Induction of erythropoiesis by hypoxia-inducible factor prolyl hydroxylase inhibitors without promotion of tumor initiation, progression, or metastasis in a VEGF-sensitive model of spontaneous breast cancer

PubMed Central

Seeley, Todd W; Sternlicht, Mark D; Klaus, Stephen J; Neff, Thomas B; Liu, David Y

2017-01-01

The effects of pharmacological hypoxia-inducible factor (HIF) stabilization were investigated in the MMTV-Neundl-YD5 (NeuYD) mouse model of breast cancer. This study first confirmed the sensitivity of this model to increased vascular endothelial growth factor (VEGF), using bigenic NeuYD;MMTV-VEGF-25 mice. Tumor initiation was dramatically accelerated in bigenic animals. Bigenic tumors were also more aggressive, with shortened doubling times and increased lung metastasis as compared to NeuYD controls. In separate studies, NeuYD mice were treated three times weekly from 7 weeks of age until study end with two different HIF prolyl hydroxylase inhibitors (HIF-PHIs), FG-4497 or roxadustat (FG-4592). In NeuYD mice, HIF-PHI treatments elevated erythropoiesis markers, but no differences were detected in tumor onset or the phenotypes of established tumors. PMID:28331872

Development of Inhibitors of the PAS-B Domain of the HIF-2α Transcription Factor

PubMed Central

Rogers, Jamie L.; Bayeh, Liela; Scheuermann, Thomas H.; Longgood, Jamie; Key, Jason; Naidoo, Jacinth; Melito, Lisa; Shokri, Cameron; Frantz, Doug E.; Bruick, Richard K.; Gardner, Kevin H.; MacMillan, John B.; Tambar, Uttam K.

2013-01-01

Hypoxia Inducible Factors (HIFs) are heterodimeric transcription factors induced in a variety of pathophysiological settings, including cancer. We describe the first detailed structure-activity-relationship study of small molecules designed to inhibit HIF-2α–ARNT heterodimerization by binding an internal cavity of the HIF-2α PAS-B domain. Through a series of biophysical characterizations of inhibitor/protein interactions (NMR and X-ray crystallography), we have established the structural requirements for artificial inhibitors of the HIF-2α–ARNT PAS-B interaction. These results may serve as a foundation for discovering therapeutic agents that function by a novel mode of action. PMID:23363003

Adenovirus-mediated HIF-1α gene transfer promotes repair of mouse airway allograft microvasculature and attenuates chronic rejection

PubMed Central

Jiang, Xinguo; Khan, Mohammad A.; Tian, Wen; Beilke, Joshua; Natarajan, Ramesh; Kosek, Jon; Yoder, Mervin C.; Semenza, Gregg L.; Nicolls, Mark R.

2011-01-01

Chronic rejection, manifested as small airway fibrosis (obliterative bronchiolitis [OB]), is the main obstacle to long-term survival in lung transplantation. Recent studies demonstrate that the airways involved in a lung transplant are relatively hypoxic at baseline and that OB pathogenesis may be linked to ischemia induced by a transient loss of airway microvasculature. Here, we show that HIF-1α mediates airway microvascular repair in a model of orthotopic tracheal transplantation. Grafts with a conditional knockout of Hif1a demonstrated diminished recruitment of recipient-derived Tie2+ angiogenic cells to the allograft, impaired repair of damaged microvasculature, accelerated loss of microvascular perfusion, and hastened denudation of epithelial cells. In contrast, graft HIF-1α overexpression induced via an adenoviral vector prolonged airway microvascular perfusion, preserved epithelial integrity, extended the time window for the graft to be rescued from chronic rejection, and attenuated airway fibrotic remodeling. HIF-1α overexpression induced the expression of proangiogenic factors such as Sdf1, Plgf, and Vegf, and promoted the recruitment of vasoreparative Tie2+ cells. This study demonstrates that a therapy that enhances vascular integrity during acute rejection may promote graft health and prevent chronic rejection. PMID:21606594

Ligand-induced perturbation of the HIF-2α:ARNT dimer dynamics

PubMed Central

Motta, Stefano

2018-01-01

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix−loop−helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses. PMID:29489822

Artificial ligand binding within the HIF2[alpha] PAS-B domain of the HIF2 transcription factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Scheuermann, Thomas H.; Tomchick, Diana R.; Machius, Mischa

2009-05-12

The hypoxia-inducible factor (HIF) basic helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim (bHLH-PAS) transcription factors are master regulators of the conserved molecular mechanism by which metazoans sense and respond to reductions in local oxygen concentrations. In humans, HIF is critically important for the sustained growth and metastasis of solid tumors. Here, we describe crystal structures of the heterodimer formed by the C-terminal PAS domains from the HIF2{alpha} and ARNT subunits of the HIF2 transcription factor, both in the absence and presence of an artificial ligand. Unexpectedly, the HIF2{alpha} PAS-B domain contains a large internal cavity that accommodates ligands identified frommore » a small-molecule screen. Binding one of these ligands to HIF2{alpha} PAS-B modulates the affinity of the HIF2{alpha}:ARNT PAS-B heterodimer in vitro. Given the essential role of PAS domains in forming active HIF heterodimers, these results suggest a presently uncharacterized ligand-mediated mechanism for regulating HIF2 activity in endogenous and clinical settings.« less

Molecular and functional evaluation of a novel HIF inhibitor, benzopyranyl 1,2,3-triazole compound

PubMed Central

Park, Kyunghye; Lee, Hye Eun; Lee, Sun Hee; Lee, Doohyun; Lee, Taeho; Lee, You Mie

2017-01-01

Hypoxia occurs in a variety of pathological events, including the formation of solid tumors. Hypoxia-inducible factor (HIF)-1α is stabilized under hypoxic conditions and is a key molecule in tumor growth and angiogenesis. Seeking to develop novel cancer therapeutics, we investigated small molecules from our in-house chemical libraries to target HIF-1α. We employed a dual-luciferase assay that uses a luciferase (Luc) reporter vector harboring five copies of hypoxia-responsive element (HRE) in the promoter. Under hypoxic conditions that increased Luc reporter activity by four-fold, we screened 144 different compounds, nine of which showed 30–50% inhibition of hypoxia-induced Luc reporter activity. Among these, “Compound 12, a benzopyranyl 1,2,3-triazole” was the most efficient at inhibiting the expression of HIF-1α under hypoxic conditions, reducing its expression by 80%. Under hypoxic conditions, the half maximal IC50 of the compound was 24 nM in HEK-293 human embryonic kidney cells, and 2 nM in A549 human lung carcinoma cells. Under hypoxic conditions, Compound 12 increased hydroxylated HIF-1α levels and HIF-1α ubiquitination, and also dose-dependently decreased HIF-1α target gene expression as well as vascular endothelial growth factor (VEGF) secretion. Furthermore, this compound inhibited VEGF-induced in vitro angiogenesis in human umbilical vein endothelial cells (HUVECs), and in vivo, it inhibited chick chorioallantoic membrane angiogenesis. In allogaft assays, cotreatment with Compound 12 and gefitinib significantly inhibited tumor growth and angiogenesis. Compound 12 can be a novel inhibitor of HIF-1α by accelerating its degradation, and shows much potential as an anti-cancer agent through its ability to suppress tumor growth and angiogenesis. PMID:27999195

Transdermal deferoxamine prevents pressure-induced diabetic ulcers

PubMed Central

Duscher, Dominik; Neofytou, Evgenios; Wong, Victor W.; Maan, Zeshaan N.; Rennert, Robert C.; Januszyk, Michael; Rodrigues, Melanie; Malkovskiy, Andrey V.; Whitmore, Arnetha J.; Galvez, Michael G.; Whittam, Alexander J.; Brownlee, Michael; Rajadas, Jayakumar; Gurtner, Geoffrey C.

2015-01-01

There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation. PMID:25535360

Differential roles of Sirt1 in HIF-1α and HIF-2α mediated hypoxic responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Haejin; Shin, Seung-Hyun; Shin, Dong Hoon

2014-01-31

Highlights: • Roles of SIRT1 in HIF-1α and HIF-2α regulations are reevaluated using specific antibodies and Gal4 reporters. • SIRT1 represses the HIF-1α-driven transcription constantly in ten cancer cell-lines. • SIRT1 regulates the HIF-2α-driven transcription cell context-dependently. • SIRT1 determines cell growth under hypoxia by regulating HIF-1α and HIF-2α activities. - Abstract: Hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) determine cancer cell fate under hypoxia. Despite the similarities of their structures, HIF-1α and HIF-2α have distinct roles in cancer growth under hypoxia, that is, HIF-1α induces growth arrest whereas HIF-2α promotes cell growth. Recently, sirtuin 1 (Sirt1) was reportedmore » to fine-tune cellular responses to hypoxia by deacetylating HIF-1α and HIF-2α. Yet, the roles of Sirt1 in HIF-1α and HIF-2α functions have been controversial. We here investigated the precise roles of Sirt1 in HIF-1α and HIF-2α regulations. Immunological analyses revealed that HIF-1α K674 and HIF-2α K741 are acetylated by PCAF and CBP, respectively, but are deacetylated commonly by Sirt1. In the Gal4 reporter systems, Sirt1 was found to repress HIF-1α activity constantly in ten cancer cell-lines but to regulate HIF-2α activity cell type-dependently. Moreover, Sirt1 determined cell growth under hypoxia depending on HIF-1α and HIF-2α. Under hypoxia, Sirt1 promoted cell proliferation of HepG2, in which Sirt1 differentially regulates HIF-1α and HIF-2α. In contrast, such an effect of Sirt1 was not shown in HCT116, in which Sirt1 inactivates both HIF-1α and HIF-2α because conflicting actions of HIF-1α and HIF-2α on cell growth may be offset. Our results provide a better understanding of the roles of Sirt1 in HIF-mediated hypoxic responses and also a basic concept for developing anticancer strategy targeting Sirt1.« less

Age-dependent Impairment of HIF-1α̣Expression in Diabetic Mice: Correction with Electroporation-facilitated Gene Therapy Increases Wound Healing, Angiogenesis, and Circulating Angiogenic Cells

PubMed Central

Liu, Lixin; Marti, Guy P.; Wei, Xiaofei; Zhang, Xianjie; Zhang, Huafeng; Liu, Ye V.; Nastai, Manuel; Semenza, Gregg L.; Harmon, John W.

2009-01-01

Wound healing is impaired in elderly patients with diabetes mellitus. We hypothesized that age-dependent impairment of cutaneous wound healing in db/db diabetic mice: (a) would correlate with reduced expression of the transcription factor hypoxia-inducible factor 1α (HIF-1α) as well as its downstream target genes; and (b) could be overcome by HIF-1α replacement therapy. Wound closure, angiogenesis, and mRNA expression in excisional skin wounds were analyzed and circulating angiogenic cells were quantified in db/db mice that were untreated or received electroporation-facilitated HIF-1α gene therapy. HIF-1α mRNA levels in wound tissue were significantly reduced in older (4–6 months) as compared to younger (1.5–2 months) db/db mice. Expression of mRNAs encoding the angiogenic cytokines vascular endothelial growth factor (VEGF), angiopoietin 1 (ANGPT1), ANGPT2, platelet derived growth factor B (PDGF-B), and placental growth factor (PLGF) was also impaired in wounds of older db/db mice. Intradermal injection of plasmid gWIZ-CA5, which encodes a constitutively active form of HIF-1α, followed by electroporation, induced increased levels of HIF-1α mRNA at the injection site on day 3 and increased levels of VEGF, PLGF, PDGF-B, and ANGPT2 mRNA on day 7. Circulating angiogenic cells in peripheral blood increased 10-fold in mice treated with gWIZ-CA5. Wound closure was significantly accelerated in db/db mice treated with gWIZ-CA5 as compared to mice treated with empty vector. Thus, HIF-1α gene therapy corrects the age-dependent impairment of HIF-1α expression, angiogenic cytokine expression, and circulating angiogenic cells that contribute to the age-dependent impairment of wound healing in db/db mice. PMID:18506785

The Acetylase/Deacetylase Couple CREB-binding Protein/Sirtuin 1 Controls Hypoxia-inducible Factor 2 Signaling*

PubMed Central

Chen, Rui; Xu, Min; Hogg, Richard T.; Li, Jiwen; Little, Bertis; Gerard, Robert D.; Garcia, Joseph A.

2012-01-01

Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors. HIF-1α plays a prominent role in hypoxic gene induction. HIF-2α target genes are more restricted but include erythropoietin (Epo), one of the most highly hypoxia-inducible genes in mammals. We previously reported that HIF-2α is acetylated during hypoxia but is rapidly deacetylated by the stress-responsive deacetylase Sirtuin 1. We now demonstrate that the lysine acetyltransferases cAMP-response element-binding protein-binding protein (CBP) and p300 are required for efficient Epo induction during hypoxia. However, despite close structural similarity, the roles of CBP and p300 differ in HIF signaling. CBP acetylates HIF-2α, is a major coactivator for HIF-2-mediated Epo induction, and is required for Sirt1 augmentation of HIF-2 signaling during hypoxia in Hep3B cells. In comparison, p300 is a major contributor for HIF-1 signaling as indicated by induction of Pgk1. Whereas CBP can bind with HIF-2α independent of the HIF-2α C-terminal activation domain via enzyme/substrate interactions, p300 only complexes with HIF-2α through the C-terminal activation domain. Maximal CBP/HIF-2 signaling requires intact CBP acetyltransferase activity in both Hep3B cells as well as in mice. PMID:22807441

HIF-1 maintains a functional relationship between pancreatic cancer cells and stromal fibroblasts by upregulating expression and secretion of Sonic hedgehog

PubMed Central

Katagiri, Tomohiro; Kobayashi, Minoru; Yoshimura, Michio; Morinibu, Akiyo; Itasaka, Satoshi; Hiraoka, Masahiro; Harada, Hiroshi

2018-01-01

Hypoxic and stroma-rich microenvironments, characteristic features of pancreatic cancers, are strongly associated with a poor prognosis. However, whether and how hypoxia increases stromal compartments remain largely unknown. Here, we investigated the potential importance of a master regulator of the cellular adaptive response to hypoxia, hypoxia-inducible factor-1 (HIF-1), in the formation of stroma-rich microenvironments of pancreatic tumors. We found that pancreatic cancer cells secreted more Sonic hedgehog protein (SHH) under hypoxia by upregulating its expression and efficiency of secretion in a HIF-1-dependent manner. Recombinant SHH, which was confirmed to activate the hedgehog signaling pathway, accelerated the growth of fibroblasts in a dose-dependent manner. The SHH protein secreted from pancreatic cancer cells under hypoxic conditions promoted the growth of fibroblasts by stimulating their Sonic hedgehog signaling pathway. These results suggest that the increased secretion of SHH by HIF-1 is potentially responsible for the formation of detrimental and stroma-rich microenvironments in pancreatic cancers, therefore providing a rational basis to target it in cancer therapy. PMID:29535824

Hypoxia Induced Factor in Chronic Kidney Disease: Friend or Foe?

