Negative regulation of HIF in skeletal muscle of elite endurance athletes: a tentative mechanism promoting oxidative metabolism.

The transcription factor hypoxia-inducible factor (HIF) has been suggested as a candidate for mediating training adaptation in skeletal muscle. However, recent evidence rather associates HIF attenuation with a trained phenotype. For example, a muscle-specific HIF deletion increases endurance performance, partly through decreased levels of pyruvate dehydrogenase kinase 1 (PDK-1). HIF activity is regulated on multiple levels: modulation of protein stability, transactivation capacity, and target gene availability. Prolyl hydroxylases (PHD1-3) induces HIF degradation, whereas factor-inhibiting HIF (FIH) and the histone deacetylase sirtuin-6 (SIRT6) repress its transcriptional activity. Together, these negative regulators introduce a mechanism for moderating HIF activity in vivo. We hypothesized that long-term training induces their expression. Negative regulators of HIF were explored by comparing skeletal muscle tissue from moderately active individuals (MA) with elite athletes (EA). In elite athletes, expression of the negative regulators PHD2 (MA 73.54 ± 9.54, EA 98.03 ± 6.58), FIH (MA 4.31 ± 0.25, EA 30.96 ± 7.99) and SIRT6 (MA 0.24 ± 0.07, EA 11.42 ± 2.22) were all significantly higher, whereas the response gene, PDK-1 was lower (MA 0.12 ± 0.03, EA 0.04 ± 0.01). Similar results were observed in a separate 6-wk training study. In vitro, activation of HIF in human primary muscle cell culture by PHD inactivation strongly induced PDK-1 (0.84 ± 0.12 vs 4.70 ± 0.63), providing evidence of a regulatory link between PHD activity and PDK-1 levels in a relevant model system. Citrate synthase activity, closely associated with aerobic exercise adaptation, increased upon PDK-1 silencing. We suggest that training-induced negative regulation of HIF mediates the attenuation of PDK-1 and contributes to skeletal muscle adaptation to exercise.

PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells.

BACKGROUND: The PIAS4 protein belongs to the family of protein inhibitors of activated STAT, but has since been implicated in various biological activities including the post-translational modification known as sumoylation. In this study, we explored the roles of PIAS4 in pancreatic tumourigenesis. METHODS: The expression levels of PIAS4 in pancreatic cancer cells were examined. Cell proliferation and invasion was studied after overexpression and gene silencing of PIAS4. The effect of PIAS4 on hypoxia signalling was investigated. RESULTS: The protein was overexpressed in pancreatic cancer cells compared with the normal pancreas. Gene silencing by PIAS4 small interfering RNA (siRNA) suppressed pancreatic cancer cell growth and overexpression of PIAS4 induced expression of genes related to cell growth. The overexpression of PIAS4 is essential for the regulation of the hypoxia signalling pathway. PIAS4 interacts with the tumour suppressor von Hippel-Lindau (VHL) and leads to VHL sumoylation, oligomerization, and impaired function. Pancreatic cancer cells (Panc0327, MiaPaCa2) treated with PIAS4 siRNA suppressed expression of the hypoxia-inducible factor hypoxia-inducible factor 1 alpha and its target genes JMJD1A, VEGF, and STAT3. CONCLUSION: Our study elucidates the role of PIAS4 in the regulation of pancreatic cancer cell growth, where the suppression of its activity represents a novel therapeutic target for pancreatic cancers.

Non-canonical Notch signaling activates IL-6/JAK/STAT signaling in breast tumor cells and is controlled by p53 and IKKα/IKKß.

