ABSTRACT
Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter's guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element-independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.
Subject(s)
Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit , Poly (ADP-Ribose) Polymerase-1 , Cell Hypoxia , Gene Knockdown Techniques , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Receptor, Notch3/metabolismABSTRACT
Skin plays an essential role, mediated in part by its remarkable vascular plasticity, in adaptation to environmental stimuli. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia-responsive transcription factor HIF-1alpha inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel-Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.
Subject(s)
Epidermis/physiology , Oxygen/metabolism , Animals , Blood Chemical Analysis , Erythropoietin/metabolism , Humans , Hypoxia-Inducible Factor 1/genetics , Hypoxia-Inducible Factor 1/metabolism , Keratinocytes/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Nitric Oxide/blood , Oxygen/blood , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
Hypoxia causes dramatic changes in gene expression profiles, and the mechanism of hypoxia-inducible transcription has been analyzed for use as a model system of stress-inducible gene regulation. In this study, changes in chromatin organization in promoters of hypoxia-inducible genes were investigated during hypoxia-reoxygenation conditions. Most of the hypoxia-inducible gene promoters were hypersensitive to DNase I under both normal and hypoxic conditions, and our data indicate an immediate recruitment of transcription factors under hypoxic conditions. In some of the hypoxia-inducible promoters, nucleosome-free DNA regions (NFRs) were established in parallel with hypoxia-induced transcription. We also show that the hypoxia-inducible formation of NFRs requires that hypoxia-inducible transcription factors (HIFs) bind to the promoters together with the transcriptional coactivator CBP. Within 1â¯h after the hypoxia exposure was ended (reoxygenation), HIF complexes were dissociated from the promoter regions. Within 24â¯h of reoxygenation, the hypoxia-induced transcription returned to basal levels and the nucleosome structure was reassembled in the hypoxia-inducible NFRs. Nucleosome reassembly required the function of the transcriptional coregulator SIN3A. Thus, reversible changes in nucleosome organization mediated by transcription factors are notable features of stress-inducible gene regulation.
Subject(s)
Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/physiopathology , Neuroblastoma/genetics , Nucleosomes/physiology , Promoter Regions, Genetic , Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Chromatin Assembly and Disassembly , Gene Expression Profiling , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Neuroblastoma/metabolism , Neuroblastoma/pathology , Tumor Cells, CulturedABSTRACT
Hypoxia causes dramatic changes in the expression profiles of genes that encode glycolytic enzymes, vascular endothelial growth factors, erythropoietin, and other factors in a tissue-specific manner through activating hypoxia-inducible transcription factors (HIFs) such as HIF1α and HIF2α. It has been elucidated that the activity of HIFs is fundamentally regulated by their protein stability in an oxygen-dependent manner. However, little is known about how stabilized HIFs regulate transcription of their target genes in hypoxic cells. Additionally, the roles of HIF3α, the third member of the HIFs, are still enigma due to its various splicing variants and the complicated phenotypes of Hif3a-gene modified mouse lines. Here, we summarize how molecular systems fine-tune hypoxia-inducible transcription with the cooperation of HIFs and their negative regulators, including IPAS, one of the HIF3α splicing variants. Since epigenetic mechanisms contribute to stress-inducible and cell-type specific gene regulation, the HIF-dependent reorganization of nucleosome structures in hypoxia-inducible gene promoters is also discussed.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Hypoxia/physiology , Gene Expression Regulation/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/metabolism , Animals , Humans , Transcription Factors/metabolismABSTRACT
Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF-1α can interact with the Notch intracellular domain and enhance Notch target gene expression. In this study, we have identified a Notch-independent mechanism that regulates the responsiveness of the Hes1 gene to hypoxia. Using P19 cells we show that silencing the Notch DNA binding partner CSL does not prevent hypoxia-dependent upregulation of Hes1 expression. In contrast to CSL, knockdown of HIF-1α or Arnt expression prevents Hes1 induction in hypoxia. Deletion analysis of the Hes1 promoter identified a minimal region near the transcription start site that is still responsive to hypoxia. In addition, we show that mutating the GA-binding protein (GABP) motif significantly reduced Hes1 promoter-responsiveness to hypoxia or to HIF-1 overexpression whereas mutation of the hypoxia-responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF-1α binds to the proximal region of the Hes1 promoter in a Notch-independent manner. Using the same experimental approach, the presence of GABPα and GABPß1 was also observed in the same region of the promoter. Loss- and gain-of-function studies demonstrated that Hes1 gene expression is upregulated by hypoxia in a GABP-dependent manner. Finally, co-immunoprecipitation assays demonstrated that HIF-1α but not HIF-2α is able to interact with either GABPα or GABPß1. These results suggest a Notch-independent mechanism where HIF-1 and GABP contribute to the upregulation of Hes1 gene expression in response to hypoxia.
