, physiology and pathology. Although initial observations showed that NO inhibits hypoxia-induced HIF-1alpha stabilization and HIF-1 transcriptional activation, later studies indicated that exposure of various cells to chemically diverse NO donors or conditions of endogenous NO formation under normoxic conditions induced HIF-1alpha accumulation, HIF-1-DNA binding, and activation of downstream target gene expression. These contrasting situations evoked by NO provide insights into basic chemical reactions, biochemical signal transduction pathways with broad implications for medicine.

Aint/Tacc3 is highly expressed in proliferating mouse tissues during development, spermatogenesis, and oogenesis.

Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.

Expression of Hypoxia-inducible Factors in the Peri-implantation Mouse Uterus Is Regulated in a Cell-specific and Ovarian Steroid Hormone-dependent Manner: EVIDENCE FOR DIFFERENTIAL FUNCTION OF HIFs DURING EARLY PREGNANCY

Increased uterine vascular permeability and angiogenesis are hallmarks of implantation and placentation. These events are profoundly influenced by vascular endothelial growth factor (VEGF). We previously showed that VEGF isoforms and VEGF receptors are expressed in the uterus, suggesting the role of VEGF in uterine vascular permeability and angiogenesis required for implantation and decidualization. We have recently shown that estrogen promotes uterine vascular permeability but inhibits angiogenesis, whereas progesterone stimulates angiogenesis with little effect on vascular permeability. However, the mechanism of differential steroid hormonal regulation of uterine angiogenesis remains unresolved. Oxygen homeostasis is essential for cell survival and is primarily mediated by hypoxia-inducible factors (HIFs). These factors are intimately associated with vascular events and induce VEGF expression by binding to the hypoxia response element in the VEGF promoter. HIFalpha isoforms function by forming heterodimers with the aryl hydrocarbon nuclear translocator (ARNT) (HIF-beta) family members. There is very limited information on the relationship among HIFs, ARNTs, and VEGF in the uterus during early pregnancy, although the role of HIFs in regulating VEGF and angiogenesis in cancers is well documented. Using molecular and physiological approaches, we here show that uterine expression of HIFs and ARNTs does not correlate with VEGF expression during the preimplantation period (days 1-4) in mice. In contrast, their expression follows the localization of uterine VEGF expression with increasing angiogenesis during the postimplantation period (days 5-8). This disparate pattern of uterine HIFs, ARNTs, and VEGF expression on days 1-4 of pregnancy suggests HIFs have multiple roles in addition to the regulation of angiogenesis during the peri-implantation period. Using pharmacological, molecular, and genetic approaches, we also observed that although progesterone primarily up-regulates uterine HIF-1alpha expression, estrogen transiently stimulates that of HIF-2alpha.

Aryl hydrocarbon receptor ligands repress T-cadherin expression in vascular smooth muscle cells

T-cadherin, a glycosylphosphatidylinositol-modified cadherin subtype, is highly expressed in cardiac and vascular tissues. Neither the functions nor regulation of T-cadherin in these tissues is understood. We have cloned rat T-cadherin cDNA encoding the full length amino acid sequence. The 5 ′ untranslated nucleotide sequences of rat, mouse, and human T-cadherin contain a conserved GCGTG motif which constitutes the invariant core sequence of dioxin- or xenobiotic-regulatory elements. These elements function as target sites for aryl hydrocarbon receptor/aryl hydrocarbon nuclear translocator (AhR/ARNT) in genes regulated by this transcription factor. Using cultures of rat aortic smooth muscle cells this study presents data revealing T-cadherin as a putative target gene for negative regulation of expression through AHR signalling. Prototypic AHR agonists benzo[ a]pyrene (BaP) or 7,12-dimethylbenzanthracene (DMBA) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) repressed T-cadherin mRNA levels. Repression was antagonized by the cognate AHR antagonist α-naphthoflavone (α-NF). Repression was insensitive to inhibitors of gene transcription (actinomycin D) or de novo protein synthesis (cycloheximide), suggesting AHR/ARNT functions directly in transcriptional repression of T-cad. Regulation of adhesion proteins through the AHR pathway may represent a novel mechanism of action by atherogenic polycyclic aromatic hydrocarbons.

Mechanism of rat UDP-glucuronosyltransferase 1A6 induction by oltipraz: evidence for a contribution of the Aryl hydrocarbon receptor pathway.