Notch signaling is frequently hyperactivated in breast cancer, but how the enhanced signaling contributes to the tumor process is less well understood. In this report, we identify the proinflammatory cytokine interleukin-6 (IL-6) as a novel Notch target in breast tumor cells. Enhanced Notch signaling upregulated IL-6 expression, leading to activation of autocrine and paracrine Janus kinase/signal transducers and activators of transcription signaling. IL-6 upregulation was mediated by non-canonical Notch signaling, as it could be effectuated by a cytoplasmically localized Notch intracellular domain and was independent of the DNA-binding protein CSL. Instead, Notch-mediated IL-6 upregulation was controlled by two proteins in the nuclear factor (NF)-κB signaling cascade, IKKα and IKKß (inhibitor of nuclear factor kappa-B kinase subunit alpha and beta, respectively), as well as by p53. Activation of IL-6 by Notch required IKKα/IKKß function, but interestingly, did not engage canonical NF-κB signaling, in contrast to IL-6 activation by inflammatory agents such as lipopolysaccharide. With regard to p53 status, IL-6 expression was upregulated by Notch when p53 was mutated or lost, and restoring wild-type p53 into p53-mutated or -deficient cells abrogated the IL-6 upregulation. Furthermore, Notch-induced transcriptomes from p53 wild-type and -mutated breast tumor cell lines differed extensively, and for a subset of genes upregulated by Notch in a p53-mutant cell line, this upregulation was reduced by wild-type p53. In conclusion, we identify IL-6 as a novel non-canonical Notch target gene, and reveal roles for p53 and IKKα/IKKß in non-canonical Notch signaling in breast cancer and in the generation of cell context-dependent diversity in the Notch signaling output.

Acute hypoxia induces apoptosis of pancreatic ß-cell by activation of the unfolded protein response and upregulation of CHOP.

The success of pancreatic ß-cells transplantation to treat type 1 diabetes has been hindered by massive ß-cell dysfunction and loss of ß-cells that follows the procedure. Hypoxia-mediated cell death has been considered one of the main difficulties that must be overcome for transplantation to be regarded as a reliable therapy. Here we have investigated the mechanisms underlying ß-cell death in response to hypoxia (1% O(2)). Our studies show that mouse insulinoma cell line 6 (Min6) cells undergo apoptosis with caspase-3 activation occurring as early as 2 h following exposure to hypoxia. Hypoxia induces endoplasmic reticulum stress in Min6 cells leading to activation of the three branches of the unfolded protein response pathway. In response to hypoxia the pro-apoptotic transcription factor C/EBP homologous protein (CHOP) is upregulated. The important role of CHOP in the apoptotic process was highlighted by the rescue of Min6 cells from hypoxia-mediated apoptosis observed in CHOP-knockdown cells. Culturing isolated pancreatic mouse islets at normoxia showed intracellular hypoxia with accumulation of hypoxia-inducible factor-1α and upregulation of CHOP, the latter one occurring as early as 4 h after isolation. Finally, we observed that pancreatic islets of type 2 db/db diabetic mice were more hypoxic than their counterpart in normoglycemic animals. This finding indicates that hypoxia-mediated apoptosis may occur in type 2 diabetes.

Non-canonical HIF-2α function drives autonomous breast cancer cell growth via an AREG-EGFR/ErbB4 autocrine loop.

Tumor progression is intrinsically tied to the clonal selection of tumor cells with acquired phenotypes allowing to cope with a hostile microenvironment. Hypoxia-inducible factors (HIFs) master the transcriptional response to local tissue hypoxia, a hallmark of solid tumors. Here, we report significantly longer patient survival in breast cancer with high levels of HIF-2α. Amphiregulin (AREG) and WNT1-inducible signaling pathway protein-2 (WISP2) expression was strongly HIF-2α-dependent and their promoters were particularly responsive to HIF-2α. The endogenous AREG promoter recruited HIF-2α in the absence of a classical HIF-DNA interaction motif, revealing a novel mechanism of gene regulation. Loss of AREG expression in HIF-2α-depleted cells was accompanied by reduced activation of epidermal growth factor (EGF) receptor family members. Apparently opposing results from patient and in vitro data point to an HIF-2α-dependent auto-stimulatory tumor phenotype that, while promoting EGF signaling in cellular models, increased the survival of diagnosed and treated human patients. Our findings suggest a model where HIF-2α-mediated autocrine growth signaling in breast cancer sustains a state of cellular self-sufficiency, thereby masking unfavorable microenvironmental growth conditions, limiting adverse selection and improving therapy efficacy. Importantly, HIF-2α/AREG/WISP2-expressing tumors were associated with luminal tumor differentiation, indicative of a better response to classical treatments. Shifting the HIF-1/2α balance toward an HIF-2-dominated phenotype could thus offer a novel approach in breast cancer therapy.

Hypoxia upregulates expression of human endosialin gene via hypoxia-inducible factor 2.

