scholarly journals RNA-seq analysis of PHD and VHL inhibitors reveals differences and similarities to the hypoxia response.

2019 ◽  
Vol 4 ◽  
pp. 17 ◽  
Author(s):  
Julianty Frost ◽  
Alessio Ciulli ◽  
Sonia Rocha
Keyword(s):  
Cellular Response ◽  
Hypoxia Inducible Factor ◽  
Gene Activation ◽  
Transcriptional Response ◽  
Dependent Manner ◽  
Rna Seq ◽  
Pharmacological Agents ◽  
Hypoxia Response ◽  
Von Hippel Lindau ◽  
Large Numbers

Background:Hypoxia-inducible factor (HIF) transcription factors are well known to control the transcriptional response to hypoxia. Given the importance of cellular response to hypoxia, a number of pharmacological agents to interfere with this pathway have been developed and entered pre-clinical or clinical trial phases. However, how similar or divergent the transcriptional response elicited by different points of interference in cells is currently unknown.Methods:We performed RNA-sequencing to analyse the similarities and differences of transcriptional response in HeLa cells treated with hypoxia or chemical agents that stabilise HIF by inhibiting components of the hypoxia signalling pathway – prolyl hydroxylase (PHD) inhibitor or von Hippel–Lindau (VHL) inhibitor.Results:This analysis revealed that hypoxia produces the highest changes in gene transcription, with activation and repression of genes being in large numbers. Treatment with the PHD inhibitor IOX2 or the VHL inhibitor VH032 led mostly to gene activation, majorly via a HIF-dependent manner. These results were also confirmed by qRT-PCR using more specific and/or efficient inhibitors, FG-4592 (PHDs) and VH298 (VHL).Conclusion:PHD inhibition and VHL inhibition mimic gene activation promoted by hypoxia via a HIF-dependent manner. However, gene repression is mostly associated with the hypoxia response and not common to the response elicited by inhibitors of the pathway.

scholarly journals The Hypoxia-Inducible Factor 2α N-Terminal and C-Terminal Transactivation Domains Cooperate To Promote Renal Tumorigenesis In Vivo

2007 ◽  
Vol 27 (6) ◽  
pp. 2092-2102 ◽  
Author(s):  
Qin Yan ◽  
Steven Bartz ◽  
Mao Mao ◽  
Lianjie Li ◽  
William G. Kaelin
Keyword(s):  
Transcriptional Activation ◽  
Hypoxia Inducible Factor ◽  
Gene Activation ◽  
Tumor Formation ◽  
Transactivation Domain ◽  
Renal Carcinogenesis ◽  
Von Hippel Lindau ◽  
Transactivation Domains ◽  
Relative Contribution

ABSTRACT Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor, consisting of an alpha subunit and a beta subunit, that controls cellular responses to hypoxia. HIFα contains two transcriptional activation domains called the N-terminal transactivation domain (NTAD) and the C-terminal transactivation domain (CTAD). HIFα is destabilized by prolyl hydroxylation catalyzed by EglN family members. In addition, CTAD function is inhibited by asparagine hydroxylation catalyzed by FIH1. Both hydroxylation reactions are linked to oxygen availability. The von Hippel-Lindau tumor suppressor protein (pVHL) is frequently mutated in kidney cancer and is part of the ubiquitin ligase complex that targets prolyl hydroxylated HIFα for destruction. Recent studies suggest that HIF2α plays an especially important role in promoting tumor formation by pVHL-defective renal carcinoma cells among the three HIFα paralogs. Here we dissected the relative contribution of the two HIF2α transactivation domains to hypoxic gene activation and renal carcinogenesis and investigated the regulation of the HIF2α CTAD by FIH1. We found that the HIF2α NTAD is capable of activating both artificial and naturally occurring HIF-responsive promoters in the absence of the CTAD. Moreover, we found that the HIF2α CTAD, in contrast to the HIF1α CTAD, is relatively resistant to the inhibitory effects of FIH1 under normoxic conditions and that, perhaps as a result, both the NTAD and CTAD cooperate to promote renal carcinogenesis in vivo.