Subject(s)
Gene Expression Regulation/physiology , Transcription Factor HES-1/genetics , Transcription, Genetic/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Hypoxia , Cell Line , Chromatin Immunoprecipitation/methods , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Promoter Regions, Genetic/genetics , Receptors, Notch/metabolism , Transcription Factor HES-1/metabolismABSTRACT
BACKGROUND: Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. METHODS: The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student's t-test and ANOVA. RESULTS: In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. CONCLUSIONS: Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis.
Subject(s)
Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Carcinogenesis/genetics , Cyclin-Dependent Kinase Inhibitor p15/biosynthesis , Genes, myc/genetics , Hypoxia-Inducible Factor 1/genetics , Kruppel-Like Transcription Factors/metabolism , Cell Line, Tumor , Chromatin Immunoprecipitation , Cyclin-Dependent Kinase Inhibitor p15/genetics , Gene Expression Regulation, Neoplastic , Humans , Immunoblotting , Immunoprecipitation , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , Transfection , Two-Hybrid System TechniquesABSTRACT
In neuroblastoma specimens, HIF-2alpha but not HIF-1alpha is strongly expressed in well-vascularized areas. In vitro, HIF-2alpha protein was stabilized at 5% O2 (resembling end capillary oxygen conditions) and, in contrast to the low HIF-1alpha activity at this oxygen level, actively transcribed genes like VEGF. Under hypoxia (1% O2), HIF-1alpha was transiently stabilized and primarily mediated acute responses, whereas HIF-2alpha protein gradually accumulated and governed prolonged hypoxic gene activation. Knockdown of HIF-2alpha reduced growth of neuroblastoma tumors in athymic mice. Furthermore, high HIF-2alpha protein levels were correlated with advanced clinical stage and high VEGF expression and predicted poor prognosis in a clinical neuroblastoma material. Our results demonstrate the relevance of HIF-2alpha in neuroblastoma progression and have general tumor biological implications.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Neuroblastoma/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Child , Female , Gene Expression Profiling , Humans , Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mice , Neoplasm Transplantation , Neuroblastoma/genetics , Neuroblastoma/pathology , Oligonucleotide Array Sequence Analysis , Oxygen/metabolism , Phenotype , Procollagen-Proline Dioxygenase/genetics , Procollagen-Proline Dioxygenase/metabolism , RNA, Messenger/metabolism , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
Chromatin compaction is a key biophysical property that influences multiple DNA transactions. Lack of chromatin accessibility is frequently used as proxy for chromatin compaction. However, we currently lack tools for directly probing chromatin compaction at individual genomic loci. To fill this gap, here we present FRET-FISH, a method combining fluorescence resonance energy transfer (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at select loci in single cells. We first validate FRET-FISH by comparing it with ATAC-seq, demonstrating that local compaction and accessibility are strongly correlated. FRET-FISH also detects expected differences in compaction upon treatment with drugs perturbing global chromatin condensation. We then leverage FRET-FISH to study local chromatin compaction on the active and inactive X chromosome, along the nuclear radius, in different cell cycle phases, and during increasing passage number. FRET-FISH is a robust tool for probing local chromatin compaction in single cells.
Subject(s)
Chromatin , Fluorescence Resonance Energy Transfer , Chromatin/genetics , Fluorescence Resonance Energy Transfer/methods , In Situ Hybridization, Fluorescence/methods , DNA/metabolism , GenomicsABSTRACT
Activation of transcription in response to low oxygen tension is mediated by the hypoxia-inducible factor-1 (HIF-1). HIF-1 is a heterodimer of two proteins: aryl hydrocarbon receptor nuclear translocator and the oxygen-regulated HIF-1 alpha. The C-terminal activation domain of HIF-1 alpha has been shown to interact with cysteine/histidine-rich region 1 (CH1) of the coactivator CBP/p300 in a hypoxia-dependent manner. However, HIF forms lacking C-terminal activation domain (naturally occurring or genetically engineered) are still able to activate transcription of target genes in hypoxia. Here, we demonstrate that the N-terminal activation domain (N-TAD) of HIF-1 alpha interacts with endogenous CBP and that this interaction facilitates its transactivation function. Our results show that interaction of HIF-1 alpha N-TAD with CBP/p300 is mediated by the CH3 region of CBP known to interact with, among other factors, p53. Using fluorescence resonance energy transfer experiments, we demonstrate that N-TAD interacts with CH3 in vivo. Coimmunoprecipitation assays using endogenous proteins showed that immunoprecipitation of CBP in hypoxia results in the recovery of a larger fraction of HIF-1 alpha than of p53. Chromatin immunoprecipitation demonstrated that at 1% O(2) CBP is recruited to a HIF-1 alpha but not to a p53 target gene. Upon activation of both pathways, lower levels of chromatin-associated CBP were detected at either target gene promoter. These results identify CBP as the coactivator directly interacting with HIF-1 alpha N-TAD and mediating the transactivation function of this domain. Thus, we suggest that in hypoxia HIF-1 alpha is a major CBP-interacting transcription factor that may compete with other CBP-dependent factors, including p53, for limiting amounts of this coactivator, underscoring the complexity in the regulation of gene expression by HIF-1 alpha.