Endosialin is a transmembrane glycoprotein selectively expressed in blood vessels and stromal fibroblasts of various human tumours. It has been functionally implicated in angiogenesis, but the factors that control its expression have remained unclear. As insufficient delivery of oxygen is a driving force of angiogenesis in growing tumours, we investigated whether hypoxia regulates endosialin expression. Here, we demonstrate that endosialin gene transcription is induced by hypoxia predominantly through a mechanism involving hypoxia-inducible factor-2 (HIF-2) cooperating with the Ets-1 transcription factor. We show that HIF-2 activates the endosialin promoter both directly, through binding to a hypoxia-response element adjacent to an Ets-binding site in the distal part of the upstream regulatory region, and indirectly, through Ets-1 and its two cognate elements in the proximal promoter. Our data also suggest that the SP1 transcription factor mediates responsiveness of the endosialin promoter to high cell density. These findings elucidate important aspects of endosialin gene regulation and provide a rational frame for future investigations towards better understanding of its biological significance.

Human mismatch repair gene, MLH1, is transcriptionally repressed by the hypoxia-inducible transcription factors, DEC1 and DEC2.

Tumor hypoxia has been reported to cause a functional loss in DNA mismatch repair (MMR) system as a result of downregulation of MMR genes, although the precise molecular mechanisms remain unclear. In this study, we focused on the downregulation of a key MMR gene, MLH1, and demonstrated that hypoxia-inducible transcription repressors, differentiated embryo chondrocytes (DEC1 and 2), participated in its transcriptional regulation via their bindings to E-box-like motif(s) in MLH1 promoter region. In all cancer cell lines examined, hypoxia increased expression of DEC1 and 2, known as hypoxia-inducible genes, but decreased MLH1 expression in an exposure time-dependent manner at both the mRNA and protein levels. Co-transfection reporter assay revealed that DEC1 and, to greater extent, DEC2 as well as hypoxia-repressed MLH1 promoter activity. We further found that the action was remarkably inhibited by trichostatin A, and identified a possible DEC-response element in the MLH1 promoter. In vitro electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that DEC1 or 2 directly bounds to the suggested element, and transient transfection assay revealed that overexpression of DEC2 repressed endogenous MLH1 expression in the cells. Hypoxia-induced DEC may impair MMR function through repression of MLH1 expression, possibly via the histone deacethylase-mediated mechanism in cancer cells.

Interactions of polybrominated diphenyl ethers with the aryl hydrocarbon receptor pathway.

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been in use as additives in various consumer products. Structural similarities of PBDEs with other polyhalogenated aromatic hydrocarbons that show affinity for the aryl hydrocarbon receptor (AhR), such as some polychlorinated biphenyls, raised concerns about their possible dioxin-like properties. We studied the ability of environmentally relevant PBDEs (BDE-47, -99, -100, -153, -154, and -183) and the "planar" congener BDE-77 to bind and/or activate the AhR in stably transfected rodent hepatoma cell lines with an AhR-responsive enhanced green fluorescent protein (AhR-EGFP) reporter gene (H1G1.1c3 mouse and H4G1.1c2 rat hepatoma). 7-Ethoxyresorufin-O-deethylation (EROD) was used as a marker for CYP1A1 activity. Dose- and bromination-specific inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced responses was measured by their ability to inhibit the induction of AhR-EGFP expression and EROD activity. Individual exposure to these PBDEs did not result in any increase in induction of AhR-EGFP or CYP1A1 activity. The lower brominated PBDEs showed the strongest inhibitory effect on TCDD-induced activities in both cell lines. While the highest brominated PBDE tested, BDE-183, inhibited EROD activity, it did not affect the induction of AhR-EGFP expression. Similar findings were observed after exposing stably transfected human hepatoma (xenobiotic response element [XRE]-HepG2) cells to these PBDEs, resulting in a small but statically significant agonistic effect on XRE-driven luciferase activity. Co-exposure with TCDD resulted again in antagonistic effects, confirming that the inhibitory effect of these PBDEs on TCDD-induced responses was not only due to direct interaction at receptor level but also at DNA-binding level. This antagonism was confirmed for BDE-99 in HepG2 cells transiently transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. In addition, a chromatin immunoprecipitation assay further confirmed that BDE-99 could bind to the AhR and activate the AhR nuclear translocation and dioxin responsive element (DRE) binding in the context of the CYP1A1 promoter. However, the transactivation function of the BDE-99-activated AhR seems to be very weak. These combined results suggest that PBDEs do bind but not activate the AhR-AhR nuclear translocator protein-XRE complex.