PubMed

Li, Weiying; Zhao, Yuliang; Fu, Ping

2017-01-01

Many studies have shown evidence that erythropoiesis-stimulating agents (ESAs), as a classic treatment for chronic kidney disease (CKD)-related anemia, have several disadvantages and may trigger various adverse events with long-term use. The hypoxia-induced factor (HIF) pathway has been intensively investigated in kidney disease, especially in CKD, as research has shown that HIF-mediated erythropoiesis might work as a potential therapeutic strategy for managing CKD-related anemia. Development of prolyl hydroxylase domain inhibitors (PHIs), as an effective HIF activator, is a valuable step toward finding a replacement for ESAs, which showed an effective erythropoiesis through a comprehensive and physiological approach by promoting erythropoietin production, increasing iron bioavailability and improving chronic inflammatory status. Heretofore no adverse events or obvious off-target effects have been reported in clinical trials of PHIs. Nevertheless, a cautious inspection with extended follow-up period is warranted to validate the safety of prolonged HIF elevation, especially considering its ambiguous role in fibrogenesis and inflammation responses and possible risks in accelerating vascular calcification and tumorigenesis. A weighed dosing strategy might be the key to circumvent the unexpected side-effect brought by pleotropic effects of HIF elevation and achieve a selective augmentation of HIF-mediated signaling pathway. New studies with longer follow-up period and adequate analysis about the risks for proinflammation, vascular calcification and tumorigenesis are needed to ensure the drugs are safe for long-term use before being widely accepted in daily clinical practice.

Targeting Renal Cell Carcinoma with a HIF-2 antagonist

PubMed Central

Chen, Wenfang; Hill, Haley; Christie, Alana; Kim, Min Soo; Holloman, Eboni; Pavia-Jimenez, Andrea; Homayoun, Farrah; Ma, Yuanqing; Patel, Nirav; Yell, Paul; Hao, Guiyang; Yousuf, Qurratulain; Joyce, Allison; Pedrosa, Ivan; Geiger, Heather; Zhang, He; Chang, Jenny; Gardner, Kevin H.; Bruick, Richard K.; Reeves, Catherine; Hwang, Tae Hyun; Courtney, Kevin; Frenkel, Eugene; Sun, Xiankai; Zojwalla, Naseem; Wong, Tai; Rizzi, James P.; Wallace, Eli M.; Josey, John A.; Xie, Yang; Xie, Xian-Jin; Kapur, Payal; McKay, Renée M.; Brugarolas, James

2016-01-01

Clear cell Renal Cell Carcinoma (ccRCC) is characterized by VHL inactivation1,2. Because no other gene is mutated as frequently, and VHL mutations are truncal3, VHL inactivation is regarded as the governing event4. VHL loss activates HIF-2, and constitutive HIF-2 restores tumorigenesis in VHL-reconstituted ccRCC cells5. HIF-2 is implicated in angiogenesis and multiple other processes6–9, but angiogenesis is the main target of drugs like sunitinib10. HIF-2, a transcription factor, has been regarded as undruggable11. A structure-based design approach identified a selective HIF-2 antagonist (PT2399) that we evaluate using a tumorgraft (TG)/PDX platform12,13. PT2399 dissociated HIF-2 (an obligatory heterodimer [HIF-2α/HIF-1β])14 in human ccRCC suppressing tumorigenesis in 56% (10/18) lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumors, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant. Resistance occurred despite HIF-2 dissociation in tumors and evidence of Hif-2 inhibition in the mouse as determined by suppression of circulating erythropoietin, a HIF-2 target15 and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumors. Illustrating drug specificity, gene expression was largely unaffected by PT2399 in resistant tumors. Sensitive tumors exhibited a distinguishing gene expression signature, and generally higher HIF-2α levels. Prolonged PT2399 treatment led to resistance. We identified a binding site and second site suppressor mutation in HIF-2α and HIF-1β respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient with a sensitive TG had disease control for >11 months with the close analogue PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCC are, unexpectedly, HIF-2 independent, and set the stage for biomarker-driven clinical trials. PMID:27595394

Hyaluronan Production Regulates Metabolic and Cancer Stem-like Properties of Breast Cancer Cells via Hexosamine Biosynthetic Pathway-coupled HIF-1 Signaling*

PubMed Central

Chanmee, Theerawut; Ontong, Pawared; Izumikawa, Tomomi; Higashide, Miho; Mochizuki, Nobutoshi; Chokchaitaweesuk, Chatchadawalai; Khansai, Manatsanan; Nakajima, Kazuki; Kakizaki, Ikuko; Kongtawelert, Prachya; Taniguchi, Naoyuki; Itano, Naoki

2016-01-01

Cancer stem cells (CSCs) represent a small subpopulation of self-renewing oncogenic cells. As in many other stem cells, metabolic reprogramming has been implicated to be a key characteristic of CSCs. However, little is known about how the metabolic features of cancer cells are controlled to orchestrate their CSC-like properties. We recently demonstrated that hyaluronan (HA) overproduction allowed plastic cancer cells to revert to stem cell states. Here, we adopted stable isotope-assisted tracing and mass spectrometry profiling to elucidate the metabolic features of HA-overproducing breast cancer cells. These integrated approaches disclosed an acceleration of metabolic flux in the hexosamine biosynthetic pathway (HBP). A metabolic shift toward glycolysis was also evident by quantitative targeted metabolomics, which was validated by the expression profiles of key glycolytic enzymes. Forced expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), an HBP rate-limiting enzyme, resembled the results of HA overproduction with regard to HIF-1α accumulation and glycolytic program, whereas GFAT1 inhibition significantly decreased HIF-1α protein level in HA-overproducing cancer cells. Moreover, inhibition of the HBP-HIF-1 axis abrogated HA-driven glycolytic enhancement and reduced the CSC-like subpopulation. Taken together, our results provide compelling evidence that HA production regulates the metabolic and CSC-like properties of breast cancer cells via HBP-coupled HIF-1 signaling. PMID:27758869

Targeting renal cell carcinoma with a HIF-2 antagonist.

PubMed

Chen, Wenfang; Hill, Haley; Christie, Alana; Kim, Min Soo; Holloman, Eboni; Pavia-Jimenez, Andrea; Homayoun, Farrah; Ma, Yuanqing; Patel, Nirav; Yell, Paul; Hao, Guiyang; Yousuf, Qurratulain; Joyce, Allison; Pedrosa, Ivan; Geiger, Heather; Zhang, He; Chang, Jenny; Gardner, Kevin H; Bruick, Richard K; Reeves, Catherine; Hwang, Tae Hyun; Courtney, Kevin; Frenkel, Eugene; Sun, Xiankai; Zojwalla, Naseem; Wong, Tai; Rizzi, James P; Wallace, Eli M; Josey, John A; Xie, Yang; Xie, Xian-Jin; Kapur, Payal; McKay, Renée M; Brugarolas, James

2016-11-03

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1β) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1β, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

Coiled-coil coactivators play a structural role mediating interactions in hypoxia-inducible factor heterodimerization

DOE PAGES

Guo, Yirui; Scheuermann, Thomas H.; Partch, Carrie L.; ...

2015-01-27

The hypoxia-inducible factor complex (HIF-α·aryl hydrocarbon receptor nuclear translocator (ARNT)) requires association with several transcription coactivators for a successful cellular response to hypoxic stress. In addition to the conventional global transcription coactivator CREB-binding protein/p300 (CBP/p300) that binds to the HIF-α transactivation domain, a new group of transcription coactivators called the coiled-coil coactivators (CCCs) interact directly with the second PER-ARNT-SIM (PAS) domain of ARNT (ARNT PAS-B). These less studied transcription coactivators play essential roles in the HIF-dependent hypoxia response, and CCC misregulation is associated with several forms of cancer. To better understand CCC protein recruitment by the heterodimeric HIF transcription factor,more » we used x-ray crystallography, NMR spectroscopy, and biochemical methods to investigate the structure of the ARNT PAS-B domain in complex with the C-terminal fragment of a coiled-coil coactivator protein, transforming acidic coiled-coil coactivator 3 (TACC3). We found that the HIF-2α PAS-B domain also directly interacts with TACC3, motivating an NMR data-derived model suggesting a means by which TACC3 could form a ternary complex with HIF-2α PAS-B and ARNT PAS-B via β-sheet/coiled-coil interactions. Furthermore, these findings suggest that TACC3 could be recruited as a bridge to cooperatively mediate between the HIF-2α PAS-B·ARNT PAS-B complex, thereby participating more directly in HIF-dependent gene transcription than previously anticipated.« less

Partial rescue of defects in Cited2-deficient embryos by HIF-1alpha heterozygosity.

PubMed

Xu, Bing; Doughman, Yongqiu; Turakhia, Mona; Jiang, Weihong; Landsettle, Chad E; Agani, Faton H; Semenza, Gregg L; Watanabe, Michiko; Yang, Yu-Chung

2007-01-01

Hypoxia inducible factor-1 (HIF-1) initiates key cellular and tissue responses to physiological and pathological hypoxia. Evidence from in vitro and structural analyses supports a critical role for Cited2 in down-regulating HIF-1-mediated transcription by competing for binding with oxygen-sensitive HIF-1alpha to transcriptional co-activators CBP/p300. We previously detected elevated expression of HIF-1 target genes in Cited2(-/-) embryonic hearts, indicating that Cited2 inhibits HIF-1 transactivation in vivo. In this study, we show for the first time that highly hypoxic cardiac regions in mouse embryos corresponded to the sites of defects in Cited2(-/-) embryos and that defects of the outflow tract, interventricular septum, cardiac vasculature, and hyposplenia were largely rescued by HIF-1alpha haploinsufficiency. The hypoxia of the outflow tract and interventricular septum peaked at E13.5 and dissipated by E15.5 in wild-type hearts, but persisted in E15.5 Cited2(-/-) hearts. The persistent hypoxia and abnormal vasculature in the myocardium of interventricular septum in E15.5 Cited2(-/-) hearts were rescued with decreased HIF-1alpha gene dosage. Accordingly, mRNA levels of HIF-1-responsive genes were reduced in Cited2(-/-) embryonic hearts by HIF-1alpha heterozygosity. These findings suggest that a precise level of HIF-1 transcriptional activity critical for normal development is triggered by differential hypoxia and regulated through feedback inhibition by Cited2.

Molecular characterization and mRNA expression of two key enzymes of hypoxia-sensing pathways in eastern oysters Crassostrea virginica (Gmelin): Hypoxia-inducible factor α (HIF-α) and HIF-prolyl hydroxylase (PHD)

PubMed Central

Piontkivska, Helen; Chung, J. Sook; Ivanina, Anna V.; Sokolov, Eugene P.; Techa, Sirinart; Sokolova, Inna M.

2010-01-01

Oxygen homeostasis is crucial for development, survival and normal function of all metazoans. A family of transcription factors called hypoxia-inducible factors (HIF) is critical in mediating the adaptive responses to reduced oxygen availability. The HIF transcription factor consists of a constitutively expressed β subunit and an oxygen-dependent α subunit; the abundance of the latter determines the activity of HIF and is regulated by a family of O2- and Fe2+-dependent enzymes prolyl hydroxylases (PHDs). Currently very little is known about the function of this important pathway and the molecular structure of its key players in hypoxia-tolerant intertidal mollusks including oysters, which are among the animal champions of anoxic and hypoxic tolerance and thus can serve as excellent models to study the role of HIF cascade in adaptations to oxygen deficiency. We have isolated transcripts of two key components of the oxygen sensing pathway - the oxygen-regulated HIF-α subunit and PHD - from an intertidal mollusk, the eastern oyster Crassostrea virginica, and determined the transcriptional responses of these two genes to anoxia, hypoxia and cadmium (Cd) stress. HIF-α and PHD homologs from eastern oysters C. virginica show significant sequence similarity and share key functional domains with the earlier described isoforms from vertebrates and invertebrates. Phylogenetic analysis shows that genetic diversification of HIF and PHD isoforms occurred within the vertebrate lineage indicating functional diversification and specialization of the oxygen-sensing pathways in this group, which parallels situation observed for many other important genes. HIF-α and PHD homologs are broadly expressed at the mRNA level in different oyster tissues and show transcriptional responses to prolonged hypoxia in the gills consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. Similarity in amino acid sequence, domain structure and transcriptional responses between HIF-α and PHD homologs from oysters and other invertebrate and vertebrate species implies the highly conserved functions of these genes throughout the evolutionary history of animals, in accordance with their critical role in oxygen sensing and homeostasis. PMID:21106446

Structure Elucidation of Verucopeptin, a HIF-1 Inhibitory Polyketide-Hexapeptide Hybrid Metabolite from an Actinomycete.

PubMed

Yoshimura, Aya; Nishimura, Shinichi; Otsuka, Saori; Hattori, Akira; Kakeya, Hideaki

2015-11-06

The transcriptional factor, hypoxia inducible factor-1 (HIF-1), is a promising target for cancer chemotherapy. From an actinomycete, verucopeptin (1) was identified as a HIF-1 signaling inhibitor. By a combination of chemical degradation and spectroscopic analyses, the absolute stereochemistry of metabolite 1 was determined to be 10R, 15S, 16S, 23S, 27S, 28R, 31S, 33S, 35R. Moreover, metabolite 1 was revealed to attenuate the HIF-1α and mTORC1 pathway, indicating that verucopeptin (1) would be a potent lead compound for anticancer chemotherapy.

Exosomes secreted from mutant-HIF-1α-modified bone-marrow-derived mesenchymal stem cells attenuate early steroid-induced avascular necrosis of femoral head in rabbit.

PubMed

Li, Haile; Liu, Danping; Li, Chen; Zhou, Shanjian; Tian, Dachuan; Xiao, Dawei; Zhang, Huan; Gao, Feng; Huang, Jianhua

2017-12-01

Mesenchymal stem cells (MSCs)-derived exosomes exhibit protective effects on damaged or diseased tissues. Hypoxia-inducible factor 1α (HIF-1α) plays a critical role in bone development. However, HIF-1α is easily biodegradable under normoxic conditions. The bone-marrow-derived mesenchymal stem cells (BMSCs) were transfected with adenovirus carrying triple point-mutations (amino acids 402, 564, and 803) in the HIF-1α coding sequence (CDS). The mutant HIF-1α can efficiently express functional proteins under normoxic conditions. To date, no study has reported the role of exosomes secreted by mutant HIF-1α modified BMSCs in the recovery of the early steroid-induced avascular necrosis of femoral head (SANFH). In this study, we firstly analyzed exosomes derived from BMSCs modified by mutant (BMSC-Exos MU ) or wild-type HIF-1α (BMSC-Exos WT ). In vitro, we investigated the osteogenic differentiation capacity of BMSCs modified by BMSC-Exos MU or BMSC-Exos WT , and the angiogenesis effects of BMSC-Exos MU and BMSC-Exos WT on human umbilical vein endothelial cells (HUVECs). Besides, the healing of the femoral head was also assessed in vivo. We found that the potential of osteogenic differentiation of BMSCs treated with BMSC-Exos MU was higher than the wild-type group in vitro. In addition, BMSC-Exos MU stimulated the proliferation, migration, and tube formation of HUVECs in a dose-dependent manner. Compared with the BMSC-Exos WT or PBS control group, the injection of BMSC-Exos MU into the necrosis region markedly accelerated the bone regeneration and angiogenesis, which were indicated by the increased trabecular reconstruction and microvascular density. Taken together, our data suggest that BMSC-Exos MU facilitates the repair of SANFH by enhancing osteogenesis and angiogenesis. © 2017 International Federation for Cell Biology.