Subject(s)
E1A-Associated p300 Protein/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit , Hypoxia/physiopathology , p300-CBP Transcription Factors/metabolism , 2,2'-Dipyridyl/pharmacology , Cell Line , Chelating Agents/pharmacology , Cysteine/metabolism , E1A-Associated p300 Protein/chemistry , HeLa Cells , Histidine/metabolism , Humans , Hypoxia/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/chemistry , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Kidney/cytology , Mutagenesis , Protein Interaction Domains and Motifs/drug effects , Protein Interaction Domains and Motifs/physiology , Protein Structure, Tertiary , Transcriptional Activation/physiology , Tumor Suppressor Protein p53/metabolism , p300-CBP Transcription Factors/chemistryABSTRACT
Cells adapt to hypoxia by a cellular response, where hypoxia-inducible factor 1alpha (HIF-1alpha) becomes stabilized and directly activates transcription of downstream genes. In addition to this "canonical" response, certain aspects of the pathway require integration with Notch signaling, i.e., HIF-1alpha can interact with the Notch intracellular domain (ICD) to augment the Notch downstream response. In this work, we demonstrate an additional level of complexity in this cross-talk: factor-inhibiting HIF-1 (FIH-1) regulates not only HIF activity, but also the Notch signaling output and, in addition, plays a role in how Notch signaling modulates the hypoxic response. We show that FIH-1 hydroxylates Notch ICD at two residues (N(1945) and N(2012)) that are critical for the function of Notch ICD as a transactivator within cells and during neurogenesis and myogenesis in vivo. FIH-1 negatively regulates Notch activity and accelerates myogenic differentiation. In its modulation of the hypoxic response, Notch ICD enhances recruitment of HIF-1alpha to its target promoters and derepresses HIF-1alpha function. Addition of FIH-1, which has a higher affinity for Notch ICD than for HIF-1alpha, abrogates the derepression, suggesting that Notch ICD sequesters FIH-1 away from HIF-1alpha. In conclusion, the data reveal posttranslational modification of the activated form of the Notch receptor and an intricate mode of cross-coupling between the Notch and hypoxia signaling pathways.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/metabolism , Receptor Cross-Talk , Receptors, Notch/metabolism , Signal Transduction , Animals , Cell Line , Chick Embryo , Humans , Hydroxylation , Mice , Mixed Function Oxygenases , Muscle Development , Proto-Oncogene Proteins/metabolism , Receptor, Notch1/metabolism , Receptor, Notch2/metabolism , Receptor, Notch3 , Receptor, Notch4 , Repressor Proteins/pharmacology , Transcription Factors/pharmacology , TransfectionABSTRACT
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. Here, we show that 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 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.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/metabolism , Receptors, Notch/physiology , Signal Transduction/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/drug effects , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Carbamates/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Line , Cell Line, Tumor , Dipeptides/pharmacology , Homeodomain Proteins/drug effects , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Myoblasts/cytology , Myoblasts/drug effects , Myoblasts/metabolism , Receptor, Notch1/metabolism , Receptors, Notch/drug effects , Receptors, Notch/genetics , Repressor Proteins/drug effects , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signal Transduction/drug effects , Stem Cells/cytology , Stem Cells/drug effects , Stem Cells/metabolism , Transcription Factor HES-1ABSTRACT
Tumour-associated expression of CA IX (carbonic anhydrase IX) is to a major extent regulated by HIF-1 (hypoxia-inducible factor-1) which is important for transcriptional activation and consists of the oxygen-regulated subunit HIF-1alpha and the partner factor ARNT [AhR (aryl hydrocarbon receptor) nuclear translocator]. We have previously observed that HIF-1alpha competes with the AhR for interaction with ARNT under conditions when both conditionally regulated factors are activated. We have therefore investigated whether TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced activation of the AhR pathway might interfere with CA IX expression. The results from the present study suggest that TCDD treatment reduces hypoxic induction of both CA IX mRNA and protein expression. Moreover, the transcriptional activity of the CA9 promoter was significantly reduced by expression of CAAhR (constitutively active AhR), which activates transcription in a ligand-independent manner. Finally, we found that ARNT is critical for both hypoxic induction and the TCDD-mediated inhibition of CA9 expression.