The utility of oltipraz as a cancer chemopreventive agent is thought to depend on the induction of enzymes involved in phase 2 xenobiotic detoxification. Although studies of some enzymes induced by oltipraz implicate a novel transcriptional activating pathway involving Nrf2 and antioxidant-response elements (AREs), the mechanism of phenol UGT induction has remained unclear. Previous work showed that UGT1A6 is transcribed from two promoters, P1 and P2, that are both induced by oltipraz in rat liver. The effect also occurs in rat hepatocytes treated with oltipraz (concentrations >3 microM). To investigate the mechanism, luciferase reporter plasmids under the control of P1 [p(-1078/+27)1A6P1-luc] or P2 [p(-1354/+65)1A6P2-luc] were transfected into rat hepatocytes and tested for inducibility. P1, but not P2, showed responsiveness to oltipraz (2- to 5-fold increase) and 3-methylcholanthrene (10- to 30-fold increase). Because P1 contained no visible AREs, the role of a xenobiotic response element (XRE) centered between bases -134 and -129 was evaluated. Mutation of the XRE core reduced the effects of both oltipraz and 3-methylcholanthrene on the P1 reporter. The 1A6 XRE conferred oltipraz responsiveness on the simian virus 40 promoter of pGL3-Promoter. Comparative effects of oltipraz and 3-methylcholanthrene on transfected cytochrome P4501A1 reporters support the general but relatively weak XRE-stimulating activity of oltipraz. The involvement of the aryl hydrocarbon receptor (AHR) and aryl hydrocarbon nuclear translocator (ARNT) in mediating the effects of oltipraz on the XRE is supported by electrophoretic mobility supershift data and AHR/ARNT overexpression studies. These data raise questions about the contribution of AHR and other secondary induction pathways in the mechanism of oltipraz.

Interaction with Nedd8, a Ubiquitin-like Protein, Enhances the Transcriptional Activity of the Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic helix-loop-helix period aryl hydrocarbon nuclear translocator single-minded (PAS) transcription factor family. This receptor has been shown to be important in various aspects of growth and development as demonstrated by AhR-null mice. A yeast two-hybrid screen of a mouse embryonic day 11 library for AhR-interacting proteins revealed Nedd8 as a candidate. The interaction was confirmed in a cell-free system and in mammalian cells by co-immunoprecipitation; however, in vitro neddylation assays showed that Nedd8 does not covalently modify AhR. Transfection of Nedd8 into a cell line stably transfected with a dioxin response element linked to a chloramphenicol acetyltransferase reporter gene demonstrated that Nedd8 amplified ligand-induced transcription. Deletion of the Gly-76 residue in the carboxyl terminus of Nedd8 abolished this effect and prevented AhR-Nedd8 interaction. Nedd8 overexpression also resulted in accumulation of AhR protein in the nucleus, independent of exogenous ligand. These studies demonstrate that the AhR interacts with Nedd8 and suggest that this interaction enhances the transcriptional activity of the receptor, perhaps involving increased nuclear accumulation or retention. Immunohistochemistry performed on embryonic day 11.5 mouse sections indicated Nedd8 and AhR localize to overlapping areas in the heart and spinal ganglia, raising the possibility that this interaction may play a role in organogenesis.

Vanadate-induced Expression of Hypoxia-inducible Factor 1α and Vascular Endothelial Growth Factor through Phosphatidylinositol 3-Kinase/Akt Pathway and Reactive Oxygen Species

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1 alpha and HIF-1 beta/aryl hydrocarbon nuclear translocator subunits. HIF-1 expression is induced by hypoxia, growth factors, and activation of oncogenes. In response to hypoxia, HIF-1 activates the expression of many genes including vascular endothelial growth factor (VEGF) and erythropoietin. HIF-1 and VEGF play an important role in angiogenesis and tumor progression. Vanadate is widely used in industry, and is a potent inducer of tumors in humans and animals. In this study, we demonstrate that vanadate induces HIF-1 activity through the expression of HIF-1alpha but not HIF-1 beta subunit, and increases VEGF expression in DU145 human prostate carcinoma cells. We also studied the signaling pathway involved in vanadate-induced HIF-1 alpha and VEGF expression and found that phosphatidylinositol 3-kinase/Akt signaling was required for HIF-1 and VEGF expression induced by vanadate, whereas mitogen-activated protein kinase pathway was not required. We also found that reactive oxygen species (ROS) were involved in vanadate-induced expression of HIF-1 and VEGF in DU145 cells. The major species of ROS responsible for the induction of HIF-1 and VEGF expression was H(2)O(2). These results suggest that the expression of HIF-1 and VEGF induced by vanadate through PI3K/Akt may be an important signaling pathway in the vanadate-induced carcinogenesis, and ROS may play an important role.