Identification of small molecule compounds that inhibit the HIF-1 signaling pathway

PubMed Central

2009-01-01

Background Hypoxia-inducible factor-1 (HIF-1) is the major hypoxia-regulated transcription factor that regulates cellular responses to low oxygen environments. HIF-1 is composed of two subunits: hypoxia-inducible HIF-1α and constitutively-expressed HIF-1β. During hypoxic conditions, HIF-1α heterodimerizes with HIF-1β and translocates to the nucleus where the HIF-1 complex binds to the hypoxia-response element (HRE) and activates expression of target genes implicated in cell growth and survival. HIF-1α protein expression is elevated in many solid tumors, including those of the cervix and brain, where cells that are the greatest distance from blood vessels, and therefore the most hypoxic, express the highest levels of HIF-1α. Therapeutic blockade of the HIF-1 signaling pathway in cancer cells therefore provides an attractive strategy for development of anticancer drugs. To identify small molecule inhibitors of the HIF-1 pathway, we have developed a cell-based reporter gene assay and screened a large compound library by using a quantitative high-throughput screening (qHTS) approach. Results The assay is based upon a β-lactamase reporter under the control of a HRE. We have screened approximate 73,000 compounds by qHTS, with each compound tested over a range of seven to fifteen concentrations. After qHTS we have rapidly identified three novel structural series of HIF-1 pathway Inhibitors. Selected compounds in these series were also confirmed as inhibitors in a HRE β-lactamase reporter gene assay induced by low oxygen and in a VEGF secretion assay. Three of the four selected compounds tested showed significant inhibition of hypoxia-induced HIF-1α accumulation by western blot analysis. Conclusion The use of β-lactamase reporter gene assays, in combination with qHTS, enabled the rapid identification and prioritization of inhibitors specific to the hypoxia induced signaling pathway. PMID:20003191

Improving access to medicines via the Health Impact Fund in India: a stakeholder analysis.

PubMed

McMullan, Patrick; Ajay, Vamadevan S; Srinivas, Ravi; Bhalla, Sandeep; Prabhakaran, Dorairaj; Banerjee, Amitava

2018-01-01

In India, 50-65% of the population face difficulties in accessing medicines. The Health Impact Fund (HIF) is a novel proposal whereby pharmaceutical companies would be paid based on the measured global health impact of their drugs. We conducted a key stakeholder analysis to explore access to medicines in India, acceptability of the HIF and potential barriers and facilitators at policy level. To conduct a stakeholder analysis of the HIF in India: to determine key stakeholder views regarding access to medicines in India; to evaluate acceptability of the HIF; and to assess potential barriers and facilitators to the HIF as a policy. In New Delhi, we conducted semi-structured interviews. There was purposive recruitment of participants with snowball sampling. Transcribed data were analysed using stakeholder analysis frameworks and directed content analysis. Participation rate was 29% (14/49). 14 semi-structured interviews were conducted among stakeholders in New Delhi. All participants highlighted access to medicines as a problem in India. There were mixed views about the HIF in terms of relevance and scaleability. Stakeholders felt it should focus on diseases with limited or no market and potentially incorporate direct investment in research. First, access to medicines is perceived to be a major problem in India by all stakeholders, but affordability is just one factor. Second, stakeholders despite considerable support for the idea of the HIF, there are major concerns about scaleability, generalisability and impact on access to medicines. Third, the HIF and other novel drug-related health policies can afford to be more radical, e.g. working outside the existing intellectual property rights regime, targeting generic as well as branded drugs, or extending to research and development. Further innovations in access to medicines must involve country-specific key stakeholders in order to increase the likelihood of their success.

Hypoxia-inducible factor 1α activates insulin-induced gene 2 (Insig-2) transcription for degradation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase in the liver.

PubMed

Hwang, Seonghwan; Nguyen, Andrew D; Jo, Youngah; Engelking, Luke J; Brugarolas, James; DeBose-Boyd, Russell A

2017-06-02

Cholesterol synthesis is a highly oxygen-consuming process. As such, oxygen deprivation (hypoxia) limits cholesterol synthesis through incompletely understood mechanisms mediated by the oxygen-sensitive transcription factor hypoxia-inducible factor 1α (HIF-1α). We show here that HIF-1α links pathways for oxygen sensing and feedback control of cholesterol synthesis in human fibroblasts by directly activating transcription of the INSIG-2 gene. Insig-2 is one of two endoplasmic reticulum membrane proteins that inhibit cholesterol synthesis by mediating sterol-induced ubiquitination and subsequent endoplasmic reticulum-associated degradation of the rate-limiting enzyme in the pathway, HMG-CoA reductase (HMGCR). Consistent with the results in cultured cells, hepatic levels of Insig-2 mRNA were enhanced in mouse models of hypoxia. Moreover, pharmacologic stabilization of HIF-1α in the liver stimulated HMGCR degradation via a reaction that requires the protein's prior ubiquitination and the presence of the Insig-2 protein. In summary, our results show that HIF-1α activates INSIG-2 transcription, leading to accumulation of Insig-2 protein, which binds to HMGCR and triggers its accelerated ubiquitination and degradation. These results indicate that HIF-mediated induction of Insig-2 and degradation of HMGCR are physiologically relevant events that guard against wasteful oxygen consumption and inappropriate cell growth during hypoxia. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Synthesis and biological evaluation of kresoxim-methyl analogues as novel inhibitors of hypoxia-inducible factor (HIF)-1 accumulation in cancer cells.

PubMed

Lee, Sanghyuck; Kwon, Oh Seok; Lee, Chang-Soo; Won, Misun; Ban, Hyun Seung; Ra, Choon Sup

2017-07-01

We designed and synthesized strobilurin analogues as hypoxia-inducible factor (HIF) inhibitors based on the molecular structure of kresoxim-methyl. Biological evaluation in human colorectal cancer HCT116 cells showed that most of the synthesized kresoxim-methyl analogues possessed moderate to potent inhibitory activity against hypoxia-induced HIF-1 transcriptional activation. Three candidates, compounds 11b, 11c, and 11d were identified as potent inhibitors against HIF-1 activation with IC 50 values of 0.60-0.94µM. Under hypoxic condition, compounds 11b, 11c, and 11d increased the intracellular oxygen contents, thereby attenuating the hypoxia-induced accumulation of HIF-1α protein. Copyright © 2017 Elsevier Ltd. All rights reserved.

Computational modeling on the recognition of the HRE motif by HIF-1: molecular docking and molecular dynamics studies.

PubMed

Sokkar, Pandian; Sathis, Vani; Ramachandran, Murugesan

2012-05-01

Hypoxia inducible factor-1 (HIF-1) is a bHLH-family transcription factor that controls genes involved in glycolysis, angiogenesis, migration, as well as invasion factors that are important for tumor progression and metastasis. HIF-1, a heterodimer of HIF-1α and HIF-1β, binds to the hypoxia responsive element (HRE) present in the promoter regions of hypoxia responsive genes, such as vascular endothelial growth factor (VEGF). Neither the structure of free HIF-1 nor that of its complex with HRE is available. Computational modeling of the transcription factor-DNA complex has always been challenging due to their inherent flexibility and large conformational space. The present study aims to model the interaction between the DNA-binding domain of HIF-1 and HRE. Experiments showed that rigid macromolecular docking programs (HEX and GRAMM-X) failed to predict the optimal dimerization of individually modeled HIF-1 subunits. Hence, the HIF-1 heterodimer was modeled based on the phosphate system positive regulatory protein (PHO4) homodimer. The duplex VEGF-DNA segment containing HRE with flanking nucleotides was modeled in the B form and equilibrated via molecular dynamics (MD) simulation. A rigid docking approach was used to predict the crude binding mode of HIF-1 dimer with HRE, in which the putative contacts were found to be present. An MD simulation (5 ns) of the HIF-1-HRE complex in explicit water was performed to account for its flexibility and to optimize its interactions. All of the conserved amino acid residues were found to play roles in the recognition of HRE. The present work, which sheds light on the recognition of HRE by HIF-1, could be beneficial in the design of peptide or small molecule therapeutics that can mimic HIF-1 and bind with the HRE sequence.

Human Intestinal Fluid Layer Separation: The Effect On Colloidal Structures & Solubility Of Lipophilic Compounds.

PubMed

Danny, Riethorst; Amitava, Mitra; Filippos, Kesisoglou; Wei, Xu; Jan, Tack; Joachim, Brouwers; Patrick, Augustijns

2018-05-23

In addition to individual intestinal fluid components, colloidal structures are responsible for enhancing the solubility of lipophilic compounds. The present study investigated the link between as well as the variability in the ultrastructure of fed state human intestinal fluids (FeHIF) and their solubilizing capacity for lipophilic compounds. For this purpose, FeHIF samples from 10 healthy volunteers with known composition and ultrastructure were used to determine the solubility of four lipophilic compounds. In light of the focus on solubility and ultrastructure, the study carefully considered the methodology of solubility determination in relation to colloid composition and solubilizing capacity of FeHIF. To determine the solubilizing capacity of human and simulated intestinal fluids, the samples were saturated with the compound of interest, shaken for 24 h, and centrifuged. When using FeHIF, solubilities were determined in the micellar layer of FeHIF, i.e. after removing the upper (lipid) layer (standard procedure), as well as in 'full' FeHIF (without removal of the upper layer). Compound concentrations were determined using HPLC-UV/fluorescence. To link the solubilizing capacity with the ultrastructure, all human and simulated fluids were imaged using transmission electron microscopy (TEM) before and after centrifugation and top layer (lipid) removal. Comparing the ultrastructure and solubilizing capacity of individual FeHIF samples demonstrated a high intersubject variability in postprandial intestinal conditions. Imaging of FeHIF after removal of the upper layer clearly showed that only micellar structures remain in the lower layer. This observation suggests that larger colloids such as vesicles and lipid droplets are contained in the upper, lipid layer. The solubilizing capacity of most FeHIF samples substantially increased with inclusion of this lipid layer. The relative increase in solubilizing capacity upon inclusion of the lipid layer was most pronounced in samples that contained mainly vesicles alongside the micelles. Current fed state simulated intestinal fluids do not contain the larger colloids observed in the lipid layer of FeHIF and can only simulate the solubilizing capacity of the micellar layer of FeHIF. While the importance of drug molecules solubilized in the micellar layer of postprandial intestinal fluids for absorption has been extensively demonstrated previously, the in-vivo relevance of drug solubilization in the lipid layer is currently unclear. In the dynamic environment of the human gastrointestinal tract, drug initially entrapped in larger postprandial colloids may become available for absorption upon lipid digestion and uptake. The current study, demonstrating the substantial solubilization of lipophilic compounds in the larger colloids of postprandial intestinal fluids, warrants further research in this field. Copyright © 2018. Published by Elsevier B.V.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedman, A.; Barnard, J.J.; Briggs, R.J.

The Heavy Ion Fusion Science Virtual National Laboratory (HIFS-VNL), a collaborationof LBNL, LLNL, and PPPL, has achieved 60-fold pulse compression of ion beams on the Neutralized Drift Compression eXperiment (NDCX) at LBNL. In NDCX, a ramped voltage pulse from an induction cell imparts a velocity"tilt" to the beam; the beam's tail then catches up with its head in a plasma environment that provides neutralization. The HIFS-VNL's mission is to carry out studies of Warm Dense Matter (WDM) physics using ion beams as the energy source; an emerging thrust is basic target physics for heavy ion-driven Inertial Fusion Energy (IFE). Thesemore » goals require an improved platform, labeled NDCX-II. Development of NDCX-II at modest cost was recently enabled by the availability of induction cells and associated hardware from the decommissioned Advanced Test Accelerator (ATA) facility at LLNL. Our initial physics design concept accelerates a ~;;30 nC pulse of Li+ ions to ~;;3 MeV, then compresses it to ~;;1 ns while focusing it onto a mm-scale spot. It uses the ATA cells themselves (with waveforms shaped by passive circuits) to impart the final velocity tilt; smart pulsers provide small corrections. The ATA accelerated electrons; acceleration of non-relativistic ions involves more complex beam dynamics both transversely and longitudinally. We are using analysis, an interactive one-dimensional kinetic simulation model, and multidimensional Warp-code simulations to develop the NDCX-II accelerator section. Both LSP and Warp codes are being applied to the beam dynamics in the neutralized drift and final focus regions, and the plasma injection process. The status of this effort is described.« less

FAM13A is associated with non-small cell lung cancer (NSCLC) progression and controls tumor cell proliferation and survival

PubMed Central

Heim, Lisanne; Trump, Sonja; Mittler, Susanne; Sopel, Nina; Andreev, Katerina; Ferrazzi, Fulvia; Ekici, Arif B.; Rieker, Ralf; Springel, Rebekka; Assmann, Vera L.; Lechmann, Matthias; Koch, Sonja; Engelhardt, Marina; Trufa, Denis I.; Sirbu, Horia; Hartmann, Arndt; Finotto, Susetta

2017-01-01

ABSTRACT Genome-wide association studies (GWAS) associated Family with sequence similarity 13, member A (FAM13A) with non-small cell lung cancer (NSCLC) occurrence. Here, we found increased numbers of FAM13A protein expressing cells in the tumoral region of lung tissues from a cohort of patients with NSCLC. Moreover, FAM13A inversely correlated with CTLA4 but directly correlated with HIF1α levels in the control region of these patients. Consistently, FAM13A RhoGAP was found to be associated with T cell effector molecules like HIF1α and Tbet and was downregulated in immunosuppressive CD4+CD25+Foxp3+CTLA4+ T cells. TGFβ, a tumor suppressor factor, as well as siRNA to FAM13A, suppressed both isoforms of FAM13A and inhibited tumor cell proliferation. RNA-Seq analysis confirmed this finding. Moreover, siRNA to FAM13A induced TGFβ levels. Finally, in experimental tumor cell migration, FAM13A was induced and TGFβ accelerated this process by inducing cell migration, HIF1α, and the FAM13A RhoGAP isoform. Furthermore, siRNA to FAM13A inhibited tumor cell proliferation and induced cell migration without affecting HIF1α. In conclusion, FAM13A is involved in tumor cell proliferation and downstream of TGFβ and HIF1α, FAM13A RhoGAP is associated with Th1 gene expression and lung tumor cell migration. These findings identify FAM13A as key regulator of NSCLC growth and progression. PMID:28197372

Targeting HIF2 in Clear Cell Renal Cell Carcinoma.