Evitar (l-Alanyl-l-Glutamine) Regulates Key Signaling Molecules in the Pathogenesis of Postoperative Tissue Fibrosis

2018 ◽  
Vol 26 (6) ◽  
pp. 724-733 ◽  
Author(s):  
Lynne M. Robertson ◽  
Nicole M. Fletcher ◽  
Michael P. Diamond ◽  
Ghassan M. Saed
Keyword(s):  
Cellular Response ◽  
Type I Collagen ◽  
Primary Cultures ◽  
Hypoxia Inducible Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Dependent Manner ◽  
Tissue Fibrosis ◽  
Hypoxia Inducible Factor 1Α ◽  
Episodic Hypoxia

Aims:Hypoxia and the resulting oxidative stress play a major role in postoperative tissue fibrosis. The objective of this study was to determine the effect of l-alanyl-l-glutamine (Ala-Gln) on key markers of postoperative tissue fibrosis: hypoxia-inducible factor (HIF) 1α and type I collagen.Methods:Primary cultures of human normal peritoneal fibroblasts (NPF) established from normal peritoneal tissue were treated with increasing doses of Ala-Gln (0, 1, 2, or 10 mM) with hypoxia ([2% O2] 0-48 hours; continuous hypoxia) or after hypoxia (0.5, 1, 2, 4 hours) and restoration of normoxia (episodic hypoxia) with immediate treatment with Ala-Gln. Hypoxia-inducible factor 1α and type 1 collagen levels were determined by enzyme-linked immunosorbent assay. Data were analyzed with 1-way analysis of variance followed by Tukey tests with Bonferroni correction.Results:Hypoxia-inducible factor 1α and type I collagen levels increased in untreated controls by 3- to 4-fold in response to continuous and episodic hypoxia in human NPF. Under continuous hypoxia, HIF-1α and type I collagen levels were suppressed by Ala-Gln in a dose-dependent manner. l-alanyl-l-glutamine treatment after episodic hypoxia also suppressed HIF-1α and type I collagen levels for up to 24 hours for all doses and up to 48 hours at the highest dose, regardless of exposure time to hypoxia.Conclusions:l-alanyl-l-glutamine significantly suppressed hypoxia-induced levels of key tissue fibrosis (adhesion) phenotype markers under conditions of continuous as well as episodic hypoxia in vitro. This effect of glutamine on molecular events involved in the cellular response to insult or injury suggests potential therapeutic value for glutamine in the prevention of postoperative tissue fibrosis.

The role of iron and 2-oxoglutarate oxygenases in signalling

2003 ◽  
Vol 31 (3) ◽  
pp. 510-515 ◽  
Author(s):  
K.S. Hewitson ◽  
L.A. McNeill ◽  
J.M. Elkins ◽  
C.J. Schofield
Keyword(s):  
Transcriptional Activation ◽  
Hypoxia Inducible Factor ◽  
Transcriptional Response ◽  
Transactivation Domain ◽  
Von Hippel Lindau ◽  
Helix Loop Helix ◽  
Hypoxic Conditions ◽  
Tumour Suppressor Protein ◽  
Dependent Modification

Sensing of ambient dioxygen levels and appropriate feedback mechanisms are essential processes for all multicellular organisms. In animals, moderate hypoxia causes an increase in the transcription levels of specific genes, including those encoding vascular endothelial growth factor and erythropoietin. The hypoxic response is mediated by hypoxia-inducible factor (HIF), an αβ heterodimeric transcription factor in which both the HIF subunits are members of the basic helix–loop–helix PAS (PER-ARNT-SIM) domain family. Under hypoxic conditions, levels of HIFα rise, allowing dimerization with HIFβ and initiating transcriptional activation. Two types of dioxygen-dependent modification to HIFα have been identified, both of which inhibit the transcriptional response. Firstly, HIFα undergoes trans-4-hydroxylation at two conserved proline residues that enable its recognition by the von Hippel-Lindau tumour-suppressor protein. Subsequent ubiquitinylation, mediated by an ubiquitin ligase complex, targets HIFα for degradation. Secondly, hydroxylation of an asparagine residue in the C-terminal transactivation domain of HIFα directly prevents its interaction with the co-activator p300. Hydroxylation of HIFα is catalysed by enzymes of the iron(II)- and 2-oxoglutarate-dependent dioxygenase family. In humans, three prolyl hydroxylase isoenzymes (PHD1–3) and an asparagine hydroxylase [factor inhibiting HIF (FIH)] have been identified. The role of 2-oxoglutarate oxygenases in the hypoxic and other signalling pathways is discussed.

scholarly journals Activation of the Hypoxia-Inducible Factor Pathway Expands Early Thymic Progenitors

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2896-2896
Author(s):  
Anita Hollenbeck ◽  
Stefanie Weber ◽  
Kathrin Händschke ◽  
Mandy Necke ◽  
Bertram Opalka ◽  
...  
Keyword(s):  
Cellular Response ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Cell Receptor ◽  
Normal Numbers ◽  
Cellular Compartment ◽  
Vhl Gene ◽  
Von Hippel Lindau

