Induction of the HIF pathway: Differential regulation by chemical hypoxia and oxygen glucose deprivation

AbstractThe Hypoxia Inducible Factor (HIF) proteins are the master regulators in the cellular response to varying oxygen levels, including hypoxia. The HIF complex is stabilized and accumulates when oxygen levels drop through inhibition of a degradative enzyme. An active HIF complex can act as a transcriptional regulator of hundreds of genes. In turn, these genes determine the response of the cell by inducing pathways which can promote survival, or result in cell death. However, little is known about the regulation of the transcriptional process. We were interested in learning more about the time dependence of transcriptional activation in order to target those pathways which could enhance cell survival after ischemia. Using mouse hippocampal organotypic cultures (HOTCs), we compared oxygen-glucose deprivation with the hypoxia mimetic cobalt, which inhibits the oxygen dependent prolyl hydroylase and blocks degradation of the HIF proteins. We demonstrated that two of the most studied HIF target genes (VEGF, EPO) as well as HIF structural genes show complex time and dose-dependent expression patterns in response to the two different insults. Understanding of these molecular responses is crucial for the development of future treatments to enhance recovery from hypoxia and stroke.

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Hypoxic Regulation of Gene Transcription and Chromatin: Cause and Effect

International Journal of Molecular Sciences ◽

10.3390/ijms21218320 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8320

Author(s):

Jessica D. Kindrick ◽

David R. Mole

Keyword(s):

Transcriptional Activation ◽

Cellular Response ◽

Target Genes ◽

Hypoxia Inducible Factor ◽

Transcriptional Response ◽

Epigenetic Landscape ◽

Low Oxygen ◽

Epigenetic Modifiers ◽

Oxygen Sensitive ◽

Cell Type Specific

Cellular responses to low oxygen (hypoxia) are fundamental to normal physiology and to the pathology of many common diseases. Hypoxia-inducible factor (HIF) is central to this by enhancing the transcriptional activity of many hundreds of genes. The cellular response to HIF is cell-type-specific and is largely governed by the pre-existing epigenetic landscape. Prior to activation, HIF-binding sites and the promoters of HIF-target genes are already accessible, in contact with each other through chromatin looping and display markers of activity. However, hypoxia also modulates the epigenetic environment, both in parallel to and as a consequence of HIF activation. This occurs through a combination of oxygen-sensitive changes in enzyme activity, transcriptional activation of epigenetic modifiers, and localized recruitment to chromatin by HIF and activated RNApol2. These hypoxic changes in the chromatin environment may both contribute to and occur as a consequence of transcriptional regulation. Nevertheless, they have the capacity to both modulate and extend the transcriptional response to hypoxia.

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Variations within oxygen-regulated gene expression in humans

Journal of Applied Physiology ◽

10.1152/japplphysiol.90578.2008 ◽

2009 ◽

Vol 106 (1) ◽

pp. 212-220 ◽

Cited By ~ 19

Author(s):

Jerome T. S. Brooks ◽

Gareth P. Elvidge ◽

Louisa Glenny ◽

Jonathan M. Gleadle ◽

Chun Liu ◽

...

Keyword(s):

Cellular Response ◽

Genetic Manipulation ◽

Hypoxia Inducible Factor ◽

Hereditary Disease ◽

Peripheral Blood Lymphocytes ◽

Cellular Level ◽

Normal Individuals ◽

Regulated Gene Expression ◽

Open Question ◽

Hif Pathway

The effects of hypoxia on gene transcription are mainly mediated by a transcription factor complex termed hypoxia-inducible factor (HIF). Genetic manipulation of animals and studies of humans with rare hereditary disease have shown that modifying the HIF pathway affects systems-level physiological responses to hypoxia. It is, however, an open question whether variations in systems-level responses to hypoxia between individuals could arise from variations within the HIF system. This study sought to determine whether variations in the responsiveness of the HIF system at the cellular level could be detected between normal individuals. Peripheral blood lymphocytes (PBL) were isolated on three separate occasions from each of 10 healthy volunteers. After exposure of PBL to eight different oxygen tensions ranging from 20% to 0.1%, the expression levels of four HIF-regulated transcripts involved in different biological pathways were measured. The profile of expression of all four transcripts in PBL was related to oxygen tension in a curvilinear manner. Double logarithmic transformation of these data resulted in a linear relationship that allowed the response to be parameterized through a gradient and intercept. Analysis of variance (ANOVA) on these parameters showed that the level of between-subject variation in the gradients of the responses that was common across all four HIF-regulated transcripts was significant ( P = 0.008). We conclude that statistically significant variation within the cellular response to hypoxia can be detected between normal humans. The common nature of the variability across all four HIF-regulated genes suggests that the source of this variation resides within the HIF system itself.