How oxygen makes its presence felt: Figure 1.

The emergence of complex multicellular organisms during evolution required systems for ensuring adequate cellular oxygenation. Any reader doubting the veracity of this statement is invited to hold his or her breath while reading the remainder of this article. For this reason, considerable effort has been devoted to understanding how cells in higher eukaryotes sense and respond to changes in oxygen availability. Over the years a number of models were put forth to explain how cells sense oxygen. Unfortunately, these models were complex and, at times, contradictory. For example, some models suggested that a decrease in intracellular reactive oxygen species (ROS) gave rise to a low oxygen (hypoxia) signal whereas other models suggested the opposite (Semenza 1999). Likewise, some models suggested that mitochondria were categorically required for delivery of a hypoxic signal whereas others did not (Chandel et al. 1998; Srinivas et al. 2001; Vaux et al. 2001). Approximately one year ago, however, a surprisingly simple and conceptually attractive picture of oxygen sensing began to emerge. At the heart of this model is a posttranslational modification, hydroxylation, that is inherently oxygen-dependent, and a transcription factor called HIF (hypoxia-inducible factor). It has been known for some time that HIF is a master regulator of genes that are activated by low oxygen levels (Semenza 2001). These genes encode proteins that play roles in the acute and chronic adaptation to oxygen deficiency. The former include proteins involved in regulating glucose uptake, glucose metabolism, and extracellular pH, which allow for continued energy generation in a hypoxic environment. The latter include proteins involved in angiogenesis and erythropoiesis, which increase blood vessel density and blood oxygen-carrying capacity, respectively. HIF is a heterodimer consisting of one of three alpha subunits (HIF-1 , HIF-2 , or HIF-3 ) and a beta subunit (HIF-1 , also called Aryl Hydrocarbon Nuclear Translocator, or ARNT). As its name suggests, HIF is only active under hypoxic conditions. This is because the alpha subunits are rapidly degraded in the presence of oxygen due to polyubiquitination by an E3 ubiquitin ligase complex that contains the von HippelLindau tumor suppressor protein (pVHL), elongin B, elongin C, Cul2, and Rbx1 (also called ROC1 or Hrt1) (Semenza 2001; Yang and Kaelin 2001). The best studied of the alpha subunits is the ubiquitous HIF-1 . pVHL binds directly to a peptidic determinant located within a region of HIF-1 called the oxygen-dependent degradation domain (ODD), which overlaps with the HIF-1 Nterminal transactivation domain (NTAD) and is sufficient to render heterologous proteins unstable in the presence of oxygen.

AINT/ERIC/TACC: An Expanding Family of Proteins with C-terminal Coiled Coil Domains

The AINT/ERIC/TACC genes encode novel proteins with a coiled coil domain at their C-terminus. The founding member of this expanding family of genes, transforming acidic coiled coil 1 (TACC1), was isolated from a BAC contig spanning the breast cancer amplicon-1 on 8pll. Transfection of cells in vitro with TACC1 resulted in anchorage-independent growth consistent with a more "neoplastic" phenotype. Database searches employing the human TACC1 sequence revealed other novel genes, TACC2 and TACC3, with substantial sequence homology particularly in the C-terminal regions encoding the coiled coil domains. TACC2, located at 10q26, is similar to anti-zuai-1 (AZU-1), a candidate breast tumour suppressor gene, and ECTACC, an endothelial cell TACC which is upregulated by erythropoietin (Epo). The murine homologue of TACC3, murine erythropoietin-induced cDNA (mERIC-1) was also found to be upregulated by Epo in the Friend virus anaemia (FVA) model by differential display-PCR. Human ERIC-1, located at 4p16.3, has been cloned and encodes an 838-amino acid protein whose N- and C-terminal regions are highly homologous to the shorter 558-amino acid murine protein, mERIC-1. In contrast, the central portions of these proteins differ markedly. The murine protein contains four 24 amino acid imperfect repeats. ARNT interacting protein (AINT), a protein expressed during embryonic development in the mouse, binds through its coiled coil region to the aryl hydrocarbon nuclear translocator protein (ARNT) and has a central portion that contains seven of the 24 amino acid repeats found in mERIC-1. Thus mERIC-1 and AINT appear to be developmentally regulated alternative transcripts of the gene. Most members of the TACC family discovered so far contain a novel nine amino acid putative phosphorylation site with the pattern [R/K]-X(3)-[E]-X(3)-Y. Genes with sequence homology to the AINT/ERIC/TACC family in other species include maskin in Xenopus, D-TACC in Drosophila and TACC4 in the rabbit. Maskin contains a peptide sequence conserved among eIF-4E binding proteins that is involved in oocyte development. D-TACC cooperates with another conserved microtubule-associated protein Msps to stabilise spindle poles during cell division. The diversity of function already attributed to this protein family, including both transforming and tumour suppressor properties, should ensure that a new and interesting narrative is about to unfold.