Development and validation of an average mammalian estrogen receptor-based QSAR model.

Development and evaluation of quantitative structure activity relationships (QSARs) for predicting estrogen receptor binding from chemical structure requires reliable algorithms for three-dimensional (3D) QSAR analysis and establishment of structurally diverse training sets of chemicals whose modes of action and measures of potency are well defined. One approach to selecting an appropriate training set is to minimize the biological variability in the model development, by using structurally restricted data sets. A second approach is to extend the structural diversity of chemicals at the cost of increased variability of biological assays. In this study, the second approach was used by organizing a training set of 151 chemicals with measured human alpha Estrogen Receptor (ERalpha), mouse uterine, rat uterine, and MCF7 cell Relative Binding Affinities (RBAs). The structurally augmented training set was submitted to a 3D pattern recognition analysis to derive a model for average mammalian ER binding affinity by employing the COmmon REactivity PAttern (COREPA) approach. Elucidation of this pattern required examination of the conformational flexibility of the compounds in an attempt to reveal areas in the multidimensional descriptor space, which are most populated by the conformers of the biologically active molecules and least populated by the inactive ones. The approach is not dependent upon a predetermined and specified toxicophore or an alignment of conformers to a lead compound. Reactivity patterns associated with mammalian ER binding affinity were obtained in terms of global nucleophilicity (E(HOMO)), interatomic distances between nucleophilic sites, and local nucleophilicity (charges or delocalizabilities) of those sites. Based on derived patterns, descriptor profiles were established for identifying and ranking compounds with RBA of > 150, 150-10, 10-1 and 1-0.1% relative to 17beta-estradiol. Specificity of reactivity profiles was found to increase gradually with increasing affinities associated with RBAs ranges under study. Using the results of this analysis, an exploratory expert system was developed for use in ranking relative mammalian ER binding affinity potential for large chemical data sets. The validity of the RBA predictions were confirmed by independent development and comparison with measured RBA values.

Comparison of acute toxicities of indolo[3,2-b]carbazole (ICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in TCDD-sensitive rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons are environmental toxicants that act via the AH receptor (AHR). In vitro studies have demonstrated that some indole derivatives present in cruciferous vegetables also bind to the AHR. One of the highest AHR binding affinities is exhibited by indolo[3,2-b]carbazole (ICZ). Since exposure to these dietary indoles is quantitatively far larger than that to halogenated aromatic compounds, their potential toxic risks have raised concern. In the present study, we compared the effects of ICZ with those of a single dose of 20 microg/kg TCDD in the most TCDD-sensitive rat strain (Long-Evans [Turku AB]) (L-E). Whereas TCDD elicited the expected toxicity syndrome, ICZ, either as a single subcutaneous dose (63.5, 127 or 508 microg/kg) or with repeated sc dosing (508 microg/kg for 5 days) failed to reproduce any toxic impacts of TCDD. Furthermore, a simultaneous ICZ treatment (63.5 or 127 microg/kg for 10 days) did not interfere with TCDD (20 microg/kg; single exposure) action. A moderate hepatic induction of CYP1A1 could be triggered by repeated intragastric administration of ICZ (127 microg/kg for 4 days, the last treatment 2.5 h prior to termination). In control experiments in a reconstituted yeast system, ICZ potently and dose-dependently activated L-E rat AHR function demonstrating that it represents a bona fide high-affinity ligand for the rat receptor in vivo. Thus, the present study does not support the view that dietary exposure to ICZ would present a hazard of AHR-mediated adverse health effects to humans.

Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression.

Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

Definition of a dioxin receptor mutant that is a constitutive activator of transcription: delineation of overlapping repression and ligand binding functions within the PAS domain.