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Key Regulatory Pathways, MicroRNAs, and Target Genes Participate in Adventitious Root Formation of Acer Rubrum L

10.21203/rs.3.rs-793059/v2 ◽

2021 ◽

Author(s):

Wenpeng Zhu ◽

Manyu Zhang ◽

Jianyi Li ◽

Hewen Zhao ◽

Kezhong Zhang ◽

...

Keyword(s):

Transcriptional Activation ◽

High Throughput Sequencing ◽

Target Genes ◽

Seedling Survival ◽

Expression Patterns ◽

Economic Value ◽

Acer Rubrum ◽

Pcr Analysis ◽

Asexual Propagation ◽

Regulatory Pathways

Abstract BackgroundAcer rubrum L. is a colorful ornamental tree with great economic value. Because this tree is difficult to root under natural conditions and the seedling survival rate is low, vegetative propagation methods are often used. Because the formation of adventitious roots (ARs) is essential for the survival of asexual propagation of A. rubrum, it is necessary to investigate the molecular regulatory mechanisms in the formation of ARs of A. ruburm. To address this knowledge gap, we sequenced the transcriptome and sRNA of the A. rubrum variety ‘Autumn Fantasy’ using high-throughput sequencing and explored changes in gene and microRNA (miRNA) expression in response to exogenous auxin treatment. ResultsWe identified 82,468 differentially expressed genes between the treated and untreated ARs, as well as 48 known and 95 novel miRNAs. We also identified 172 target genes of the known miRNAs using degradome sequencing. Two regulatory pathways (ubiquitin mediated proteolysis and plant hormone signal transduction), Ar-miR160a and the target gene ArARF10 were shown to be involved in the auxin response. We further investigated the expression patterns and regulatory roles of ArARF10 through subcellular localization, transcriptional activation, plant transformation, qRT-PCR analysis, and GUS staining. ConclusionsDifferential expression patterns indicated the Ar-miR160a-ArARF10 interaction might play a significant role in the regulation of AR formation in A. rubrum. Our study provided new insights into mechanisms underlying the regulation of AR formation in A. rubrum.

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Abstract 396: Inhibition of Prolyl Hydroxylase Domain-containing Protein Downregulates Vascular Angiotensin II Type 1 Receptor

Hypertension ◽

10.1161/hyp.60.suppl_1.a396 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Toshihiro Ichiki

Keyword(s):

Cellular Response ◽

Target Genes ◽

Interstitial Fibrosis ◽

Critical Role ◽

Hypoxia Inducible Factor ◽

Renin Angiotensin System ◽

Prolyl Hydroxylase ◽

Ang Ii ◽

Prolyl Hydroxylase Domain

Background: Prolyl hydroxylase domain-containing protein (PHD) mediates hydroxylation of hypoxia-inducible factor (HIF)-1α and thereby induces proteasomal degradation of HIF-1α. Inhibition of PHD by hypoxia or hypoxia mimetics such as cobalt chloride (CoCl2) stabilizes HIF-1 and increases the expression of target genes such as vascular endothelial growth factor (VEGF). Although hypoxia activates the systemic renin angiotensin system (RAS), the role of PHD in regulating RAS remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin (Ang) II type 1 receptor (AT1R) and its signaling. Methods and Results: Hypoxia (1% O2), CoCl2 (100-300 μmol/L), and dimethyloxalylglycine (0.25-1.0 mmol/L), all known to inhibit PHD, reduced AT1R expression by 37.7±7.6, 39.6±8.4-69.7±9.9, and 13.4±6.1-25.2±7.0%, respectively (p<0.01) in cultured vascular smooth muscle cell. The same stimuli increased the expression of nuclear HIF-1α and VEGF (p<0.05), suggesting that PHD activity is inhibited. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT1R expression by 55.3±6.0% (p<0.01). CoCl2 decreased AT1R mRNA through transcriptional and posttranscriptional mechanisms (p<0.01 and <0.05, respectively). CoCl2 and PHD2 knockdown diminished Ang II-induced ERK phosphorylation (P<0.01). Over-expression of the constitutively active HIF-1α did not impact the AT1R gene promoter activity. Oral administration of CoCl2 (14 mg/kg/day) to C57BL/6J mice receiving Ang II infusion (490 ng/kg/min) for 4 weeks significantly reduced the expression of AT1R in the aorta by 60.9±11.3% (p<0.05) and attenuated coronary perivascular fibrosis by 85% (p<0.01) without affecting blood pressure. However, CoCl2 did not affect Ang II-induced renal interstitial fibrosis. Conclusion: PHD inhibition downregulates AT1R expression independently of HIF-1α, reduces the cellular response to Ang II, and attenuates profibrotic effect of Ang II on the coronary arteries. PHD inhibition may be beneficial for the treatment of cardiovascular diseases, in which activation of RAS plays a critical role.