PubMed

Cho, Hyejin; Kaelin, William G

2016-01-01

Inactivation of the von Hippel-Lindau tumor-suppressor protein (pVHL) is the signature "truncal" event in clear cell renal cell carcinoma, which is the most common form of kidney cancer. pVHL is part of a ubiquitin ligase the targets the α subunit of the hypoxia-inducible factor (HIF) transcription factor for destruction when oxygen is available. Preclinical studies strongly suggest that deregulation of HIF, and particularly HIF2, drives pVHL-defective renal carcinogenesis. Although HIF2α was classically considered undruggable, structural and chemical work by Rick Bruick and Kevin Gardner at University of Texas Southwestern laid the foundation for the development of small molecule direct HIF2α antagonists (PT2385 and the related tool compound PT2399) by Peloton Therapeutics that block the dimerization of HIF2α with its partner protein ARNT1. These compounds inhibit clear cell renal cell carcinoma growth in preclinical models, and PT2385 has now entered the clinic. Nonetheless, the availability of such compounds, together with clustered regularly interspaced short palindromic repeat (CRISPR)-based gene editing approaches, has revealed a previously unappreciated heterogeneity among clear cell renal carcinomas and patient-derived xenografts with respect to HIF2 dependence, suggesting that predictive biomarkers will be needed to optimize the use of such agents in the clinic. © 2016 Cho and Kaelin; Published by Cold Spring Harbor Laboratory Press.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukiyama, Fuyo; Nakai, Yumi; Yoshida, Masataka

Catechins have recently been reported to increase the cellular content of the hypoxia-inducible factor (HIF)-1{alpha} within mammalian cells. These catechins have a gallate moiety as a common structure. We now report that n-propyl gallate (nPG) also increases the HIF-1{alpha} protein in the rat heart-derived H9c2 cells. The increase was dose-dependent and reached a maximum at 2-4 h after the addition of nPG to the cells. nPG did not change the HIF-1{alpha} mRNA level, showing that the increase is a posttranscriptional event. Although nPG did not inhibit the HIF prolyl hydroxylase, gallate, the hydrolysis product of nPG, inhibited the enzyme completelymore » at submillimolar concentrations. Model building studies on the human HIF prolyl hydroxylase 2 showed that the two phenolate oxygen atoms of gallate form a chelate with the active site Fe{sup 2+}, while the carboxyl group of gallate forms a strong ionic/hydrogen bonding interaction with Arg383, explaining why nPG, which has an esterified carboxyl group, is unable to inhibit the hydroxylase. Together with the observation that gallate was detected in the H9c2 cells treated with nPG, these results suggest that nPG incorporated into the cells is hydrolyzed and the released gallate inhibits the HIF prolyl hydroxylase, thereby reducing the HIF degradation rate and increasing the HIF-1{alpha} content.« less

Improving access to medicines via the Health Impact Fund in India: a stakeholder analysis

PubMed Central

McMullan, Patrick; Ajay, Vamadevan S.; Srinivas, Ravi; Bhalla, Sandeep; Prabhakaran, Dorairaj; Banerjee, Amitava

2018-01-01

ABSTRACT Background: In India, 50–65% of the population face difficulties in accessing medicines. The Health Impact Fund (HIF) is a novel proposal whereby pharmaceutical companies would be paid based on the measured global health impact of their drugs. We conducted a key stakeholder analysis to explore access to medicines in India, acceptability of the HIF and potential barriers and facilitators at policy level. Objectives: To conduct a stakeholder analysis of the HIF in India: to determine key stakeholder views regarding access to medicines in India; to evaluate acceptability of the HIF; and to assess potential barriers and facilitators to the HIF as a policy. Methods: In New Delhi, we conducted semi-structured interviews. There was purposive recruitment of participants with snowball sampling. Transcribed data were analysed using stakeholder analysis frameworks and directed content analysis. Results: Participation rate was 29% (14/49). 14 semi-structured interviews were conducted among stakeholders in New Delhi. All participants highlighted access to medicines as a problem in India. There were mixed views about the HIF in terms of relevance and scaleability. Stakeholders felt it should focus on diseases with limited or no market and potentially incorporate direct investment in research. Conclusions: First, access to medicines is perceived to be a major problem in India by all stakeholders, but affordability is just one factor. Second, stakeholders despite considerable support for the idea of the HIF, there are major concerns about scaleability, generalisability and impact on access to medicines. Third, the HIF and other novel drug-related health policies can afford to be more radical, e.g. working outside the existing intellectual property rights regime, targeting generic as well as branded drugs, or extending to research and development. Further innovations in access to medicines must involve country-specific key stakeholders in order to increase the likelihood of their success. PMID:29495950

Laser Cooling for Heavy-Ion Fusion (HIF)

NASA Astrophysics Data System (ADS)

Ho, D. D.-M.; Brandon, S.; Lee, Y.

1997-05-01

A critical requirement for HIF is the ability to focus space-charge dominated beams onto a millimeter-size spot. However, chromatic aberration can result in a substantial fraction of the beam ions falling outside the spot radius. Because of the space-charge force, correcting the chromatic aberration using sextupoles is impractical. Success in laser cooling of low-current ion beams in storage rings leads us to explore the application of laser cooling to HIF. Basic scheme: After the beams have been accelerated to the desired energy by the recirculating induction linac, we let the beams coast around at constant energy. For efficient interaction between the laser and the beam ions, we use Ba+ beams. We use two lasers to pump the transitions in the Ba+ for generating the laser force FL. There is also an auxiliary force Fa, which is in the opposition direction of FL, provided by the induction cores. The momentum spread along the beam can be compressed by FL and Fa. We will present preliminary PIC simulations using the PIC code CONDOR. Potential difficulties caused by velocity space instabilities will be discussed.

Down-regulation of Jab1, HIF-1alpha, and VEGF by Moloney murine leukemia virus-ts1 infection: a possible cause of neurodegeneration.

PubMed

Lungu, Gina F; Stoica, George; Wong, Paul K Y

2008-05-01

Moloney murine leukemia virus-temperature sensitive (MoMuLV-ts1)-mediated neuronal death is a result of both loss of glial support and release of cytokines and neurotoxins from ts1-infected glial cells. Here the authors propose vascular endothelial growth factor (VEGF) down-regulation as another contributory factor in neuronal degeneration induced by ts1 infection. To determine how ts1 affects VEGF expression in ts1-infected brain, the authors examined the expression of several proteins that are important in regulating the expression of VEGF. The authors found significant decreases in Jun-activating domain-binding protein 1 (Jab1), hypoxia-inducible factor (HIF)-1alpha, and VEGF levels and increases in p53 protein levels in ts1-infected brains compared to noninfected control brains. The authors suggest that a decrease Jab1 expression in ts1 infection leads to accumulation of p53, which binds to HIF-1alpha to accelerate its degradation. A rapid degradation of HIF-1alpha leads to decreased VEGF production and secretion. Considering that endothelial cells are the most conspicuous in virus replication and production in ts1 infection, but are not killed by the infection, the authors examined the expression of these proteins using infected and noninfected mouse cerebrovascular endothelial (CVE) cells. The ts1- infected CVE cells showed decreased Jab1, HIF-1alpha, and VEGF mRNA and protein levels and increased p53 protein levels compared with noninfected cells, consistent with the results found in vivo. These results confirm that ts1 infection results in insufficient secretion of VEGF from endothelial cells and may result in decreased neuroprotection. This study suggested that ts1-mediated neuropathology in mice may result from changes in expression and activity of Jab1, p53, and HIF-1alpha, with a final target on VEGF expression and neuronal degeneration.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Yirui; Scheuermann, Thomas H.; Partch, Carrie L.

The hypoxia-inducible factor complex (HIF-α·aryl hydrocarbon receptor nuclear translocator (ARNT)) requires association with several transcription coactivators for a successful cellular response to hypoxic stress. In addition to the conventional global transcription coactivator CREB-binding protein/p300 (CBP/p300) that binds to the HIF-α transactivation domain, a new group of transcription coactivators called the coiled-coil coactivators (CCCs) interact directly with the second PER-ARNT-SIM (PAS) domain of ARNT (ARNT PAS-B). These less studied transcription coactivators play essential roles in the HIF-dependent hypoxia response, and CCC misregulation is associated with several forms of cancer. To better understand CCC protein recruitment by the heterodimeric HIF transcription factor,more » we used x-ray crystallography, NMR spectroscopy, and biochemical methods to investigate the structure of the ARNT PAS-B domain in complex with the C-terminal fragment of a coiled-coil coactivator protein, transforming acidic coiled-coil coactivator 3 (TACC3). We found that the HIF-2α PAS-B domain also directly interacts with TACC3, motivating an NMR data-derived model suggesting a means by which TACC3 could form a ternary complex with HIF-2α PAS-B and ARNT PAS-B via β-sheet/coiled-coil interactions. Furthermore, these findings suggest that TACC3 could be recruited as a bridge to cooperatively mediate between the HIF-2α PAS-B·ARNT PAS-B complex, thereby participating more directly in HIF-dependent gene transcription than previously anticipated.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedman, A; Barnard, J J; Briggs, R J

The Heavy Ion Fusion Science Virtual National Laboratory (HIFS-VNL), a collaboration of LBNL, LLNL, and PPPL, has achieved 60-fold pulse compression of ion beams on the Neutralized Drift Compression eXperiment (NDCX) at LBNL. In NDCX, a ramped voltage pulse from an induction cell imparts a velocity 'tilt' to the beam; the beam's tail then catches up with its head in a plasma environment that provides neutralization. The HIFS-VNL's mission is to carry out studies of warm dense matter (WDM) physics using ion beams as the energy source; an emerging thrust is basic target physics for heavy ion-driven inertial fusion energymore » (IFE). These goals require an improved platform, labeled NDCX-II. Development of NDCX-II at modest cost was recently enabled by the availability of induction cells and associated hardware from the decommissioned advanced test accelerator (ATA) facility at LLNL. Our initial physics design concept accelerates an {approx} 30 nC pulse of Li{sup +} ions to {approx} 3 MeV, then compresses it to {approx} 1 ns while focusing it onto a mm-scale spot. It uses the ATA cells themselves (with waveforms shaped by passive circuits) to impart the final velocity tilt; smart pulsers provide small corrections. The ATA accelerated electrons; acceleration of non-relativistic ions involves more complex beam dynamics both transversely and longitudinally. We are using an interactive one-dimensional kinetic simulation model and multidimensional Warp-code simulations to develop the NDCX-II accelerator section. Both LSP and Warp codes are being applied to the beam dynamics in the neutralized drift and final focus regions, and the plasma injection process. The status of this effort is described.« less

STAT3 or USF2 Contributes to HIF Target Gene Specificity

PubMed Central

Pawlus, Matthew R.; Wang, Liyi; Murakami, Aya; Dai, Guanhai; Hu, Cheng-Jun

2013-01-01

The HIF1- and HIF2-mediated transcriptional responses play critical roles in solid tumor progression. Despite significant similarities, including their binding to promoters of both HIF1 and HIF2 target genes, HIF1 and HIF2 proteins activate unique subsets of target genes under hypoxia. The mechanism for HIF target gene specificity has remained unclear. Using siRNA or inhibitor, we previously reported that STAT3 or USF2 is specifically required for activation of endogenous HIF1 or HIF2 target genes. In this study, using reporter gene assays and chromatin immuno-precipitation, we find that STAT3 or USF2 exhibits specific binding to the promoters of HIF1 or HIF2 target genes respectively even when over-expressed. Functionally, HIF1α interacts with STAT3 to activate HIF1 target gene promoters in a HIF1α HLH/PAS and N-TAD dependent manner while HIF2α interacts with USF2 to activate HIF2 target gene promoters in a HIF2α N-TAD dependent manner. Physically, HIF1α HLH and PAS domains are required for its interaction with STAT3 while both N- and C-TADs of HIF2α are involved in physical interaction with USF2. Importantly, addition of functional USF2 binding sites into a HIF1 target gene promoter increases the basal activity of the promoter as well as its response to HIF2+USF2 activation while replacing HIF binding site with HBS from a HIF2 target gene does not change the specificity of the reporter gene. Importantly, RNA Pol II on HIF1 or HIF2 target genes is primarily associated with HIF1α or HIF2α in a STAT3 or USF2 dependent manner. Thus, we demonstrate here for the first time that HIF target gene specificity is achieved by HIF transcription partners that are required for HIF target gene activation, exhibit specific binding to the promoters of HIF1 or HIF2 target genes and selectively interact with HIF1α or HIF2α protein. PMID:23991099

Development of the 2-MV Injector for HIF

NASA Astrophysics Data System (ADS)

Bieniosek, F. M.; Kwan, J. W.; Henestroza, E.; Kim, C.

2001-05-01

The 2-MV Injector consists of a 17-cm-diameter surface ionization source, an extraction diode, and an electrostatic quadrupole (ESQ) accelerator, with maximum current of 0.8 A of potassium beam at 2 MeV. Previous performance of the Injector produced a beam with adequate current and emittance but with a hollow profile at the end of the ESQ section. We have examined the profile of the beam as it leaves the diode. The measured nonuniform beam density distribution qualitatively agrees with EGUN simulation. Implications for emittance growth in the post acceleration and transport phase will be investigated.

Inhibition of HIF-2.alpha. heterodimerization with HIF1.beta. (ARNT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bruick, Richard K.; Caldwell, Charles G.; Frantz, Doug E.

2017-09-12

Provided is a method of inhibiting heterodimerization of HIF-2.alpha. to HIF1.beta. (ARNT) comprising binding certain small molecules to the HIF-2.alpha. PAS-B domain cavity but not to HIF1.alpha. and inhibiting HIF-2.alpha. heterodimerization to HIF1.beta. (ARNT) but not inhibiting HIF1.alpha. heterodimerization to HIF1.beta. (ARNT). Those certain small molecules are also referenced synonymously as HIF2-HDI and HIF2.alpha. heterodimerization inhibitors and also simply as certain small molecules.

Effect of HIF-1a/VEGF signaling pathway on plasma progesterone and ovarian prostaglandin F₂a secretion during luteal development of pseudopregnant rats.

PubMed

Pan, X Y; Zhang, Z H; Wu, L X; Wang, Z C

2015-08-03

The corpus luteum is a temporary endocrine structure in mammals that plays an important role in the female reproductive cycle and is formed from a ruptured and ovulated follicle with rapid angiogenesis. Vascular endothelial growth factor (VEGF) is thought to be vital in normal and abnormal angiogenesis in the ovary, but the molecular regulation of luteal VEGF expression during corpus luteum development in vivo is still poorly understood at present. Therefore, we examined whether hypoxia-inducible factor-1a (HIF-1a) is induced and regulates VEGF expression and luteal function in vivo using a pseudopregnant rat model treated with a small-molecule inhibitor of HIF-1a, echinomycin. Corpus luteum development in the pseudopregnant rat ovary was determined after measuring plasma progesterone concentration and ovarian prostaglandin F2a content to reflect changes in HIF-1a and VEGF on different days of this developmental process. At day 7, the corpus luteum was formed and the expression of HIF- 1a/VEGF reached a maximum, while a significant decrease in HIF-1a/ VEGF expression was observed when luteolysis occurred at day 13. Additionally, echinomycin blocked luteal development by inhibiting VEGF expression mediated by HIF-1a and following luteal function by detecting the progesterone changes at day 7. These results demonstrated that HIF-1a-mediated VEGF expression might be an important mechanism regulating ovarian luteal development in mammals in vivo, which may provide new strategies for fertility control and for treating some types of ovarian dysfunction, such as polycystic ovarian syndrome, ovarian hyperstimulation syndrome, and ovarian neoplasia.

Synthetic biology: ensuring the greatest global value.

PubMed

Hollis, Aidan

2013-09-01

Synthetic biology (SynBio) has tremendous, transformative potential. Like other technologies, it can be used for good or ill. Currently, the structure of the allocation of potential benefits and risks is biased in favor of richer countries. The underlying problem is simple: most risks from SynBio are universal and affect both the rich and the poor with equal force; but benefits from SynBio can be expected to accrue chiefly to the rich. The risk/benefit balance is therefore skewed in a way that may lead to inefficient and unfair decisions. One potential solution is presented in this paper, using the principles that underlie the Health Impact Fund (HIF). The HIF is designed to reward companies based on assessed health impact, no matter where it occurs in the world, so that extending the life of a poor person is as profitable as extending the life of a rich person. This paper considers both the potential benefits and costs of SynBio; examines how the current global pharmaceutical industry is structured; introduces the HIF proposal; and finally explores how the principles underlying the HIF could be used productively with SynBio for global health.

Donepezil, an acetylcholinesterase inhibitor against Alzheimer's dementia, promotes angiogenesis in an ischemic hindlimb model.

PubMed

Kakinuma, Yoshihiko; Furihata, Mutsuo; Akiyama, Tsuyoshi; Arikawa, Mikihiko; Handa, Takemi; Katare, Rajesh G; Sato, Takayuki

2010-04-01

Our recent studies have indicated that acetylcholine (ACh) protects cardiomyocytes from prolonged hypoxia through activation of the PI3K/Akt/HIF-1alpha/VEGF pathway and that cardiomyocyte-derived VEGF promotes angiogenesis in a paracrine fashion. These results suggest that a cholinergic system plays a role in modulating angiogenesis. Therefore, we assessed the hypothesis that the cholinergic modulator donepezil, an acetylcholinesterase inhibitor utilized in Alzheimer's disease, exhibits beneficial effects, especially on the acceleration of angiogenesis. We evaluated the effects of donepezil on angiogenic properties in vitro and in vivo, using an ischemic hindlimb model of alpha7 nicotinic receptor-deleted mice (alpha7 KO) and wild-type mice (WT). Donepezil activated angiogenic signals, i.e., HIF-1alpha and VEGF expression, and accelerated tube formation in human umbilical vein endothelial cells (HUVECs). ACh and nicotine upregulated signal transduction with acceleration of tube formation, suggesting that donepezil promotes a common angiogenesis pathway. Moreover, donepezil-treated WT exhibited rich capillaries with enhanced VEGF and PCNA endothelial expression, recovery from impaired tissue perfusion, prevention of ischemia-induced muscular atrophy with sustained surface skin temperature in the limb, and inhibition of apoptosis independent of the alpha7 receptor. Donepezil exerted comparably more effects in alpha7 KO in terms of angiogenesis, tissue perfusion, biochemical markers, and surface skin temperature. Donepezil concomitantly elevated VEGF expression in intracardiac endothelial cells of WT and alpha7 KO and further increased choline acetyltransferase (ChAT) protein expression, which is critical for ACh synthesis in endothelial cells. The present study concludes that donepezil can act as a therapeutic tool to accelerate angiogenesis in cardiovascular disease patients. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor.