2] Detection of oxygen-sensing properties of mitochondria

The chapter describes two techniques described to detect the role of mitochondria in regulating the hypoxic generation of reactive oxygen species (ROS) and activation of hypoxia inducible factor 1 ( HIF-1). In the first method, the generation of ROS and the activation of HIF-1 are examined in cells depleted of mtDNA (p° cells). The second method examines the generation of ROS and the activation of HIF-1 in cells treated with mitochondrial inhibitors. Accompanying these methods are protocols for detecting nuclear HIF-1 α protein levels by immunoblotting and detecting intracellular ROS by the oxidation of 2', 7'-dichlorofluorescin (DCFH). In theory, the same principles and methods can be applied in examining a different hypoxic response, such as activation of kinases or other transcription factors. Currently, there are two redox-dependent models to explain oxygen sensing in mammalian cells. Another model assumes that an oxygen sensor located in the cytosol detects decreases in oxygen tension by decreasing the generation of ROS. In an alternative model, it is proposed that mitochondria detect decreases in oxygen concentration and respond by increasing the generation of ROS that are required and sufficient to activate HIF-1. At the cellular level, one of the most crucial responses to hypoxia is the activation of the transcription factor HIF-1. HIF-1 is a heterodimer of two basic helix loop-helix/PAS proteins: HIF-1 α and the aryl hydrocarbon nuclear translocator (ARNT or HIF-1 β).

Okadaic acid increases ARNT homodimer transactivation potential.

The human aryl hydrocarbon nuclear translocator (hARNT) protein belongs to the family of basic helix-loop-helix (bHLH) PAS transcription factors and regulates a range of cellular processes by either homodimerizing or heterodimerizing with other bHLH-PAS proteins. hARNT has been shown to be almost exclusively phosphorylated on serine residues. However, regulation of hARNT with respect to phosphorylation remains poorly understood. The phosphatase inhibitor okadaic acid was used to explore whether a change in hARNT phosphorylation status could influence hARNT homodimer activity. The hARNT homodimer has been shown to bind to E boxes and E-box binding factors are believed to be important in the regulation of cell differentiation and proliferation. Okadaic acid significantly increased hARNT-mediated class B, E-box-driven reporter activity in COS-1 cells, transiently expressing hARNT without affecting hARNT protein levels. This alteration in hARNT-mediated class B, E-box-driven reporter activity correlates with an observed increase in [32P]orthophosphate incorporation into hARNT. Treatment with okadaic acid resulted in a 12-fold increase in [32P]orthophosphate incorporation into hARNT that was transiently expressed in COS-1 cells; an increase in the number of tryptic phosphopeptides generated from hARNT digests on two-dimensional phosphopeptide maps was also observed. Despite the significant increase in [32P]orthophosphate incorporation into hARNT, serine remained the predominantly phosphorylated residue. Clearly, increased serine phosphorylation does not appear to negatively regulate hARNT homodimerization or transactivation potential. These results demonstrate that increased hARNT homodimer signaling in COS-1 cells may result from a direct change in hARNT phosphorylation status.

Aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) activity is unaltered by phosphorylation of a periodicity/ARNT/single-minded (PAS)-region serine residue.