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Role of the Hypoxia-Inducible Factor Pathway in Normal and Osteoarthritic Meniscus and in Mice after Destabilization of the Medial Meniscus

Cartilage ◽

10.1177/1947603520958143 ◽

2020 ◽

pp. 194760352095814

Author(s):

Austin V. Stone ◽

Richard F. Loeser ◽

Michael F. Callahan ◽

Margaret A. McNulty ◽

David L. Long ◽

...

Keyword(s):

Gene Expression ◽

Target Genes ◽

Medial Meniscus ◽

Transforming Growth Factor ◽

Hypoxia Inducible Factor ◽

Early Time ◽

Wild Type ◽

Inflammatory Stimuli ◽

Hif Pathway

Objective Meniscus injury and the hypoxia-inducible factor (HIF) pathway are independently linked to osteoarthritis pathogenesis, but the role of the meniscus HIF pathway remains unclear. We sought to identify and evaluate HIF pathway response in normal and osteoarthritic meniscus and to examine the effects of Epas1 (HIF-2α) insufficiency in mice on early osteoarthritis development. Methods Normal and osteoarthritic human meniscus specimens were obtained and used for immunohistochemical evaluation and cell culture studies for the HIF pathway. Meniscus cells were treated with pro-inflammatory stimuli, including interleukins (IL)-1β, IL-6, transforming growth factor (TGF)-α, and fibronectin fragments (FnF). Target genes were also evaluated with HIF-1α and HIF-2α (Epas1) overexpression and knockdown. Wild-type ( n = 36) and Epas1+/− ( n = 30) heterozygous mice underwent destabilization of the medial meniscus (DMM) surgery and were evaluated at 2 and 4 weeks postoperatively for osteoarthritis development using histology. Results HIF-1α and HIF-2α immunostaining and gene expression did not differ between normal and osteoarthritic meniscus. While pro-inflammatory stimulation significantly increased both catabolic and anabolic gene expression in the meniscus, HIF-1α and Epas1 expression levels were not significantly altered. Epas1 overexpression significantly increased Col2a1 expression. Both wild-type and Epas1+/− mice developed osteoarthritis following DMM surgery. There were no significant differences between genotypes at either time point. Conclusion The HIF pathway is likely not responsible for osteoarthritic changes in the human meniscus. Additionally, Epas1 insufficiency does not protect against osteoarthritis development in the mouse at early time points after DMM surgery. The HIF pathway may be more important for protection against catabolic stress.

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Hypoxia and HIF Signaling: One Axis with Divergent Effects

International Journal of Molecular Sciences ◽

10.3390/ijms21165611 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5611 ◽

Cited By ~ 2

Author(s):

Chiara Corrado ◽

Simona Fontana

Keyword(s):

Cellular Response ◽

Target Genes ◽

Hypoxia Inducible Factor ◽

Biological Processes ◽

Oxygen Homeostasis ◽

Tissue Responses ◽

Liver And Kidney ◽

Heterodimeric Complex ◽

Complex Activities

The correct concentration of oxygen in all tissues is a hallmark of cellular wellness, and the negative regulation of oxygen homeostasis is able to affect the cells and tissues of the whole organism. The cellular response to hypoxia is characterized by the activation of multiple genes involved in many biological processes. Among them, hypoxia-inducible factor (HIF) represents the master regulator of the hypoxia response. The active heterodimeric complex HIF α/β, binding to hypoxia-responsive elements (HREs), determines the induction of at least 100 target genes to restore tissue homeostasis. A growing body of evidence demonstrates that hypoxia signaling can act by generating contrasting responses in cells and tissues. Here, this dual and controversial role of hypoxia and the HIF signaling pathway is discussed, with particular reference to the effects induced on the complex activities of the immune system and on mechanisms determining cell and tissue responses after an injury in both acute and chronic human diseases related to the heart, lung, liver, and kidney.