PubMed

Knowles, Helen J; Schaefer, Karl-Ludwig; Dirksen, Uta; Athanasou, Nicholas A

2010-07-16

Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. HIF-1alpha and HIF-2alpha immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. 17/56 Ewing's tumours were HIF-1alpha-positive, 15 HIF-2alpha-positive and 10 positive for HIF-1alpha and HIF-2alpha. Expression of HIF-1alpha and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1alpha and HIF-2alpha in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2alpha in Ewing's. Downstream transcription was HIF-1alpha-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by >or= 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Co-localisation of HIF-1alpha and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

Cadmium exposure inhibits branching morphogenesis and causes alterations consistent with HIF-1α inhibition in human primary breast organoids.

PubMed

Rocco, Sabrina A; Koneva, Lada; Middleton, Lauren Y M; Thong, Tasha; Solanki, Sumeet; Karram, Sarah; Nambunmee, Kowit; Harris, Craig; Rozek, Laura S; Sartor, Maureen A; Shah, Yatrik M; Colacino, Justin A

2018-05-07

Developmental cadmium exposure in vivo disrupts mammary gland differentiation, while exposure of breast cell lines to cadmium causes invasion consistent with the epithelial-mesenchymal transition (EMT). The effects of cadmium on normal human breast stem cells have not been measured. Here, we quantified the effects of cadmium exposure on reduction mammoplasty patient-derived breast stem cell proliferation and differentiation. Using the mammosphere assay and organoid formation in 3D hydrogels, we tested two physiologically relevant doses of cadmium, 0.25μM and 2.5μM, and tested for molecular alterations using RNA-seq. We functionally validated our RNA-seq findings with a HIF-1α activity reporter line and pharmaceutical inhibition of HIF-1α in organoid formation assays. 2.5μM cadmium reduced primary mammosphere formation and branching structure organoid formation rates by 33% and 87%, respectively. Despite no changes in mammosphere formation, 0.25μM cadmium inhibited branching organoid formation in hydrogels by 73%. RNA-seq revealed cadmium downregulated genes associated with extracellular matrix formation and EMT, while upregulating genes associated with metal response including metallothioneins and zinc transporters. In the RNA-seq data, cadmium downregulated HIF-1α target genes including LOXL2, ZEB1, and VIM. Cadmium significantly inhibited HIF-1α activity in a luciferase assay, and the HIF-1α inhibitor acriflavine ablated mammosphere and organoid formation. These findings show that cadmium, at doses relevant to human exposure, inhibited human mammary stem cell proliferation and differentiation, potentially through disruption of HIF-1α activity.

Delayed Administration of a Single Dose of Lithium Promotes Recovery from AKI

PubMed Central

Bao, Hui; Ge, Yan; Wang, Zhen; Zhuang, Shougang; Dworkin, Lance; Peng, Ai

2014-01-01

Evidence suggests that glycogen synthase kinase 3β (GSK3β) contributes to AKI; however, its role in post-AKI kidney repair remains uncertain. Here, delayed treatment with a single dose of lithium, a selective inhibitor of GSK3β and a US Food and Drug Administration–approved mood stabilizer, accelerated recovery of renal function, promoted repopulation of renal tubular epithelia, and improved kidney repair in murine models of cisplatin- and ischemia/reperfusion-induced AKI. These effects associated with reduced GSK3β activity and elevated expression of proproliferative molecules, including cyclin D1, c-Myc, and hypoxia-inducible factor 1α (HIF-1α), in renal tubular epithelia. In cultured renal tubular cells, cisplatin exposure led to transient repression of GSK3β activity followed by a prolonged upregulation of activity. Rescue treatment with lithium inhibited GSK3β activity, enhanced nuclear expression of cyclin D1, c-Myc, and HIF-1α, and boosted cellular proliferation. Similarly, ectopic expression of a kinase-dead mutant of GSK3β enhanced the expression of cyclin D1, c-Myc, and HIF-1α and amplified cellular proliferation after cisplatin injury, whereas forced expression of a constitutively active mutant of GSK3β abrogated the effects of lithium. Mechanistically, GSK3β colocalized and physically interacted with cyclin D1, c-Myc, and HIF-1α in tubular cells. In silico analysis revealed that cyclin D1, c-Myc, and HIF-1α harbor putative GSK3β consensus phosphorylation motifs, implying GSK3β-directed phosphorylation and subsequent degradation of these molecules. Notably, cotreatment with lithium enhanced the proapoptotic effects of cisplatin in cultured colon cancer cells. Collectively, our findings suggest that pharmacologic targeting of GSK3β by lithium may be a novel therapeutic strategy to improve renal salvage after AKI. PMID:24408869

Reduced Perinatal Leptin Availability May Contribute to Adverse Metabolic Programming in a Rat Model of Uteroplacental Insufficiency.

PubMed

Nüsken, Eva; Wohlfarth, Maria; Lippach, Gregor; Rauh, Manfred; Schneider, Holm; Dötsch, Jörg; Nüsken, Kai-Dietrich

2016-05-01

Leptin availability in perinatal life critically affects metabolic programming. We tested the hypothesis that uteroplacental insufficiency and intrauterine stress affect perinatal leptin availability in rat offspring. Pregnant rats underwent bilateral uterine vessel ligation (LIG; n = 14), sham operation (SOP; n = 12), or no operation (controls, n = 14). Fetal livers (n = 180), placentas (n = 180), and maternal blood were obtained 4 hours (gestational day [E] 19), 24 hours (E20), and 72 hours (E22) after surgery. In the offspring, we took blood samples on E22 (n = 44), postnatal day (P) 1 (n = 29), P2 (n = 16), P7 (n = 30), and P12 (n = 30). Circulating leptin (ELISA) was significantly reduced in LIG (E22, P1, P2) and SOP offspring (E22). Postnatal leptin surge was delayed in LIG but was accelerated in SOP offspring. Placental leptin gene expression (quantitative RT-PCR) was reduced in LIG (E19, E20, E22) and SOP (E20, E22). Hepatic leptin receptor (Lepr-a, mediating leptin degradation) gene expression was increased in LIG fetuses (E20, E22) only. Surprisingly, hypoxia-inducible factors (Hif; Western blot) were unaltered in placentas and were reduced in the livers of LIG (Hif1a, E20; Hif2a, E19, E22) and SOP (Hif2a, E19) fetuses. Gene expression of prolyl hydroxylase 3, a factor expressed under hypoxic conditions contributing to Hif degradation, was increased in livers of LIG (E19, E20, E22) and SOP (E19) fetuses and in placentas of LIG and SOP (E19). In summary, reduced placental leptin production, increased fetal leptin degradation, and persistent perinatal hypoleptinemia are present in intrauterine growth restriction offspring, especially after uteroplacental insufficiency, and may contribute to perinatal programming of leptin resistance and adiposity in later life.

DPPIV promotes endometrial carcinoma cell proliferation, invasion and tumorigenesis

PubMed Central

Yang, Xiaoqing; Zhang, Xinhua; Wu, Rongrong; Huang, Qicheng; Jiang, Yao; Qin, Jianbing; Yao, Feng; Jin, Guohua; Zhang, Yuquan

2017-01-01

Dipeptidyl peptidase IV (DPPIV), also known as CD26, is a 110-kDa cell surface glycoprotein expressed in various tissues. DPPIV reportedly plays a direct role in the progression of several human malignancies. DPPIV specific inhibitors are employed as antidiabetics and could potentially be repurposed to enhance anti-tumor immunotherapies. In the present study, we investigated the correlation between DPPIV expression and tumor progression in endometrial carcinoma (EC). DPPIV overexpression altered cell morphology and stimulated cell proliferation, invasion and tumorigenesis in vitro and in vivo. These effects were abrogated by DPPIV knockdown or pharmacological inhibition using sitagliptin. DPPIV overexpression increased hypoxia-inducible factor 1a (HIF-1a) and vascular endothelial growth factor A (VEGFA) expression to promote HIF-1a-VEGFA signaling. Our results indicated that DPPIV accelerated endometrial carcinoma progression and that sitagliptin may be an effective anti-EC therapeutic. PMID:28060721

DPPIV promotes endometrial carcinoma cell proliferation, invasion and tumorigenesis.

PubMed

Yang, Xiaoqing; Zhang, Xinhua; Wu, Rongrong; Huang, Qicheng; Jiang, Yao; Qin, Jianbing; Yao, Feng; Jin, Guohua; Zhang, Yuquan

2017-01-31

Dipeptidyl peptidase IV (DPPIV), also known as CD26, is a 110-kDa cell surface glycoprotein expressed in various tissues. DPPIV reportedly plays a direct role in the progression of several human malignancies. DPPIV specific inhibitors are employed as antidiabetics and could potentially be repurposed to enhance anti-tumor immunotherapies. In the present study, we investigated the correlation between DPPIV expression and tumor progression in endometrial carcinoma (EC). DPPIV overexpression altered cell morphology and stimulated cell proliferation, invasion and tumorigenesis in vitro and in vivo. These effects were abrogated by DPPIV knockdown or pharmacological inhibition using sitagliptin. DPPIV overexpression increased hypoxia-inducible factor 1a (HIF-1a) and vascular endothelial growth factor A (VEGFA) expression to promote HIF-1a-VEGFA signaling. Our results indicated that DPPIV accelerated endometrial carcinoma progression and that sitagliptin may be an effective anti-EC therapeutic.

PHD-2 Suppression in Mesenchymal Stromal Cells Enhances Wound Healing.

PubMed

Ko, Sae Hee; Nauta, Allison C; Morrison, Shane D; Hu, Michael S; Zimmermann, Andrew S; Chung, Michael T; Glotzbach, Jason P; Wong, Victor W; Walmsley, Graham G; Peter Lorenz, H; Chan, Denise A; Gurtner, Geoffrey C; Giaccia, Amato J; Longaker, Michael T

2018-01-01

Cell therapy with mesenchymal stromal cells is a promising strategy for tissue repair. Restoration of blood flow to ischemic tissues is a key step in wound repair, and mesenchymal stromal cells have been shown to be proangiogenic. Angiogenesis is critically regulated by the hypoxia-inducible factor (HIF) superfamily, consisting of transcription factors targeted for degradation by prolyl hydroxylase domain (PHD)-2. The aim of this study was to enhance the proangiogenic capability of mesenchymal stromal cells and to use these modified cells to promote wound healing. Mesenchymal stromal cells harvested from mouse bone marrow were transduced with short hairpin RNA (shRNA) against PHD-2; control cells were transduced with scrambled shRNA (shScramble) construct. Gene expression quantification, human umbilical vein endothelial cell tube formation assays, and wound healing assays were used to assess the effect of PHD knockdown mesenchymal stromal cells on wound healing dynamics. PHD-2 knockdown mesenchymal stromal cells overexpressed HIF-1α and multiple angiogenic factors compared to control (p < 0.05). Human umbilical vein endothelial cells treated with conditioned medium from PHD-2 knockdown mesenchymal stromal cells exhibited increased formation of capillary-like structures and enhanced migration compared with human umbilical vein endothelial cells treated with conditioned medium from shScramble-transduced mesenchymal stromal cells (p < 0.05). Wounds treated with PHD-2 knockdown mesenchymal stromal cells healed at a significantly accelerated rate compared with wounds treated with shScramble mesenchymal stromal cells (p < 0.05). Histologic studies revealed increased blood vessel density and increased cellularity in the wounds treated with PHD-2 knockdown mesenchymal stromal cells (p < 0.05). Silencing PHD-2 in mesenchymal stromal cells augments their proangiogenic potential in wound healing therapy. This effect appears to be mediated by overexpression of HIF family transcription factors and up-regulation of multiple downstream angiogenic factors.

Emotional Arousal and Regulation: Further Evidence of the Validity of the "How I Feel" Questionnaire for Use With School-Age Children.

PubMed

Ciucci, Enrica; Baroncelli, Andrea; Grazzani, Ilaria; Ornaghi, Veronica; Caprin, Claudia

2016-03-01

The ability to understand and manage emotional experience is critical to children's health. This study confirmed the validity of the How I Feel (HIF) Questionnaire, a measure of children's emotional arousal and regulation, exploring its associations with measures of emotional and social functioning. The sample was comprised of 1379 Italian students (aged 8 to 12 years) who attended schools interested in the study aims. Participants completed the 30-item HIF scale, and measures of emotional self-efficacy and social desirability (SD). Factor structures were assessed using confirmatory factor analysis. Invariance by age and gender, internal consistency, temporal stability, and concurrent validity were also tested. A 3-factor model was identified: frequency and intensity of (1) positive emotion--PE (8 items, α = .82), (2) negative emotion--NE (12 items, α = .86), and (3) positive and negative emotion control--EC (10 items, α = .77). This factor structure was invariant across age and gender groups. The HIF displayed moderate longitudinal stability over a 15-month period and a low social desirability effect. Positive emotion was positively associated with social acceptance and visibility, and negatively with social withdrawal. The HIF is a reliable and valid measure for research and school intervention promoting students' emotional and mental health. © 2016, American School Health Association.

Structures and mechanisms of antitumor agents: xestoquinones uncouple cellular respiration and disrupt HIF signaling in human breast tumor cells.

PubMed

Du, Lin; Mahdi, Fakhri; Datta, Sandipan; Jekabsons, Mika B; Zhou, Yu-Dong; Nagle, Dale G

2012-09-28

The organic extract of a marine sponge, Petrosia alfiani, selectively inhibited iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in a human breast tumor T47D cell-based reporter assay. Bioassay-guided fractionation yielded seven xestoquinones (1-7) including three new compounds: 14-hydroxymethylxestoquinone (1), 15-hydroxymethylxestoquinone (2), and 14,15-dihydroxestoquinone (3). Compounds 1-7 were evaluated for their effects on HIF-1 signaling, mitochondrial respiration, and tumor cell proliferation/viability. The known metabolites adociaquinones A (5) and B (6), which possess a 3,4-dihydro-2H-1,4-thiazine-1,1-dioxide moiety, potently and selectively inhibited iron chelator-induced HIF-1 activation in T47D cells, each with an IC(50) value of 0.2 μM. Mechanistic studies revealed that adociaquinones promote oxygen consumption without affecting mitochondrial membrane potential. Compound 1 both enhances respiration and decreases mitochondrial membrane potential, suggesting that it acts as a protonophore that uncouples mitochondrial respiration.