Abstract Early thymic progenitors enter the thymus and are exposed to regional hypoxia while they develop in a step-wise manner to mature functional T-cells. Therefore, hypoxia might represent an important component of the highly specialized thymic microenvironment. On the molecular level the hypoxia-inducible factor pathway controls the cellular response to hypoxia. In this pathway, the von-Hippel-Lindau protein (pVHL) continuously mediates the destruction of the transcription factor hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. Under hypoxia HIF-1α degradation is inhibited leading to the activation of HIF-1α target genes. Others used lck-Cre transgene-mediated conditional in vivo deletion of the Vhl gene to study the role of the oxygen-sensing pathway in developing thymocytes and found normal numbers of early double-negative (DN; CD4-CD8-) thymocytes (Biju et al., Mol Cell Biol, 2004). However, lck-Cre deletion initiates at the DN3 (CD25+CD44-) stage leaving the Vhl locus of very early DN1 (CD25-CD44+), DN2 (CD25+CD44+) and DN3 thymocytes unaltered. Therefore, we here used the ubiquitous hematopoietic deleter strain vav-Cre to investigate the role of pVHL in very early thymocytes (vav-Cre;VhlloxP;loxP mice). Using a PCR-based strategy we confirmed complete deletion of the Vhl gene in this model. We observed unaltered DN1 and DN2 progenitor numbers, however in contrast to the published lck-cre-mediated system we consistently observed an up to twofold expansion of the DN3 cellular compartment. As the hypoxia-inducible factor pathway was shown to modulate NOTCH1 signaling we studied Notch1 expression on Vhl-deficient thymocytes. Strikingly, Notch1 expression was significantly increased on expanded Vhl null DN3 thymocytes. At the DN3 developmental stage selection of cells with an accurately re-arranged T-cell receptor β-locus occurs. Thus, we analyzed pre- and post-β-selection DN3 cells by CD28 staining. Interestingly, we found both pre- and post-β-selection DN3 subpopulations expanded. In order to investigate whether the progenitor expansion is mediated by the lack of HIF-1α inhibition in the Vhl-deficient context we studied DN3 thymocytes in a conditional hematopoietic HIF-1α gain-of-function model (vav-Cre;HIF1dPA). Overexpression of HIF-1α, which is insensitive to pVHL-mediated degradation in vav-Cre;HIF1dPAmice, also resulted in expanded DN3 thymocytes. In summary, we describe novel conditional models to genetically alter the hypoxia-inducible factor pathway within very early thymic progenitors. Genetic Vhl loss led to an expansion of DN3 thymocytes. This DN3 expansion is most likely due to the absence of HIF-1α-inhibition, because HIF-1α overexpression phenocopied the Vhl-deficient DN3 thymocyte expansion. Disclosures Dührsen: Celgene: Honoraria, Research Funding.

scholarly journals Hydroxylation of Actin Impairs Cell Motility Through Blocking its’ Histidine Methylation

2021 ◽  
Author(s):  
Zhenzhen Zi ◽  
Lin Yuan ◽  
Qing Ding ◽  
Chiho Kim ◽  
Xu-dong Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Motility ◽  
Actin Polymerization ◽  
Hypoxia Inducible Factor ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Von Hippel Lindau ◽  
Oxygen Conditions ◽  
Prolyl Hydroxylation

AbstractProtein hydroxylation is a post translational modification happens on various amino acids, which is catalyzed by the oxoglutarate and oxygen dependent dioxygenases. The best characterized hydroxylated protein is the hypoxia inducible factor (HIF), which is degraded by VHL/elongin C/elongin B/cullin 2/RBX1 (VCB/CR) E3 complex under normal oxygen conditions. Hypoxia or inhibitors (including FG4592 and MK8617) of PHDs stabilize HIF1a and regulate its downstream targets. Prolyl hydroxylase, including PHD2 and PHD3 has been reported in regulating actin polymerization and cell motility. Here, we found MK8617 regulated cell motility in Von Hippel Lindau (VHL) dependent manner. Through the protein hydroxylation proteome experiment upon MK8617 treatment, we identified Pro70 in actin could be hydroxylated and near to His73, which has been reported be methylated and stabilize actin polymerization. Using biochemical assay, we found that binding of VHL with hydroxylated actin (Pro70) decrease the His73 methylation by blocking the interaction of actin with SETD3, the His73 methyltransferase, and further regulated actin polymerization and cell motility. In summary, our study revealed that hypoxia and deficiencies in the VHL, in a HIF independent and prolyl hydroxylation dependent manner, regulate actin polymerization and cell motility through the PTM (Post Translational Modifications) crosstalk.