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The chromatin remodeler ISWI regulates the cellular response to hypoxia: role of FIH

Molecular Biology of the Cell ◽

10.1091/mbc.e11-02-0163 ◽

2011 ◽

Vol 22 (21) ◽

pp. 4171-4181 ◽

Cited By ~ 22

Author(s):

Andrew Melvin ◽

Sharon Mudie ◽

Sonia Rocha

Keyword(s):

Rna Polymerase Ii ◽

Chromatin Remodeling ◽

Cellular Response ◽

Target Genes ◽

Hypoxia Inducible Factor ◽

Prolyl Hydroxylases ◽

Additional Information ◽

Protein Levels ◽

Survival Factor

The hypoxia-inducible factor (HIF) is a master regulator of the cellular response to hypoxia. Its levels and activity are controlled by dioxygenases called prolyl-hydroxylases and factor inhibiting HIF (FIH). To activate genes, HIF has to access sequences in DNA that are integrated in chromatin. It is known that the chromatin-remodeling complex switch/sucrose nonfermentable (SWI/SNF) is essential for HIF activity. However, no additional information exists about the role of other chromatin-remodeling enzymes in hypoxia. Here we describe the role of imitation switch (ISWI) in the cellular response to hypoxia. We find that unlike SWI/SNF, ISWI depletion enhances HIF activity without altering its levels. Furthermore, ISWI knockdown only alters a subset of HIF target genes. Mechanistically, we find that ISWI is required for full expression of FIH mRNA and protein levels by changing RNA polymerase II loading to the FIH promoter. Of interest, exogenous FIH can rescue the ISWI-mediated upregulation of CA9 but not BNIP3, suggesting that FIH-independent mechanisms are also involved. Of importance, ISWI depletion alters the cellular response to hypoxia by reducing autophagy and increasing apoptosis. These results demonstrate a novel role for ISWI as a survival factor during the cellular response to hypoxia.

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Key Regulatory Pathways, microRNAs, and Target Genes Participate in Adventitious Root Formation of Acer Rubrum L.

10.21203/rs.3.rs-793059/v1 ◽

2021 ◽

Author(s):

Wenpeng Zhu ◽

Manyu Zhang ◽

Jianyi Li ◽

Hewen Zhao ◽

Kezhong Zhang ◽

...

Keyword(s):

Transcriptional Activation ◽

High Throughput Sequencing ◽

Target Genes ◽

Seedling Survival ◽

Expression Patterns ◽

Economic Value ◽

Acer Rubrum ◽

Pcr Analysis ◽

Asexual Propagation ◽

Regulatory Pathways

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

Blood ◽

10.1182/blood.v124.21.2896.2896 ◽

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.

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Expression patterns of vHNF1 and HNF1 homeoproteins in early postimplantation embryos suggest distinct and sequential developmental roles

Development ◽

10.1242/dev.116.3.783 ◽

1992 ◽

Vol 116 (3) ◽

pp. 783-797 ◽

Cited By ~ 8

Author(s):

S. Cereghini ◽

M.O. Ott ◽

S. Power ◽

M. Maury

Keyword(s):

Transcriptional Activation ◽

Target Genes ◽

Fetal Liver ◽

Expression Patterns ◽

Yolk Sac ◽

Control Element ◽

Visceral Endoderm ◽

Mouse Development ◽

F9 Cells

The homeoproteins HNF1 (LFB1/HNF1-A) and vHNF1 (LFB3/HNF1 beta) interact with an essential control element of a group of liver-specific genes. During development, these putative target genes are initially expressed in the visceral endoderm of the yolk sac and subsequently in fetal liver. To assess the possible involvement of HNF1 and/or vHNF1 as transcriptional regulators in the early steps of visceral endoderm differentiation, we have analyzed the expression pattern of both factors both in vitro during differentiation of murine F9 embryonal carcinoma cells and in vivo during early postimplantation mouse development. We show here that differentiation of F9 cells into either visceral or parietal endoderm is accompanied by a sharp induction in vHNF1 mRNA and protein. By contrast, only low levels of aberrantly sized HNF1 transcripts, but not DNA-binding protein, are found in F9 cells and its differentiated derivatives. At 6–7.5 days of gestation, high levels of vHNF1 mRNA are present in the visceral extraembryonic endoderm, which co-localize with transcripts of the transthyretin gene. HNF1 transcripts are first detected in the yolk sac roughly two embryonic days later, after the developmental onset of transcription of target genes. As development proceeds, discrepancies are observed between the level of transcripts of both vHNF1 and HNF1 and their respective nuclear binding proteins, notably in the yolk sac and embryonic kidney. In addition, we show that two alternative spliced isoforms of vHNF1 mRNA, vHNF-A and vHNF1-B, are expressed in both embryonic and adult tissues. Taken together, these data suggest that vHNF1 participates as a regulatory factor in the initial transcriptional activation of the target genes in the visceral endoderm of the yolk sac, whereas the later appearance of HNF1 could be required for maintenance of their expression. Our results also provide evidence of a posttranscriptional level of control of vHNF1 and HNF1 gene expression during development, in addition to the spatial restriction in transcription.

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