Novel mechanism of cardiac protection by valsartan: synergetic roles of TGF-β1 and HIF-1α in Ang II-mediated fibrosis after myocardial infarction

PubMed Central

Sui, Xizhong; Wei, Hongchao; Wang, Dacheng

2015-01-01

Transforming growth factor (TGF)-β1 is a known factor in angiotensin II (Ang II)-mediated cardiac fibrosis after myocardial infarction (MI). Hypoxia inducible factor-1 (Hif-1α) was recently demonstrated to involve in the tissue fibrosis and influenced by Ang II. However, whether Hif-1α contributed to the Ang II-mediated cardiac fibrosis after MI, and whether interaction or synergetic roles between Hif-1α and TGF-β pathways existed in the process was unclear. In vitro, cardiac cells were incubated under hypoxia or Ang II to mimic ischaemia. In vivo, valsartan was intravenously injected into Sprague–Dawley rats with MI daily for 1 week; saline and hydralazine (another anti-hypertensive agent like valsartan) was used as control. The fibrosis-related proteins were detected by Western blotting. Cardiac structure and function were assessed with multimodality methods. We demonstrated in vitro that hypoxia would induce the up-regulation of Ang II, TGF-β/Smad and Hif-1α, which further induced collagen accumulation. By blocking with valsartan, a blocker of Ang II type I (AT1) receptor, we confirmed that the up-regulation of TGF-β/Smad and Hif-1α was through the Ang II-mediated pathway. By administering TGF-β or dimethyloxalylglycine, we determined that both TGF-β/Smad and Hif-1α contributed to Ang II-mediated collagen accumulation and a synergetic effect between them was observed. Consistent with in vitro results, valsartan significantly attenuated the expression of TGF-β/Smad, Hif-1α and fibrosis-related protein in rats after MI. Heart function, infarcted size, wall thickness as well as myocardial vascularization of ischaemic hearts were also significantly improved by valsartan compared with saline and hydralazine. Our study may provide novel insights into the mechanisms of Ang II-induced cardiac fibrosis as well as into the cardiac protection of valsartan. PMID:25823960

Evaluation of Hypoxia Inducible Factor-1α and Glucose Transporter-1 Expression in Non Melanoma Skin Cancer: An Immunohistochemical Study

PubMed Central

Seleit, Iman; Bakry, Ola Ahmed; Ragab, Rania Abdel Aziz; Al-Shiemy, shimaa Ahmed

2017-01-01

Introduction Hypoxia Inducible Factor-1 (HIF-1) is a mediator enabling cell adaptation to hypoxia. It plays its role mainly through transcription of many target genes including Glucose Transporter-1 (GLUT-1) gene. Aim The present work aimed at evaluating the pattern and distribution of HIF-1α and GLUT-1 in each case and control. Materials and Methods A case-control and retrospective study was conducted on archival blocks diagnosed from pathology department as, Basal Cell Carcinoma (BCC, 20 cases), cutaneous Squamous Cell Carcinoma (SCC, 20 cases) and 20 normal site-matched skin biopsies from age and gender-matched healthy subjects as a control. Evaluation of both HIF-1α and GLUT1 expression using standard immunohistochemical techniques was performed on cut sections from selected paraffin embedded blocks. Results HIF-1α was expressed in 90%, 35% and 100% of normal skin, BCC and SCC tumour islands respectively. It was up regulated in both BCC and SCC compared with normal skin (p= 0.001, p<0.001 respectively). GLUT-1 was expressed in 100%, 70% and 100% of normal skin, BCC and SCC tumour islands respectively. It was down regulated in Non Melanoma Skin Cancer (NMSC) cases compared with normal skin (p=0.004). HIF-1α and GLUT-1 localization in tumour nests was central, peripheral or central and peripheral. Both HIF-1α and GLUT-1 showed variable expression in stroma, adnexa and inflammatory cells. No significant correlation was found between Histo (H) score or expression percentage values of HIF-1α and those of GLUT-1 in tumour islands or in overlying epidermis either in BCC or SCC. Conclusion HIF-1α may have a role in NMSC pathogenesis through adaptation to hypoxia which results from excessive proliferation. GLUT-1 down regulation in NMSC may be explained by its consumption by proliferating tumour cells. The expression of HIF-1α and GLUT-1 in normal epidermis, stromal and adnexal structures needs further research. PMID:28764171

Hypoxia-inducible factor-1β (HIF-1β) is upregulated in a HIF-1α-dependent manner in 518A2 human melanoma cells under hypoxic conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mandl, Markus, E-mail: mmandl@mail.austria.com; Kapeller, Barbara; Lieber, Roman

2013-04-26

Highlights: •HIF-1β is a hypoxia-responsive protein in 518A2 human melanoma cells. •HIF-1β is upregulated in a HIF-1α-dependent manner under hypoxic conditions. •HIF-1β is not elevated due to heterodimerization with HIF-1α per se. •HIF-1β inducibility has a biological relevance as judged in Het-CAM model. -- Abstract: Solid tumors include hypoxic areas due to excessive cell proliferation. Adaptation to low oxygen levels is mediated by the hypoxia-inducible factor (HIF) pathway promoting invasion, metastasis, metabolic alterations, chemo-resistance and angiogenesis. The transcription factor HIF-1, the major player within this pathway consists of HIF-1α and HIF-1β. The alpha subunit is continuously degraded under normoxia andmore » becomes stabilized under reduced oxygen supply. In contrast, HIF-1β is generally regarded as constitutively expressed and being present in excess within the cell. However, there is evidence that the expression of this subunit is more complex. The aim of this study was to investigate the role of HIF-1β in human melanoma cells. Among a panel of five different cell lines, in 518A2 cells exposed to the hypoxia-mimetic cobalt chloride HIF-1β was rapidly elevated on protein level. Knockdown experiments performed under cobalt chloride-exposure and hypoxia revealed that this effect was mediated by HIF-1α. The non-canonical relationship between these subunits was further confirmed by pharmacologic inhibition of HIF-1α and by expression of a dominant-negative HIF mutant. Overexpression of HIF-1α showed a time delay in HIF-1β induction, thus arguing for HIF-1β de novo synthesis rather than protein stabilization by heterodimerization. A Hen’s egg test-chorioallantoic membrane model of angiogenesis and invasion indicated a local expression of HIF-1β and implies a biological relevance of these findings. In summary, this study demonstrates the HIF-1α-dependent regulation of HIF-1β under hypoxic conditions for the first time. The results indicate a novel cell specific mechanism which might prevent HIF-1β to become a limiting factor.« less

Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development.

PubMed

Lange, Clemens A K; Luhmann, Ulrich F O; Mowat, Freya M; Georgiadis, Anastasios; West, Emma L; Abrahams, Sabu; Sayed, Haroon; Powner, Michael B; Fruttiger, Marcus; Smith, Alexander J; Sowden, Jane C; Maxwell, Patrick H; Ali, Robin R; Bainbridge, James W B

2012-07-01

Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.

High Energy Density Physics and Exotic Acceleration Schemes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cowan, T.; /General Atomics, San Diego; Colby, E.

2005-09-27

The High Energy Density and Exotic Acceleration working group took as our goal to reach beyond the community of plasma accelerator research with its applications to high energy physics, to promote exchange with other disciplines which are challenged by related and demanding beam physics issues. The scope of the group was to cover particle acceleration and beam transport that, unlike other groups at AAC, are not mediated by plasmas or by electromagnetic structures. At this Workshop, we saw an impressive advancement from years past in the area of Vacuum Acceleration, for example with the LEAP experiment at Stanford. And wemore » saw an influx of exciting new beam physics topics involving particle propagation inside of solid-density plasmas or at extremely high charge density, particularly in the areas of laser acceleration of ions, and extreme beams for fusion energy research, including Heavy-ion Inertial Fusion beam physics. One example of the importance and extreme nature of beam physics in HED research is the requirement in the Fast Ignitor scheme of inertial fusion to heat a compressed DT fusion pellet to keV temperatures by injection of laser-driven electron or ion beams of giga-Amp current. Even in modest experiments presently being performed on the laser-acceleration of ions from solids, mega-amp currents of MeV electrons must be transported through solid foils, requiring almost complete return current neutralization, and giving rise to a wide variety of beam-plasma instabilities. As keynote talks our group promoted Ion Acceleration (plenary talk by A. MacKinnon), which historically has grown out of inertial fusion research, and HIF Accelerator Research (invited talk by A. Friedman), which will require impressive advancements in space-charge-limited ion beam physics and in understanding the generation and transport of neutralized ion beams. A unifying aspect of High Energy Density applications was the physics of particle beams inside of solids, which is proving to be a very important field for diverse applications such as muon cooling, fusion energy research, and ultra-bright particle and radiation generation with high intensity lasers. We had several talks on these and other subjects, and many joint sessions with the Computational group, the EM Structures group, and the Beam Generation group. We summarize our groups' work in the following categories: vacuum acceleration schemes; ion acceleration; particle transport in solids; and applications to high energy density phenomena.« less

Differential regulation of HIF-1α and HIF-2α in neuroblastoma: Estrogen-related receptor alpha (ERRα) regulates HIF2A transcription and correlates to poor outcome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamidian, Arash; Stedingk, Kristoffer von; Munksgaard Thorén, Matilda

2015-06-05

Hypoxia-inducible factors (HIFs) are differentially regulated in tumor cells. While the current paradigm supports post-translational regulation of the HIF-α subunits, we recently showed that hypoxic HIF-2α is also transcriptionally regulated via insulin-like growth factor (IGF)-II in the childhood tumor neuroblastoma. Here, we demonstrate that transcriptional regulation of HIF-2α seems to be restricted to neural cell-derived tumors, while HIF-1α is canonically regulated at the post-translational level uniformly across different tumor forms. Enhanced expression of HIF2A mRNA at hypoxia is due to de novo transcription rather than increased mRNA stability, and chemical stabilization of the HIF-α proteins at oxygen-rich conditions unexpectedly leadsmore » to increased HIF2A transcription. The enhanced HIF2A levels do not seem to be dependent on active HIF-1. Using a transcriptome array approach, we identified members of the Peroxisome proliferator-activated receptor gamma coactivator (PGC)/Estrogen-related receptor (ERR) complex families as potential regulators of HIF2A. Knockdown or inhibition of one of the members, ERRα, leads to decreased expression of HIF2A, and high expression of the ERRα gene ESRRA correlates with poor overall and progression-free survival in a clinical neuroblastoma material consisting of 88 tumors. Thus, targeting of ERRα and pathways regulating transcriptional HIF-2α are promising therapeutic avenues in neuroblastoma. - Highlights: • Transcriptional control of HIF-2α is restricted to neural cell-derived tumors. • Enhanced transcription of HIF2A is not due to increased mRNA stability. • Chemical stabilization of the HIF-α subunits leads to increased HIF2A transcription. • ERRα regulates HIF2A mRNA expression in neuroblastoma. • High expression of ESRRA correlates to poor outcome in neuroblastoma.« less

Mitochondrial Respiration Inhibitors Suppress Protein Translation and Hypoxic Signaling via the Hyperphosphorylation and Inactivation of Translation Initiation Factor eIF2α and Elongation Factor eEF2

PubMed Central

Li, Jun; Mahdi, Fakhri; Du, Lin; Datta, Sandipan; Nagle, Dale G.; Zhou, Yu-Dong

2011-01-01

Over 20000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 µM and 0.068 µM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods. PMID:21875114

Unraveling the role of hypoxia-inducible factor (HIF)-1α and HIF-2α in the adaption process of human microvascular endothelial cells (HMEC-1) to hypoxia: Redundant HIF-dependent regulation of macrophage migration inhibitory factor.

PubMed

Hahne, Martin; Schumann, Peggy; Mursell, Mathias; Strehl, Cindy; Hoff, Paula; Buttgereit, Frank; Gaber, Timo

2018-03-01

Hypoxia driven angiogenesis is a prominent feature of tissue regeneration, inflammation and tumor growth and is regulated by hypoxia-inducible factor (HIF)-1 and -2. The distinct functions of HIFs in the hypoxia-induced angiogenesis and metabolic switch of endothelial cells are still unknown and therefore aim of this study. We investigated the role of HIF-1 and -2 in the adaptation of immortalized human microvascular endothelial cells (HMEC-1) to hypoxic conditions (1% O 2 ) in terms of angiogenesis, cytokine secretion, gene expression and ATP/ADP-ratio using shRNA-mediated reduction of the oxygen sensitive α-subunits of either HIF-1 or HIF-2 or the combination of both. Reduction of HIF-1α diminished cellular energy, hypoxia-induced glycolytic gene expression, and angiogenesis not altering pro-angiogenic factors. Reduction of HIF-2α diminished hypoxia-induced pro-angiogenic factors, enhanced anti-angiogenic factors and attenuated angiogenesis not altering glycolytic gene expression. Reduction of both HIFs reduced cell survival, gene expression of glycolytic enzymes and pro-angiogenic factors as compared to the corresponding control. Finally, we identified the macrophage migration inhibitory factor (MIF) to be redundantly regulated by HIF-1 and HIF-2 and to be essential in the process of hypoxia-driven angiogenesis. Our results demonstrate a major impact of HIF-1 and HIF-2 on hypoxia-induced angiogenesis indicating distinct but also overlapping functions of HIF-1 and HIF-2. These findings open new possibilities for therapeutic approaches by specifically targeting the HIF-1 and HIF-2 or their target MIF. Copyright © 2017 Elsevier Inc. All rights reserved.

Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

PubMed

Sena, Johnny A; Wang, Liyi; Heasley, Lynn E; Hu, Cheng-Jun

2014-09-01

Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. ©2014 American Association for Cancer Research.

20(S)-Protopanaxadiol enhances angiogenesis via HIF-1α-mediated VEGF secretion by activating p70S6 kinase and benefits wound healing in genetically diabetic mice

PubMed Central

Zhang, Er-Yun; Gao, Bo; Shi, Hai-Lian; Huang, Ling-Fang; Yang, Li; Wu, Xiao-Jun; Wang, Zheng-Tao

2017-01-01

Impaired angiogenesis is one of the crucial factors that impede the wound healing process in diabetic foot ulcers (DFUs). In this study, we found that 20(S)-protopanaxadiol (PPD), an aglycone of ginsenosides in Panax notoginseng, stimulated angiogenesis and benefited wound healing in genetically diabetic mice. In HUVECs, PPD promoted cell proliferation, tube formation and VEGF secretion accompanied by increased nuclear translocalization of HIF-1α, which led to elevated VEGF mRNA expression. PPD activated both PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways in HUVECs, which were abrogated by LY294002 and PD98059. Furthermore, these two pathways had crosstalk through p70S6K, as LY294002, PD98059 and p70S6K siRNA abolished the angiogenic responses of PPD. In the excisional wound splinting model established in db/db diabetic mice, PPD (0.6, 6 and 60 mg ml−1) accelerated wound closure, which was reflected by a significantly reduced wound area and epithelial gaps, as well as elevated VEGF expression and capillary formation. In addition, PPD activated PI3K/Akt/ERK signaling pathways, as well as enhanced p70S6K activity and HIF-1α synthesis in the wounds. Overall, our results revealed that PPD stimulated angiogenesis via HIF-1α-mediated VEGF expression by activating p70S6K through PI3K/Akt/mTOR and Raf/MEK/ERK signaling cascades, which suggests that the compound has potential use in wound healing therapy in patients suffering from DFUs. PMID:29075038

The transition state structure for binding between TAZ1 of CBP and the disordered Hif-1α CAD.

PubMed

Lindström, Ida; Andersson, Eva; Dogan, Jakob

2018-05-18

Intrinsically disordered proteins (IDPs) are common in eukaryotes. However, relatively few experimental studies have addressed the nature of the rate-limiting transition state for the coupled binding and folding reactions involving IDPs. By using site-directed mutagenesis in combination with kinetics measurements we have here characterized the transition state for binding between the globular TAZ1 domain of CREB binding protein and the intrinsically disordered C-terminal activation domain of Hif-1α (Hif-1α CAD). A total of 17 Hif-1α CAD point-mutations were generated and a Φ-value binding analysis was carried out. We found that native hydrophobic binding interactions are not formed at the transition state. We also investigated the effect the biologically important Hif-1α CAD Asn-803 hydroxylation has on the binding kinetics, and found that the whole destabilization effect due the hydroxylation is within the dissociation rate constant. Thus, the rate-limiting transition state is "disordered-like", with native hydrophobic binding contacts being formed cooperatively after the rate-limiting barrier, which is clearly shown by linear free energy relationships. The same behavior was observed in a previously characterized TAZ1/IDP interaction, which may suggest common features for the rate-limiting transition state for TAZ1/IDP interactions.