The aryl hydrocarbon nuclear translocator (ARNT) protein belongs to the family of basic helix-loop-helix (HLH)-periodicity/ARNT/single-minded [Per/ARNT/Sim (PAS)] transcription factors and regulates a range of cellular processes by either homodimerizing or heterodimerizing with other basic HLH-PAS proteins. To date, it has been shown that both the HLH and PAS domains are required for aryl hydrocarbon receptor (AhR) ARNT heterodimerization and that phosphorylation of ARNT is also required for this heterodimerization. Presently, regulation of ARNT with respect to phosphorylation is poorly understood. In an earlier study, murine ARNT was shown to be a phosphoprotein, to display charge heterogeneity, and to have a shift in its predominant isoforms after heterodimerization with the AhR. It was hypothesized that this shift may represent a change in ARNT phosphorylation status. Metabolic [(32)P]orthophosphate labeling of human ARNT-transfected COS-1 cells, in conjunction with phosphoamino acid analysis, Edman degradation, and phosphopeptide mapping, demonstrated that ARNT is predominantly phosphorylated on serine residues and that serine 348 (S348) in the PAS domain is phosphorylated. Alanine and glutamic acid substitutions were used to demonstrate that loss of phosphorylation at this site did not influence AhR-mediated xenobiotic response elements-driven or ARNT-mediated class B E-box-driven signaling. Additionally, the phosphorylation pattern of ARNT was unaltered after AhR heterodimerization. Although phosphorylation of S348 did not modulate AhR-ARNT or ARNT-ARNT signaling, phosphorylation of this PAS-region serine residue may be important in other ARNT-mediated gene expression systems.

Identification and expression of alternatively spliced aryl hydrocarbon nuclear translocator 2 (ARNT2) cDNAs from zebrafish with distinct functions

In order to further establish zebrafish as a vertebrate model for studying the mechanism of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) toxicity it is necessary to characterize the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator (AhR/ARNT) signaling pathways in this species. In this study, three zfARNT2 cDNAs were isolated, expressed, and characterized and named zfARNT2b, zfARNT2c, and zfARNT2a. zfARNT2b, zfARNT2c, and zfARNT2a encode proteins with theoretical molecular weights of 81, 79, and 45 kDa, respectively. zfARNT2b and zfARNT2a proteins are identical over the first 403 amino acids but differ in their C-terminal domains as a result of alternative mRNA splicing. zfARNT2c is nearly identical to zfARNT2b, with the exception of an in frame 15 amino acid deletion adjacent to the basic region of zfARNT2c. Using quantitative RT-PCR methods the tissue distribution of each zfARNT2 isoform was determined. In COS-7 cells expressing zfARNT2b and zfAhR2, 10 nM TCDD causes a nine-fold induction of a dioxin responsive reporter gene. In COS-7 cells expressing zfARNT2a or zfARNT2c, TCDD does not induce reporter gene expression. In contrast, all three zfARNT2 proteins induce reporter gene activity under control of hypoxia responsive elements when cotransfected with the zebrafish endothelial specific PAS protein 1. DNA gel shift analysis suggests that the decreased function of zfARNT2a is due to inefficient binding of zfARNT2a/zfAhR2 complexes to dioxin responsive elements. These results also indicate that alternative mRNA splicing results in formation of ARNT proteins with distinct functional properties.

Effect of Dioxin on the Gene Promoters

Effect of and other polychlorinated polyaromatic compounds on mammalian gene's expression is reviewed. The attention is focused on molecular mechanisms of gene promoter activation. We describe first the classical activation pathway: activation of aryl hydrocarbon (AhR) by agonists, the association of activated AhR with Aromatic hydrocarbon nuclear translocator (Arnt) and the activation by this dimer of the response elements of several genes. We then discuss the interaction of proteins implicated in AhR activation such as hsp90 and Arnt in the cross talk with other cellular regulatory pathways. Action of polychlorinated polyaromatic compounds on genes by mechanisms not involving dioxin receptor is also examined.

Analysis of aryl hydrocarbon receptor-mediated signaling during physiological hypoxia reveals lack of competition for the aryl hydrocarbon nuclear translocator transcription factor.