scholarly journals Nuclear Hormone Receptor NHR-49 controls a HIF-1-independent hypoxia adaptation pathway inCaenorhabditis elegans

2021 ◽  
Author(s):  
Kelsie R. S. Doering ◽  
Xuanjin Cheng ◽  
Luke Milburn ◽  
Ramesh Ratnappan ◽  
Arjumand Ghazi ◽  
...  
Keyword(s):  
Hormone Receptor ◽  
Hypoxia Inducible Factor ◽  
Nuclear Hormone Receptor ◽  
Negative Regulator ◽  
Peroxisome Proliferator ◽  
Rna Seq ◽  
Peroxisome Proliferator Activated Receptor ◽  
Interacting Protein ◽  
Hypoxia Response ◽  
Synthetic Lethal

AbstractThe response to insufficient oxygen (hypoxia) is orchestrated by the conserved Hypoxia-Inducible Factor (HIF). However, HIF-independent hypoxia response pathways exist that act in parallel to HIF to mediate the physiological hypoxia response. Here, we describe a HIF-independent hypoxia response pathway controlled byCaenorhabditis elegansNuclear Hormone Receptor NHR-49, an orthologue of mammalian Peroxisome Proliferator-Activated Receptor alpha (PPARα). We show thatnhr-49is required for worm survival in hypoxia and is synthetic lethal withhif-1in this context, demonstrating that these factors act independently. RNA-seq analysis shows that in hypoxianhr-49regulates a set of genes that arehif-1-independent, including autophagy genes that promote hypoxia survival. We further show that Nuclear Hormone Receptornhr-67is a negative regulator and Homeodomain-interacting Protein Kinasehpk-1is a positive regulator of the NHR-49 pathway. Together, our experiments define a new, essential hypoxia response pathway that acts in parallel to the well-known HIF-mediated hypoxia response.

scholarly journals Properties of Switch-like Bioregulatory Networks Studied by Simulation of the Hypoxia Response Control System

2004 ◽  
Vol 15 (7) ◽  
pp. 3042-3052 ◽  
Author(s):  
Kurt W. Kohn ◽  
Joseph Riss ◽  
Olga Aprelikova ◽  
John N. Weinstein ◽  
Yves Pommier ◽  
...  
Keyword(s):  
Cellular Response ◽  
Synthesis Rate ◽  
Growth Factor Signaling ◽  
Simulation Studies ◽  
Response Control ◽  
Hypoxia Response ◽  
Von Hippel Lindau ◽  
Response Characteristics ◽  
Interaction Map ◽  
Conserved Core

A complex bioregulatory network could be more easily comprehended if its essential function could be described by a small “core” subsystem, and if its response characteristics were switch-like. We tested this proposition by simulation studies of the hypoxia response control network. We hypothesized that a small subsystem governs the basics of the cellular response to hypoxia and that this response has a sharp oxygen-dependent transition. A molecular interaction map of the network was prepared, and an evolutionarily conserved core subsystem was extracted that could control the activity of hypoxia response promoter elements on the basis of oxygen concentration. The core subsystem included the hypoxia-inducible transcription factor (HIFα:ARNT heterodimer), proline hydroxylase, and the von Hippel-Lindau protein. Simulation studies showed that the same core subsystem can exhibit switch-like responses both to oxygen level and to HIFα synthesis rate, thus suggesting a mechanism for hypoxia response promoter element-dependent responses common to both hypoxia and growth factor signaling. The studies disclosed the mechanism responsible for the sharp transitions. We show how parameter sets giving switch-like behavior can be found and how this type of behavior provides a foundation for quantitative studies in cells.

scholarly journals Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization

2017 ◽  
Vol 114 (37) ◽  
pp. 9948-9953 ◽  
Author(s):  
Andrea Vettori ◽  
David Greenald ◽  
Garrick K. Wilson ◽  
Margherita Peron ◽  
Nicola Facchinello ◽  
...  
Keyword(s):  
Cross Talk ◽  
Cellular Response ◽  
Isolated Hepatocytes ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Transcriptional Responses ◽  
Human Liver Tissue ◽  
Von Hippel Lindau ◽  
Response To Stress

Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src–mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL.