Hypoxia-inducible factor stabilizers and other small-molecule erythropoiesis-stimulating agents in current and preventive doping analysis.

PubMed

Beuck, Simon; Schänzer, Wilhelm; Thevis, Mario

2012-11-01

Increasing the blood's capacity for oxygen transport by erythropoiesis-stimulating agents (ESAs) constitutes a prohibited procedure of performance enhancement according to the World Anti-Doping Agency (WADA). The advent of orally bio-available small-molecule ESAs such as hypoxia-inducible factor (HIF) stabilizers in the development of novel anti-anaemia therapies expands the list of potential ESA doping techniques. Here, the erythropoiesis-stimulating properties and doping relevance of experimental HIF-stabilizers, such as cobaltous chloride, 3,4-dihydroxybenzoic acid or GSK360A, amongst others, are discussed. The stage of clinical trials is reviewed for the anti-anaemia drug candidates FG-2216, FG-4592, GSK1278863, AKB-6548, and BAY85-3934. Currently available methods and strategies for the determination of selected HIF stabilizers in sports drug testing are based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). For the support of further analytical assay development, patents claiming distinct compounds for the use in HIF-mediated therapies are evaluated and exemplary molecular structures of HIF stabilizers presented. Moreover, data concerning the erythropoiesis-enhancing effects of the GATA inhibitors K7174 and K11706 as well as the lipidic small-molecule ESA PBI-1402 are elucidated the context of doping analysis. Copyright © 2012 John Wiley & Sons, Ltd.

The protective effect of Hif3a RNA interference and HIF-prolyl hydroxylase inhibition on cardiomyocytes under anoxia-reoxygenation.

PubMed

Drevytska, T; Gonchar, E; Okhai, I; Lynnyk, O; Mankovska, I; Klionsky, D; Dosenko, V

2018-06-01

The aim of this study was to investigate the molecular mechanisms underlying the protective effects of hypoxia-inducible factor (HIF) signaling pathway activation in cardiomyocytes under anoxia-reoxygenation (A/R) injury. In this study, rat neonatal cardiomyocytes were pretreated with anti-Hif3A/Hif-3α siRNA or HIF-prolyl hydroxylase inhibitor prior to A/R injury. Our results showed that both HIF3A silencing and HIF-prolyl hydroxylase inhibition effectively increased the cell viability during A/R, led to changes in mRNA expression of HIF1-target genes, and reduced the loss of mitochondrial membrane potential (Δψ m ). Furthermore, application of anti-Hif3a siRNA led to an increase in mRNA expression of Epo, Igf1, Slc2a1/Glut-1, and Slc2a4/Glut-4. Similar results were observed with HIF-prolyl hydroxylase inhibition, which additionally upregulated the mRNA expression of Epor, Tert, and Pdk1. Hif3a RNA-interference and application of HIF-prolyl hydroxylase inhibitor during A/R modelling led to an increase of Δψ m on 11.5 and 11.9 mV respectively, compared to the control groups. Thus, Hif3a RNA interference and HIF-prolyl hydroxylase inhibition protect cardiomyocytes against A/R injury via the HIF signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

Contribution of HIF-1alpha or HIF-2alpha to erythropoietin expression: in vivo evidence based on chromatin immunoprecipitation.

PubMed

Yeo, Eun-Jin; Cho, Young-Suk; Kim, Myung-Suk; Park, Jong-Wan

2008-01-01

Circulating erythropoietin (EPO) is mainly produced by the kidneys and mediates erythrogenesis in bone marrow and nonhematopoietic cell survival. EPO is also produced in other tissues where it functions as a paracrine. Moreover, the hypoxic induction of EPO is known to be mediated by HIF-1alpha and HIF-2alpha, but it remains obscure as to which of these two mediators mainly contributes to EPO expression. Thus, we designed in vivo experiments to evaluate the contributions made by HIF-1alpha and HIF-2alpha to EPO expression. In mice exposed to mild whole body hypoxia, HIF-1alpha and HIF-2alpha were both induced in all tissues examined. However, EPO mRNA was expressed in kidney and brain, but not in liver and lung. Likewise, chromatin immunoprecipitation (CHIP) analyses demonstrated that HIF-1alpha or HIF-2alpha binding to the EPO gene increased under hypoxic conditions only in kidney and brain. A comparison of CHIP data and EPO mRNA levels suggested that, during mild hypoxia, renal EPO transcription is induced equally by HIF-1alpha and HIF-2alpha, but that brain EPO is mainly induced during hypoxia by HIF-2alpha. Thus, HIF-1alpha and HIF-2alpha appear to contribute to EPO expression tissue specifically.

Adult hematopoietic stem cells lacking Hif-1α self-renew normally.

PubMed

Vukovic, Milica; Sepulveda, Catarina; Subramani, Chithra; Guitart, Amélie V; Mohr, Jasmine; Allen, Lewis; Panagopoulou, Theano I; Paris, Jasmin; Lawson, Hannah; Villacreces, Arnaud; Armesilla-Diaz, Alejandro; Gezer, Deniz; Holyoake, Tessa L; Ratcliffe, Peter J; Kranc, Kamil R

2016-06-09

The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed β subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance. © 2016 by The American Society of Hematology.

Adult hematopoietic stem cells lacking Hif-1α self-renew normally

PubMed Central

Vukovic, Milica; Sepulveda, Catarina; Subramani, Chithra; Guitart, Amélie V.; Mohr, Jasmine; Allen, Lewis; Panagopoulou, Theano I.; Paris, Jasmin; Lawson, Hannah; Villacreces, Arnaud; Armesilla-Diaz, Alejandro; Gezer, Deniz; Holyoake, Tessa L.; Ratcliffe, Peter J.

2016-01-01

The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed β subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α–deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α–deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance. PMID:27060169

Hypoxic preconditioning protects photoreceptors against light damage independently of hypoxia inducible transcription factors in rods.

PubMed

Kast, Brigitte; Schori, Christian; Grimm, Christian

2016-05-01

Hypoxic preconditioning protects photoreceptors against light-induced degeneration preserving retinal morphology and function. Although hypoxia inducible transcription factors 1 and 2 (HIF1, HIF2) are the main regulators of the hypoxic response, photoreceptor protection does not depend on HIF1 in rods. Here we used rod-specific Hif2a single and Hif1a;Hif2a double knockout mice to investigate the potential involvement of HIF2 in rods for protection after hypoxic preconditioning. To identify potential HIF2 target genes in rods we determined the retinal transcriptome of hypoxic control and rod-specific Hif2a knockouts by RNA sequencing. We show that rods do not need HIF2 for hypoxia-induced increased survival after light exposure. The transcriptomic analysis revealed a number of genes that are potentially regulated by HIF2 in rods; among those were Htra1, Timp3 and Hmox1, candidates that are interesting due to their connection to human degenerative diseases of the retina. We conclude that neither HIF1 nor HIF2 are required in photoreceptors for protection by hypoxic preconditioning. We hypothesize that HIF transcription factors may be needed in other cells to produce protective factors acting in a paracrine fashion on photoreceptor cells. Alternatively, hypoxic preconditioning induces a rod-intrinsic response that is independent of HIF transcription factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evolutionary Conserved Regulation of HIF-1β by NF-κB

PubMed Central

van Uden, Patrick; Kenneth, Niall S.; Webster, Ryan; Müller, H. Arno; Mudie, Sharon; Rocha, Sonia

2011-01-01

Hypoxia Inducible Factor-1 (HIF-1) is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs) and Factor Inhibiting HIF (FIH) respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1β mRNA and protein. In addition, we found that NF-κB–mediated changes in HIF-1β result in modulation of HIF-2α protein. HIF-1β overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1β (tango) and HIF-α (sima) levels and activity (Hph/fatiga, ImpL3/ldha) in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF–related pathologies including ageing, ischemia, and cancer. PMID:21298084

Purifying Properly Folded Cysteine-rich, Zinc Finger Containing Recombinant Proteins for Structural Drug Targeting Studies: the CH1 Domain of p300 as a Case Example

PubMed Central

Kim, Yong Joon; Kaluz, Stefan; Mehta, Anil; Weinert, Emily; Rivera, Shannon; Van Meir, Erwin G.

2017-01-01

The transcription factor Hypoxia-Inducible Factor (HIF) complexes with the coactivator p300, activating the hypoxia response pathway and allowing tumors to grow. The CH1 and CAD domains of each respective protein form the interface between p300 and HIF. Small molecule compounds are in development that target and inhibit HIF/p300 complex formation, with the goal of reducing tumor growth. High resolution NMR spectroscopy is necessary to study ligand interaction with p300-CH1, and purifying high quantities of properly folded p300-CH1 is needed for pursuing structural and biophysical studies. p300-CH1 has 3 zinc fingers and 9 cysteine residues, posing challenges associated with reagent compatibility and protein oxidation. A protocol has been developed to overcome such issues by incorporating zinc during expression and streamlining the purification time, resulting in a high yield of optimally folded protein (120 mg per 4 L expression media) that is suitable for structural NMR studies. The structural integrity of the final recombinant p300-CH1 has been verified to be optimal using onedimensional 1H NMR spectroscopy and circular dichroism. This protocol is applicable for the purification of other zinc finger containing proteins. PMID:28966947

Hypoxia Increases Sirtuin 1 Expression in a Hypoxia-inducible Factor-dependent Manner*

PubMed Central

Chen, Rui; Dioum, Elhadji M.; Hogg, Richard T.; Gerard, Robert D.; Garcia, Joseph A.

2011-01-01

Hypoxia-inducible factors (HIFs) are stress-responsive transcriptional regulators of cellular and physiological processes involved in oxygen metabolism. Although much is understood about the molecular machinery that confers HIF responsiveness to oxygen, far less is known about HIF isoform-specific mechanisms of regulation, despite the fact that HIF-1 and HIF-2 exhibit distinct biological roles. We recently determined that the stress-responsive genetic regulator sirtuin 1 (Sirt1) selectively augments HIF-2 signaling during hypoxia. However, the mechanism by which Sirt1 maintains activity during hypoxia is unknown. In this report, we demonstrate that Sirt1 gene expression increases in a HIF-dependent manner during hypoxia in Hep3B and in HT1080 cells. Impairment of HIF signaling affects Sirt1 deacetylase activity as decreased HIF-1 signaling results in the appearance of acetylated HIF-2α, which is detected without pharmacological inhibition of Sirt1. We also find that Sirt1 augments HIF-2 mediated, but not HIF-1 mediated, transcriptional activation of the isolated Sirt1 promoter. These data in summary reveal a bidirectional link of HIF and Sirt1 signaling during hypoxia. PMID:21345792

The acetate/ACSS2 switch regulates HIF-2 stress signaling in the tumor cell microenvironment.

PubMed

Chen, Rui; Xu, Min; Nagati, Jason S; Hogg, Richard T; Das, Alok; Gerard, Robert D; Garcia, Joseph A

2015-01-01

Optimal stress signaling by Hypoxia Inducible Factor 2 (HIF-2) during low oxygen states or hypoxia requires coupled actions of a specific coactivator/lysine acetyltransferase, Creb binding protein (CBP), and a specific deacetylase, Sirtuin 1 (SIRT1). We recently reported that acetylation of HIF-2 by CBP also requires a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (ACSS2). In this study, we demonstrate that ACSS2/HIF-2 signaling is active not only during hypoxia, but also during glucose deprivation. Acetate levels increase during stress and coincide with maximal HIF-2α acetylation and CBP/HIF-2α complex formation. Exogenous acetate induces HIF-2α acetylation, CBP/HIF-2α complex formation, and HIF-2 signaling. ACSS2 and HIF-2 are required for maximal colony formation, proliferation, migration, and invasion during stress. Acetate also stimulates flank tumor growth and metastasis in mice in an ACSS2 and HIF-2 dependent manner. Thus, ACSS2/CBP/SIRT1/HIF-2 signaling links nutrient sensing and stress signaling with cancer growth and progression in mammals.

The Acetate/ACSS2 Switch Regulates HIF-2 Stress Signaling in the Tumor Cell Microenvironment

PubMed Central

Chen, Rui; Xu, Min; Nagati, Jason S.; Hogg, Richard T.; Das, Alok; Gerard, Robert D.; Garcia, Joseph A.

2015-01-01

Optimal stress signaling by Hypoxia Inducible Factor 2 (HIF-2) during low oxygen states or hypoxia requires coupled actions of a specific coactivator/lysine acetyltransferase, Creb binding protein (CBP), and a specific deacetylase, Sirtuin 1 (SIRT1). We recently reported that acetylation of HIF-2 by CBP also requires a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (ACSS2). In this study, we demonstrate that ACSS2/HIF-2 signaling is active not only during hypoxia, but also during glucose deprivation. Acetate levels increase during stress and coincide with maximal HIF-2α acetylation and CBP/HIF-2α complex formation. Exogenous acetate induces HIF-2α acetylation, CBP/HIF-2α complex formation, and HIF-2 signaling. ACSS2 and HIF-2 are required for maximal colony formation, proliferation, migration, and invasion during stress. Acetate also stimulates flank tumor growth and metastasis in mice in an ACSS2 and HIF-2 dependent manner. Thus, ACSS2/CBP/SIRT1/HIF-2 signaling links nutrient sensing and stress signaling with cancer growth and progression in mammals. PMID:25689462

HIF-1α and VEGF expression correlates with thrombus remodeling in cases of intravascular papillary endothelial hyperplasia

PubMed Central

Kim, Sunzoo; Jun, Jae Hun; Kim, Jeongshik; Kim, Do Won; Jang, Yong Hyun; Lee, Weon Ju; Chung, Ho Yun; Lee, Seok-Jong

2013-01-01

Intravascular papillary endothelial hyperplasia (IPEH) is histopathologically characterized by endothelium-lined papillary structures encircling an acellular fibrin core. The process of IPEH pathogenesis is unclear. The purpose of our study was to identify histopathological and immunohistochemical characteristics of IPEH to better understand the pathogenesis of this disease. After reviewing microscopic and medical records from Kyungpook National University Hospital, we selected 16 cases of IPEH. Masson’s trichrome and immunohistochemical staining as well as hematoxylin-eosin staining for 16 cases of IPH were performed. Immunohistochemical studies included CD31, CD68, mast cell tryptase, hypoxia-inducible factor-1 (HIF-1α), and vascular endothelial growth factor (VEGF). Sections from all our cases showed three distinct histological regions including a papillary portion with hyalinized fibrous or fibroblastic cores, an area containing an unorganized thrombus, and organization area with an ingrowth of endothelial cells, myofibroblasts, and fibroblasts. In the organization area, HIF-1α-positive cells were identified in the loose connective tissue. Endothelial cells forming vascular channels were negative for HIF-1α while VEGF was highly expressed in both interstitial mononuclear and endothelial cells. In the papillary portion, the cellular cores were strongly positive for both HIF-1α and VEGF, but the acellular cores were negative. Our investigation confirmed that IPEH is a reactive lesion that incidentally arises during the organization process of older thrombi. It was also found that HIF-1α and VEGF expression was dependent on the thrombus remodeling stage in cases of IPEH. PMID:24294378

HIF-1-driven skeletal muscle adaptations to chronic hypoxia: molecular insights into muscle physiology.