Subject(s)
Carbonic Anhydrases/metabolism , Cell Hypoxia/physiology , Receptors, Aryl Hydrocarbon/physiology , Animals , Antigens, Neoplasm/genetics , Binding Sites , Blotting, Western , Carbonic Anhydrase IX , Carbonic Anhydrases/genetics , Cell Hypoxia/genetics , Cell Line, Tumor , Chromatin Immunoprecipitation , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Polychlorinated Dibenzodioxins/pharmacology , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effectsABSTRACT
Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or amplify dsRNA sequences. When combined with reverse transcription-polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As examples, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells.
Subject(s)
RNA, Double-Stranded/analysis , Bacterial Toxins/genetics , DNA Ligases/metabolism , Escherichia coli Proteins/genetics , Genetic Techniques , HIV Long Terminal Repeat , Polynucleotide 5'-Hydroxyl-Kinase/metabolism , RNA, Double-Stranded/metabolism , RNA, Messenger/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Substrate SpecificityABSTRACT
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
ABSTRACT
Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated α subunit (HIF-1α) and constitutively expressed ß subunit (HIF-1ß) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1α stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1α and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1α and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.
Subject(s)
Cell Nucleus/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Nuclear Proteins/metabolism , Animals , Cell Culture Techniques , Cell Hypoxia , HumansABSTRACT
Hypoxia-inducible factors (HIFs) play a central role in the transcriptional response to changes in oxygen availability. Stability of HIFs is regulated by multi-step reactions including recognition by the von Hippel-Lindau tumour suppressor protein (pVHL) in association with an E3 ligase complex. Here we show that pVHL physically interacts with fatty acid synthase (FASN), displacing the E3 ubiquitin ligase complex. This results in HIF-α protein stabilization and activation of HIF target genes even in normoxia such as during adipocyte differentiation. 25-hydroxycholesterol (25-OH), an inhibitor of FASN expression, also inhibited HIF target gene expression in cultured cells and in mouse liver. Clinically, FASN is frequently upregulated in a broad variety of cancers and has been reported to have an oncogenic function. We found that upregulation of FASN correlated with induction of many HIF target genes, notably in a malignant subtype of prostate tumours. Therefore, pVHL-FASN interaction plays a regulatory role for HIFs and their target gene expression.
ABSTRACT
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is known as a mediator of toxic responses. Recently, it was shown that the AhR has dual functions. Besides being a transcription factor, it also possesses an intrinsic E3 ubiquitin ligase function that targets, e.g., the steroid receptors for proteasomal degradation. The aim of this study was to identify the molecular switch that determines whether the AhR acts as a transcription factor or an E3 ubiquitin ligase. To do this, we used the breast cancer cell line MCF7, which expresses a functional estrogen receptor alpha (ERα) signaling pathway. Our data suggest that aryl hydrocarbon receptor nuclear translocator (ARNT) plays an important role in the modulation of the dual functions of the AhR. ARNT knockdown dramatically impaired the transcriptional activation properties of the ligand-activated AhR but did not affect its E3 ubiquitin ligase function. The availability of ARNT itself is modulated by another basic helix-loop-helix (bHLH)-Per-ARNT-SIM (PAS) protein, the repressor of AhR function (AhRR). MCF7 cells overexpressing the AhRR showed lower ERα protein levels, reduced responsiveness to estradiol, and reduced growth rates. Importantly, when these cells were used to produce estrogen-dependent xenograft tumors in SCID mice, we also observed lower ERα protein levels and a reduced tumor mass, implying a tumor-suppressive-like function of the AhR in MCF7 xenograft tumors.