scholarly journals USP37 promotes deubiquitination of HIF2α in kidney cancer

2020 ◽  
Vol 117 (23) ◽  
pp. 13023-13032
Author(s):  
Kai Hong ◽  
Lianxin Hu ◽  
Xijuan Liu ◽  
Jeremy M. Simon ◽  
Travis S. Ptacek ◽  
...  
Keyword(s):  
Clear Cell ◽  
Hypoxia Inducible Factor ◽  
Three Dimensional ◽  
Xenograft Model ◽  
Dependent Manner ◽  
Potential Therapeutic Target ◽  
Cell Renal Cell Carcinoma ◽  
Von Hippel Lindau ◽  
Orthotopic Xenograft Model ◽  
Factor Α

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of tumor suppressor Von Hippel Lindau (VHL) function, which leads to accumulation of hypoxia inducible factor α (including HIF1α and HIF2α). HIF2α was previously reported to be one of the major oncogenic drivers in ccRCC, however, its therapeutic targets remain challenging. Here we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and discovered that ubiquitin-specific peptidase 37 (USP37) is a DUB that binds HIF2α and promotes HIF2α deubiquitination. As a result, USP37 promotes HIF2α protein stability in an enzymatically dependent manner, and depletion of USP37 leads to HIF2α down-regulation in ccRCC. Functionally, USP37 depletion causes decreased cell proliferation measured by MTS, two-dimensional (2D) colony formation as well as three-dimensional (3D) anchorage- independent growth. USP37 is also essential for maintaining kidney tumorigenesis in an orthotopic xenograft model and its depletion leads to both decreased primary kidney tumorigenesis and spontaneous lung metastasis. Our results suggest that USP37 is a potential therapeutic target in ccRCC.

scholarly journals OR-026 Exercise induces HIF-1α redistribution in the small intestine

2018 ◽  
Vol 1 (2) ◽  
Author(s):  
Die Wu ◽  
Wei Cao ◽  
Beibei Luo ◽  
Dao Xiang ◽  
Peijie Chen
Keyword(s):  
Blood Flow ◽  
Small Intestine ◽  
Hypoxia Inducible Factor ◽  
Transcriptional Response ◽  
Positive Control ◽  
Time Dependent ◽  
Dependent Manner ◽  
Long Time ◽  
Normoxic Control ◽  
Exercise Induced

Objective Intestinal epithelial cells are positioned between an anaerobic lumen and a highly metabolic lamina propria, affected by reduced blood flow and tissue hypoxia. Exercise induces blood flow redistribution, leading to hypoperfusion and gastrointestinal (GI) compromise. The hypoxia-inducible factor (HIF) 1α is pivotal in the transcriptional response to oxygen flux. In this study, we hypothesized that exercise induces GI system hypoxia and accumulates HIF-1α. Methods (1) ROSA26 ODD-Luc/+ mouse model (ODD-Luc) was used to detect HIF-1α expression in the intestine (female, 8-week, n=6/group). ODD-Luc mice were randomized into 4 groups: stayed in 21% O2 as the normoxic control (C), exercise (E), injected HIF-1α inhibitor PX-478 before swimming (PS), placed in the chamber containing 9% O2 for 4 hours as the positive control (PC). (2) Exercise models were conducted by volume: Moderate Exercise (ME): mice voluntarily swam for 30 min; Heavy-intensity Exercise (HE): mice swam for 1.5 hours with 5% body weight loads attached to their tails; Long-time Exercise (LE): mice voluntarily swam for 3 hours or till fatigue. Results (1) Exercise increased HIF-1α in the abdominal area. The luciferase activities boosted after exercise, compared to the controls (ME v.s. C, P<0.05; HE v.s. C, P<0.05; LE v.s. C, P<0.05) but no differences among three exercise groups (ME v.s. HE, P>0.99; ME v.s. LE, P>0.99; HE v.s. LE, P>0.99); (2) Exercise altered HIF-1α distribution in the small intestine in a time-dependent manner. The expression of HIF-1α was significantly increased after exercise and gradually reduced to the rest level. The photons increased at the 0th hour after exercise compared to that of the normoxic control (P<0.01). The level of photons then reduced over time, while the 2nd, 4th and 6th hour post-exercise were still greater than that of the normoxic control  (2nd hour v.s. C, P<0.01; 4th hour v.s. C, P<0.01; 6th hour v.s. C, P<0.05), and returned to normal after 24 hours (24th hour v.s. C, P>0.99). Conclusions Exercise induced the distribution of HIF-1α in the small intestine. The expression of HIF-1α is shown in a time-dependent manner after exercise.

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