The aryl hydrocarbon nuclear translocator (ARNT) protein functions as a transcription factor after dimerization with other basic helix-loop-helix proteins. Thus, dimerization of ARNT within one pathway may limit the availability of this protein to others. To investigate this issue, aryl hydrocarbon receptor (AHR)-mediated signaling was investigated in mouse (Hepa-1), rat (H4IIE), and human (HepG2) hepatoma cell lines undergoing physiologically induced hypoxia (<1% O(2)). Basal levels of ARNT protein were not affected by hypoxia in any cell line, and ARNT remained exclusively nuclear. Furthermore, quantitative Western blotting revealed that the concentration of ARNT sequestered during hypoxia represented a small fraction of the total ARNT protein pool (12 and 15% in Hepa-1 and H4 cells, respectively). When the AHR-mediated signaling pathway was activated during hypoxia by 2,3,7,8-tetrachlorodibenzo-p-dioxin, the induction of P4501A1 protein was reduced by 55% without changes in the level of mRNA in Hepa-1 cells, whereas the levels of induction of both P4501A1 protein and CYP1A1 mRNA were reduced by >80% in the H4 cell line. Importantly, gel mobility shift analysis and Western blotting showed that the same level of AHR/ARNT complexes could be detected in cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin during hypoxia and normoxia. These data suggest that the effects of hypoxia on AHR-mediated gene regulation occur distal to the formation of AHR/ARNT complexes and imply that functional interference between hypoxia and AHR-mediated signaling does not occur through competition for ARNT protein.

Differential gene expression in wild-type and arnt-defective mouse hepatoma (Hepa1c1c7) cells.

The aryl hydrocarbon nuclear translocator (Arnt) is a basic helix-loop-helix (per/Arnt/Ahr/sim) PAS-containing protein that can heterodimerize with the aryl hydrocarbon receptor (AhR), the hypoxia-inducible factor-1 alpha, and other PAS-containing proteins to form transcriptionally active complexes. To identify the genes whose expression is modulated by Arnt, we used the technique of differential display to compare the expression of genes in wild-type and Arnt-defective (BPRc1) mouse hepatoma (Hepa1c1c7) cells. Here we report two gene products whose expression was reduced in BPRc1 cells (a WW domain-binding, protein-like factor and one unknown gene product) when compared to wild-type cells, and two that were elevated (Steel factor and a serpin-like protein). Comparison of the relative expression of these gene products between two independently-derived, Arnt-defective cell lines, as well as in BPRc1 cells in which Arnt expression was restored by a stably integrated Arnt-expression plasmid, revealed that each gene was expressed in an Arnt-independent manner. Our results clearly demonstrate that gene expression in the variant cell clones is distinctly different from that of the parental wild-type Hepa1c1c7 cells from which they were derived and involves genes in addition to, and unrelated to, that of Arnt. The identification of these differentially expressed gene products suggests that caution should be exercised when using these variant cell lines to confirm the role of the AhR/Arnt-signaling pathway in a given cellular response.

Hypoxia-induced activation of HIF-1: role of HIF-1α-Hsp90 interaction

The protein chaperone heat shock protein 90 (Hsp90) is a major regulator of different transcription factors such as MyoD, a basic helix loop helix (bHLH) protein, and the bHLH-Per-aryl hydrocarbon nuclear translocator (ARNT)-Sim (PAS) factors Sim and aryl hydrocarbon receptor (Ahr). The transcription factor hypoxia-inducible factor-1α (HIF-1α), involved in the response to hypoxia, also belongs to the bHLH-PAS family. This work was aimed to investigate the putative role of Hsp90 in HIF-1 activation by hypoxia. Using a EGFP-HIF-1α fusion protein, co-immunoprecipitation experiments evidenced that the chimeric protein expressed in COS-7 cells interacts with Hsp90 in normoxia but not in hypoxia. We also demonstrated that Hsp90 interacts with the bHLH-PAS domain of HIF-1α. Moreover, Hsp90 is not co-translocated with HIF-1α into the nucleus. At last, we showed that Hsp90 activity is essential for HIF-1 activation in hypoxia since it is inhibited in the presence of geldanamycin. These results indicate that Hsp90 is a major regulator in HIF-1α activation.

Aromatic Hydrocarbon Nuclear Translocator as a Common Component for the Hypoxia- and Dioxin-induced Gene Expression

Aromatic Hydrocarbon Nuclear Translocator as a Common Component for the Hypoxia- and Dioxin-induced Gene Expression

Induction of Endothelial PAS Domain Protein-1 by Hypoxia: Characterization and Comparison With Hypoxia-Inducible Factor-1α

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1α and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1α is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1α, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1α responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1α in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1α nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1α transactivation) of the VEGF promoter than the LDH-A promoter.

Induction of Endothelial PAS Domain Protein-1 by Hypoxia: Characterization and Comparison With Hypoxia-Inducible Factor-1α

Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1α and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1α is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1α, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1α responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1α in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1α nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1α transactivation) of the VEGF promoter than the LDH-A promoter.