Subject(s)
Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Breast Neoplasms/metabolism , Estrogen Receptor alpha/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Mice, SCID , Receptors, Aryl Hydrocarbon/genetics , Signal Transduction , Transcription Factors/genetics , Transcriptional Activation , Tumor Cells, Cultured , Ubiquitin-Protein Ligases/genetics , Xenograft Model Antitumor AssaysABSTRACT
The dioxin/aryl hydrocarbon receptor functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding primarily drug-metabolizing enzymes. Expression of a constitutively active mutant of the aryl hydrocarbon receptor (CA-AhR) in transgenic mice results in development of stomach tumours, correlating with increased mortality. We have used suppression subtractive hybridization techniques followed by macroarray analysis to elucidate which genes are differentially expressed during this process. In the glandular stomach of CA-AhR mice, we observed decreased mRNA expression of osteopontin (OPN), a noncollagenous protein of bone matrix that is also involved in several important functions including regulation of cytokine production, macrophage accumulation, cell motility and adhesion. Downregulated expression of OPN during tumour development was confirmed by RT-PCR and RNA blot analysis. Immunohistochemical analysis showed that this decrease was confined to the corpus region, correlating with the restricted localization of the tumours. Decreased OPN mRNA expression was also observed in other organs of CA-AhR mice. Taken together, these results show that OPN is negatively regulated by the dioxin receptor, and that downregulation of its expression correlates with development of stomach tumours in mice expressing a constitutively active mutant of dioxin receptor.
Subject(s)
Down-Regulation , Gene Expression Regulation, Neoplastic , Hydrocarbons/chemistry , Neoplasms, Experimental/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Sialoglycoproteins/biosynthesis , Stomach Neoplasms/metabolism , Animals , Bone and Bones/metabolism , Female , Gastric Mucosa/metabolism , Genotype , Humans , Immunohistochemistry , Male , Mice , Mice, Transgenic , Models, Genetic , Mutation , Neoplasms, Experimental/genetics , Oligonucleotide Array Sequence Analysis , Osteopontin , RNA/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sialoglycoproteins/metabolism , Time Factors , Tissue DistributionABSTRACT
Premature senescence in vitro has been attributed to oxidative stress leading to a DNA damage response. In the absence of oxidative damage that occurs at atmospheric oxygen levels, proliferation of untransformed cells continues for extended periods of time. We have investigated the role of the hypoxia-inducible factor 1alpha (HIF1alpha) transcription factor in preventing senescence in aerobic and hypoxic conditions. Using embryonic fibroblasts from a conditional HIF1alpha knockout mouse, we found that loss of HIF1alpha under aerobic conditions significantly accelerated the onset of cellular senescence, and decreased proliferation under hypoxia. Furthermore, we identify the macrophage migration inhibitory factor (MIF) as a crucial effector of HIF1alpha that delays senescence. Inhibition of MIF phenocopies loss of HIF1alpha. Our findings highlight a novel role for HIF1alpha under aerobic conditions, and identify MIF as a target responsible for this function.
Subject(s)
Cellular Senescence/physiology , Fibroblasts/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Intramolecular Oxidoreductases/physiology , Macrophage Migration-Inhibitory Factors/physiology , Aerobiosis , Animals , Cell Hypoxia , Fibroblasts/radiation effects , Gamma Rays , Gene Expression Regulation , Intramolecular Oxidoreductases/genetics , Macrophage Migration-Inhibitory Factors/genetics , Mice , Mice, Knockout , Transcription, GeneticABSTRACT
EBNA-3 is one of the Epstein-Barr virus (EBV)-encoded nuclear antigens that is indispensable for immunoblastic transformation and sustained proliferation of B-lymphocytes. The molecular mechanisms responsible for the function of EBNA-3 are poorly understood. We previously found that EBNA-3 interacts with an immunophilin-like protein XAP2/ARA9/AIP, which in mammalian cells is known to interact with the latent aryl hydrocarbon receptor (AhR). AhR is a ligand-inducible transcription factor that mediates cellular responses to environmental pollutants, such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD). In this study, we show that EBNA-3 interacts specifically with AhR. The stability of this interaction is determined by the activation state of AhR and its association with XAP2. We and others have demonstrated that XAP2 retains the nonactivated AhR in the cell cytoplasm. However, in the presence of TCDD, the effect of XAP2 on the intracellular localization of AhR was counter-acted by EBNA-3, resulting in nuclear translocation of the AhR. In addition, EBNA-3 enhanced transactivation function by the ligand-activated AhR in cells, as assessed by reporter gene assays. Our data suggested that EBNA-3 plays a role in facilitating the ligand-dependent AhR activation process. Following activation of the AhR, we also observed that EBNA-3 counteracted the inhibitory effect of TCDD on the growth of EBV-carrying lymphoblasts. Taken together, our studies revealed a novel interaction between EBV- and AhR-dependent cellular pathways that control cell proliferation and survival.