The intracellular dioxin (aryl hydrocarbon) receptor is a ligand-activated transcription factor that mediates the adaptive and toxic responses to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and structurally related congeners. Whereas the ligand-free receptor is characterized by its association with the molecular chaperone hsp90, exposure to ligand initiates a multistep activation process involving nuclear translocation, dissociation from the hsp90 complex, and dimerization with its partner protein Arnt. In this study, we have characterized a dioxin receptor deletion mutant lacking the minimal ligand-binding domain of the receptor. This mutant did not bind ligand and localized constitutively to the nucleus. However, this protein was functionally inert since it failed to dimerize with Arnt and to bind DNA. In contrast, a dioxin receptor deletion mutant lacking the minimal PAS B motif but maintaining the N-terminal half of the ligand-binding domain showed constitutive dimerization with Arnt, bound DNA, and activated transcription in a ligand-independent manner. Interestingly, this mutant showed a more potent functional activity than the dioxin-activated wild-type receptor in several different cell lines. In conclusion, the constitutively active dioxin receptor may provide an important mechanistic tool to investigate receptor-mediated regulatory pathways in closer detail.

A mouse model for adenovirus gene delivery.

The cellular attachment receptor for adenovirus (Ad), Coxsackie adenovirus receptor (CAR), required for delivery of Ad into primary cells, is not present on all cell types, thus restricting Ad-gene delivery systems. To circumvent this constrain, a transgenic mouse has been generated that expresses a truncated human CAR in all tissues analyzed. These mice allowed efficient in vitro infections at low multiplicities into lymphoid, myeloid, and endothelial cells. Furthermore, in vivo administration of Ad-vectors results in infection of macrophages, lymphocytes, and endothelial cells. In addition, tail vein injection resulted in targeting of virus into previously inaccessible areas, such as the lung and the capillaries of the brain. The CAR transgenic mice will be useful for rapid functional genomic analysis in vivo, for testing the efficacy of gene therapy procedures or as a source of easily transducible cells.

Monitoring of in vitro and in vivo translation of green fluorescent protein and its fusion proteins by fluorescence correlation spectroscopy.

BACKGROUND: Because the process of protein translation is an event of sparse molecules, the measurement requires high sensitivity. One of the candidates for studying the molecules is fluorescence correlation spectroscopy (FCS), which gleans quantitative information from fluctuating fluorescence signals in a diluted solution. METHODS: Using FCS, the translation products of expression plasmid for green fluorescent protein (GFP) and its fusion proteins were measured in vitro and in vivo. RESULTS: In in vitro translation, the number of products increased linearly for 90 min upon concentration of the plasmid. The autocorrelation function for GFP was fitted with a one-component model with a diffusion time of 0.18 ms, which was identical to the value expected from the molecular weight. In the cases of GFP- tagged hypoxia-inducible factor-1 alpha and glucocorticoid receptor, each fitting result was significantly improved with a two-component model. The slow component with a diffusion time of 6 ms appeared to be related to the ribosome or polysome. In response to the addition of dexamethasone, the nuclear translocation from cytosol clearly induced the decrease in number of molecules in the focal point. CONCLUSIONS: FCS permits monitoring of the number of molecules translated in vitro and in vivo, the translation rate, and the molecular weight.

The hsp90 chaperone complex regulates intracellular localization of the dioxin receptor.

The molecular chaperone complex hsp90-p23 interacts with the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/Per-Arnt-Sim domain transcription factor. Whereas biochemical and genetic evidence indicates that hsp90 is important for maintenance of a high-affinity ligand binding conformation of the dioxin receptor, the role of hsp90-associated proteins in regulation of the dioxin receptor function remains unclear. Here we demonstrate that the integrity of the hsp90 complex characterized by the presence of the hsp90-associated cochaperone p23 and additional cochaperone proteins is important for regulation of the intracellular localization of the dioxin receptor by two mechanisms. First, in the absence of ligand, the dioxin receptor-hsp90 complex was associated with the immunophilin-like protein XAP2 to mediate cytoplasmic retention of the dioxin receptor. Second, upon exposure to ligand, the p23-associated hsp90 complex mediated interaction of the dioxin receptor with the nuclear import receptor protein pendulin and subsequent nuclear translocation of the receptor. Interestingly, these two modes of regulation target two distinct functional domains of the dioxin receptor. Whereas the nuclear localization signal-containing and hsp90-interacting bHLH domain of the receptor regulates ligand-dependent nuclear import, the interaction of the p23-hsp90-XAP2 complex with the ligand binding domain of the dioxin receptor was essential to mediate cytoplasmic retention of the ligand-free receptor form. In conclusion, these data suggest a novel role of the hsp90 molecular chaperone complex in regulation of the intracellular localization of the dioxin receptor.

Aryl hydrocarbon receptor/dioxin receptor in human monocytes and macrophages.

Aryl hydrocarbon receptor (AhR) belongs to the bHLH/PAS transcription factor family and is activated by various polycyclic or halogenated aromatic hydrocarbons, e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3MC). In the present study, we showed that in U937 cells and human macrophages AhR, with its partner cofactor Arnt, is expressed and CYP1A1 mRNA expression is induced in the presence of AhR ligand 3MC. Moreover, we showed that AhR, associating with Arnt, binds to target DNA sequences and activates transcription. Since part of AhR is activated into DNA binding species in the absence of exogenous ligand and competitive AhR antagonist alpha-naphthoflavone inhibits this activation process with reducing CYP1A1 mRNA expression levels, the presence of endogenous ligand is indicated.

Characterization and developmental expression of xSim, a Xenopus bHLH/PAS gene related to the Drosophila neurogenic master gene single-minded.

We have isolated a novel gene from Xenopus, denominated xSim, which encodes a protein of 760 amino acids containing a basic helix-loop-helix (bHLH) motif contiguous to a PAS domain characteristic of an emerging family of transcriptional regulators so called bHLH/PAS. xSim shares a strong amino acid sequence identity with the Drosophila Single-minded (dSim) and with the murine Sim1 and Sim2 proteins. Phylogenetic analysis reveals that xSim gene is an ortholog gene of the mSim2 gene. Spatio-temporal analysis shows a maternal and a zygotic expression of xSim throughout early Xenopus development. In situ hybridization assays reveal that the transcripts are enriched in the animal hemisphere until blastula stage and extend to the marginal zone at early gastrula stage. As development proceeds, xSim is mainly restricted to the central nervous system.

The immunophilin-like protein XAP2 regulates ubiquitination and subcellular localization of the dioxin receptor.

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent transcription factor that induces expression of a number of genes encoding drug metabolizing enzymes. The nonactivated form of the dioxin receptor is associated with heat shock protein (hsp) 90, the co-chaperone p23, and the immunophilin-like protein XAP2. Whereas hsp90 has a role in maintenance of the high-affinity ligand binding conformation of the dioxin receptor complex, and p23 stabilizes receptor-hsp90 interaction, the exact role of XAP2 is largely unknown. Here we show that XAP2 protected the ligand-free form of receptor against ubiquitination, resulting in increased dioxin receptor protein levels. Upon exposure to ligand, nuclear translocation of the dioxin receptor was markedly delayed by XAP2, indicating an additional role of XAP2 in regulation of the subcellular localization of the receptor by a mechanism of cytoplasmic retention. In order to mediate these effects, XAP2 required stable association with the hsp90-p23 molecular chaperone complex. The association of XAP2 as well as p23 with the dioxin receptor was determined by the functional state of hsp90. These data indicate a novel mode of regulation of dioxin receptor signaling by the hsp90-dependent molecular chaperone machinery.

Mechanism of regulation of the hypoxia-inducible factor-1 alpha by the von Hippel-Lindau tumor suppressor protein.

In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha. The region of VHL mediating interaction with HIF-1 alpha overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1 alpha and subsequent degradation. Interestingly, the VHL binding site within HIF-1 alpha overlapped with one of the minimal transactivation domains. Protection of HIF-1 alpha against degradation by VHL was a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1 alpha during this process. Finally, stabilization of HIF-1 alpha protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.

Mechanistic aspects--the dioxin (aryl hydrocarbon) receptor.

The Ah receptor mediates the toxicological responses of 2,3,7,8-TCDD and related compounds. Receptor-deficient animals were shown to be resistant to the toxic effects of dioxin, although there is also evidence for the existence of a receptor-independent pathway for dioxin-induced toxicity. In the cytosol the receptor is present in a non-activated ligand binding conformation. Association with Arnt in the nucleus turns the receptor complex into a ligand activated form. The physiological role of the receptor is not yet understood; however, the conservation of the receptor in a wide range of animal species (including humans) suggests a fundamental role in cellular physiology.