PubMed

Favier, F B; Britto, F A; Freyssenet, D G; Bigard, X A; Benoit, H

2015-12-01

Skeletal muscle is a metabolically active tissue and the major body protein reservoir. Drop in ambient oxygen pressure likely results in a decrease in muscle cells oxygenation, reactive oxygen species (ROS) overproduction and stabilization of the oxygen-sensitive hypoxia-inducible factor (HIF)-1α. However, skeletal muscle seems to be quite resistant to hypoxia compared to other organs, probably because it is accustomed to hypoxic episodes during physical exercise. Few studies have observed HIF-1α accumulation in skeletal muscle during ambient hypoxia probably because of its transient stabilization. Nevertheless, skeletal muscle presents adaptations to hypoxia that fit with HIF-1 activation, although the exact contribution of HIF-2, I kappa B kinase and activating transcription factors, all potentially activated by hypoxia, needs to be determined. Metabolic alterations result in the inhibition of fatty acid oxidation, while activation of anaerobic glycolysis is less evident. Hypoxia causes mitochondrial remodeling and enhanced mitophagy that ultimately lead to a decrease in ROS production, and this acclimatization in turn contributes to HIF-1α destabilization. Likewise, hypoxia has structural consequences with muscle fiber atrophy due to mTOR-dependent inhibition of protein synthesis and transient activation of proteolysis. The decrease in muscle fiber area improves oxygen diffusion into muscle cells, while inhibition of protein synthesis, an ATP-consuming process, and reduction in muscle mass decreases energy demand. Amino acids released from muscle cells may also have protective and metabolic effects. Collectively, these results demonstrate that skeletal muscle copes with the energetic challenge imposed by O2 rarefaction via metabolic optimization.

Vascular Endothelial Growth Factor-dependent Spinogenesis Underlies Antidepressant-like Effects of Enriched Environment*

PubMed Central

Huang, Yu-Fei; Yang, Chih-Hao; Huang, Chiung-Chun; Hsu, Kuei-Sen

2012-01-01

Current antidepressant treatments remain limited by poor efficacy and a slow onset of action. Increasing evidence demonstrates that enriched environment (EE) treatment can promote structural and behavioral plasticity in the brain and dampen stress-induced alterations of neuroplasticity. Here, we have examined whether short term exposure to EE is able to produce antidepressant-like effects. Our results show that housing adult mice in an EE cage for 7 days led to antidepressant-like behavioral profiles and a significant increase in the number of dendritic spines in hippocampal CA1 pyramidal neurons. These EE-induced antidepressant-like effects are primarily attributed to increased vascular endothelial growth factor (VEGF) expression through a hypoxia-inducible factor-1α (HIF-1α)-mediated transcriptional mechanism. Blockade of HIF-1α synthesis by lentiviral infection with HIF-1α small hairpin RNAs completely blocked the increase in expression of VEGF and the antidepressant-like effects induced by EE. Moreover, no significant antidepressant-like effects were observed with EE treatment in VEGF receptor 2 (Flk-1) knock-out mice. The increase in HIF-1α expression in the hippocampus induced by EE was associated with a decrease in endogenous levels of microRNA-107 (miR-107). Overexpression of miR-107 in the hippocampus completely blocked EE-induced HIF-1α expression and the antidepressant-like effects. These results support a model in which the down-regulation of miR-107, acting through HIF-1α, mediates VEGF-dependent spinogenesis to underlie the EE-induced antidepressant-like effects. PMID:23074224

Discovery of novel inhibitors disrupting HIF-1α/von Hippel–Lindau interaction through shape-based screening and cascade docking

PubMed Central

Xue, Xin; Zhao, Ning-Yi; Yu, Hai-Tao; Sun, Yuan; Kang, Chen; Huang, Qiong-Bin; Sun, Hao-Peng

2016-01-01

Major research efforts have been devoted to the discovery and development of new chemical entities that could inhibit the protein–protein interaction between HIF-1α and the von Hippel–Lindau protein (pVHL), which serves as the substrate recognition subunit of an E3 ligase and is regarded as a crucial drug target in cancer, chronic anemia, and ischemia. Currently there is only one class of compounds available to interdict the HIF-1α/pVHL interaction, urging the need to discover chemical inhibitors with more diversified structures. We report here a strategy combining shape-based virtual screening and cascade docking to identify new chemical scaffolds for the designing of novel inhibitors. Based on this strategy, nine active hits have been identified and the most active hit, 9 (ZINC13466751), showed comparable activity to pVHL with an IC50 of 2.0 ± 0.14 µM, showing the great potential of utilizing these compounds for further optimization and serving as drug candidates for the inhibition of HIF-1α/von Hippel–Lindau interaction. PMID:27994971

Amniotic fluid HIF1α and exosomal HIF1α in cervical insufficiency patients with physical examination-indicated cerclage.

PubMed

Song, Ji Eun; Park, Seok Ju; Lee, Keun Young; Lee, Wang Jae

2018-02-15

Hypoxia inducible factor 1α (HIF1α) has been reported to activate inflammatory cascade. Recently, exosomes have been known to have pivotal roles in intercellular communication. The aim of this study was to compare the concentration of amniotic fluid (AF) HIF1α, exosomal HIF1α, and inflammatory cytokines such as interleukin 1α (IL1α), interleukin 1β (IL1β), interleukin 6 (IL6), and tumor necrosis factor α (TNFα) between physical examination-indicated cerclage (PEIC) and control group. We also investigated the associations between biomarkers and amniocentesis-to-delivery interval and the correlations of inflammatory cytokines, HIF1α, and exosomal HIF1α. Case-control study was performed. Cases are defined as 16 patients who underwent PEIC and controls are 19 women who underwent amniocentesis for confirming chromosomal abnormalities. The concentration of IL1α, IL1β, IL6, TNFα, HIF1α, and exosomal HIF1α were measured using enzyme-linked immunosorbent assay (ELISA). Exosomes were confirmed by tumor susceptibility Gene 101 (TSG 101) and transmission electron microscopy (TEM). The mean HIF1α in PEIC group was higher than control group (PEIC, 15.03 ± 9.60-pg/mL versus control, 2.96 ± 1.99 pg/mL; p < .01). There were significant differences in inflammatory cytokines between two groups. A significant difference in exosomal HIF1α was shown between two groups (PEIC, 27.97 ± 28.61-µg/mL versus control, 12.42 ± 8.20 µg/mL; p < .01). HIF1α, IL1α, IL6, TNFα, and exosomal HIF1α showed significantly negative association with cerclage-to-delivery interval. However, IL1β was not associated with cerclage-to-delivery interval. HIF1α was positively correlated with exosomal HIF1α (rho = 0.93, p < .01). Both HIF1α and exosomal HIF1α were significantly associated with TNFα (rho = 0.94, p < .01; rho = 0.97, p < .01). Both HIF-1α and exosomal HIF1α had positive correlation with IL1α (rho = 0.96, p < .01; rho = 0.91, p < .01). However, IL1β showed no correlations with HIF1α and exosomal HIF1α. A positive correlation between HIF-1α and IL6 was observed (rho = 0.58, p = .01.) Exosomal HIF1α also had correlation with IL6 (rho = 0.52, p = .03). This study demonstrated that amniotic fluid (AF) HIF1α and AF exosomal HIF1α were higher in physical examination-indicated cerclage (PEIC) group than control group. AF HIF1α and AF exosomal HIF1α were associated with shorter amniocentesis-to-delivery interval. More importantly, they had positive correlations with AF inflammatory cytokines such as IL1α, IL6, and TNFα. Our results may indicate that AF HIF1α and AF exosomes interact with AF inflammatory cytokines and contribute inflammatory cascade in complicated pregnancies.

Oxygen sensing in intestinal mucosal inflammation.

PubMed

Flück, Katharina; Fandrey, Joachim

2016-01-01

Hypoxia is a hallmark of chronically inflamed tissue. Hypoxia develops from vascular dysfunction and increased oxygen consumption by infiltrating leukocytes. With respect to inflammatory bowel disease (IBD), hypoxia is likely to be of particular importance: Impairment of the intestinal barrier during IBD allows anoxia from the lumen of the gut to spread to formerly normoxic tissue. In addition, disturbed perfusion of inflamed tissue and a higher oxygen demand of infiltrating immune cells lead to low oxygen levels in inflamed mucosal tissue. Here, cells become hypoxic and must now adapt to this condition. The hypoxia inducible factor (HIF)-1 complex is a key transcription factor for cellular adaption to low oxygen tension. HIF-1 is a heterodimer formed by two subunits: HIF-α (either HIF-1α or HIF-2α) and HIF-1β. Under normoxic conditions, hydroxylation of the HIF-α subunit by specific oxygen-dependent prolyl hydroxylases (PHDs) leads to ubiquitin proteasome-dependent degradation. Under hypoxic conditions, however, PHD activity is inhibited; thus, HIF-α can translocate into the nucleus, dimerize with HIF-1β, and bind to hypoxia-responsive elements of HIF-1 target genes. So far, most studies have addressed the function of HIF-1α in intestinal epithelial cells and the effect of HIF stabilization by PHD inhibitors in murine models of colitis. Furthermore, the role of HIF-1α in immune cells becomes more and more important as T cells or dendritic cells for which HIF-1 is of critical importance are highly involved in the pathogenesis of IBD. This review will summarize the function of HIF-1α and the therapeutic prospects for targeting the HIF pathway in intestinal mucosal inflammation.

Intermittent Induction of HIF-1α Produces Lasting Effects on Malignant Progression Independent of Its Continued Expression

PubMed Central

Choi, Hyunsung; Gillespie, David L.; Berg, Shauna; Rice, Christopher; Couldwell, Sandrine; Gu, Jie; Colman, Howard; Jensen, Randy L.; Huang, L. Eric

2015-01-01

Dysregulation of hypoxia-inducible transcription factors HIF-1α and HIF-2α correlates with poor prognosis in human cancers; yet, divergent and sometimes opposing activities of these factors in cancer biology have been observed. Adding to this complexity is that HIF-1α apparently possesses tumor-suppressing activities, as indicated by the loss-of-function mutations or even homozygous deletion of HIF1A in certain human cancers. As a step towards understanding this complexity, we employed 8-week intermittent induction of a stable HIF-1α variant, HIF1α(PP), in various cancer cell lines and examined the effects on malignant progression in xenografts of immunocompromised mice in comparison to those of HIF2α(PP). Although 8-week treatment led to eventual loss of HIF1α(PP) expression, treated osteosarcoma U-2 OS cells acquired tumorigenicity in the subcutaneous tissue. Furthermore, the prior treatment resulted in widespread invasion of malignant glioma U-87 MG cells in the mouse brain and sustained growth of U-118 MG glioma cells. The lasting effects of HIF-1α on malignant progression are specific because neither HIF2α(PP) nor β-galactosidase yielded similar effects. By contrast, transient expression of HIF1α(PP) in U-87 MG cells or constitutive expression of HIF1α(PP) but not HIF2α(PP) in a patient-derived glioma sphere culture inhibited tumor growth and spread. Our results indicate that intermittent induction of HIF-1α produces lasting effects on malignant progression even at its own expense. PMID:25893706

Novel mechanism of cardiac protection by valsartan: synergetic roles of TGF-β1 and HIF-1α in Ang II-mediated fibrosis after myocardial infarction.

PubMed

Sui, Xizhong; Wei, Hongchao; Wang, Dacheng

2015-08-01

Transforming growth factor (TGF)-β1 is a known factor in angiotensin II (Ang II)-mediated cardiac fibrosis after myocardial infarction (MI). Hypoxia inducible factor-1 (Hif-1α) was recently demonstrated to involve in the tissue fibrosis and influenced by Ang II. However, whether Hif-1α contributed to the Ang II-mediated cardiac fibrosis after MI, and whether interaction or synergetic roles between Hif-1α and TGF-β pathways existed in the process was unclear. In vitro, cardiac cells were incubated under hypoxia or Ang II to mimic ischaemia. In vivo, valsartan was intravenously injected into Sprague-Dawley rats with MI daily for 1 week; saline and hydralazine (another anti-hypertensive agent like valsartan) was used as control. The fibrosis-related proteins were detected by Western blotting. Cardiac structure and function were assessed with multimodality methods. We demonstrated in vitro that hypoxia would induce the up-regulation of Ang II, TGF-β/Smad and Hif-1α, which further induced collagen accumulation. By blocking with valsartan, a blocker of Ang II type I (AT1) receptor, we confirmed that the up-regulation of TGF-β/Smad and Hif-1α was through the Ang II-mediated pathway. By administering TGF-β or dimethyloxalylglycine, we determined that both TGF-β/Smad and Hif-1α contributed to Ang II-mediated collagen accumulation and a synergetic effect between them was observed. Consistent with in vitro results, valsartan significantly attenuated the expression of TGF-β/Smad, Hif-1α and fibrosis-related protein in rats after MI. Heart function, infarcted size, wall thickness as well as myocardial vascularization of ischaemic hearts were also significantly improved by valsartan compared with saline and hydralazine. Our study may provide novel insights into the mechanisms of Ang II-induced cardiac fibrosis as well as into the cardiac protection of valsartan. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

HIF1α in Tumorigenesis of Adenoid Cystic Carcinoma.

PubMed

Lim, Yun-Sung; Cha, Wonjae; Park, Min-Woo; Jeong, Woo-Jin; Ahn, Soon-Hyun

2017-02-01

Tumor hypoxia induces hypoxia-inducible factor-1α (HIF1α), which can influence tumorigenesis and metastasis. We evaluated the expression of HIF1α and the effect of HIF1α inhibitors in adenoid cystic carcinoma (ACC). HIF1α expression was demonstrated in ACC cell lines (ACC2 and ACCM). The effect of HIF1α inhibitors was evaluated. A systemic metastasis model was developed. The number of metastatic pulmonary nodules were analyzed. The ACCM cell line demonstrated greater HIF1α expression and invasion than ACC2. The expression of HIF1α and invasion of ACC cells were blocked by HIF1α siRNA. HIF1α inhibitors 17-N-allylamino-17-demethoxygeldanamycin (17AAG) and echinomycin inhibited cell invasion. 17AAG inhibited metastasis in the animal model, although not statistically significantly. HIF1α siRNA and 17AAG and echinomycin blocked invasion by ACC2 and ACCM cells. 17AAG exhibited therapeutic potential for inhibition of metastasis. Our results provide positive evidence that HIF1α is a promising research pathway for therapy of ACC. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Emodin Decreases Hepatic Hypoxia-Inducible Factor-1[Formula: see text] by Inhibiting its Biosynthesis.

PubMed

Ma, Feifei; Hu, Lijuan; Yu, Ming; Wang, Feng

2016-01-01

Hypoxia-inducible factor-1 (HIF-1) is an [Formula: see text] dimeric transcription factor. Because HIF-1[Formula: see text] is instable with oxygen, HIF-1 is scarce in normal mammalian cells. However, HIF-1[Formula: see text] is expressed in pathological conditions such as cancer and obesity. Inhibiting HIF-1[Formula: see text] may be of therapeutic value for these pathologies. Here, we investigated whether emodin, derived from the herb of Rheum palmatum L, which is also known as Chinese rhubarb, and is native to China, regulates HIF-1[Formula: see text] expression. Male C57BL/6 mice without or with diet-induced obesity were treated with emodin for two weeks, while control mice were treated with vehicle. HIF-1[Formula: see text] expression was determined by Western blot. We found that emodin inhibited obesity-induced HIF-1[Formula: see text] expression in liver and skeletal muscle but did not regulate HIF-1[Formula: see text] expression in the kidneys or in intra-abdominal fat. In vitro, emodin inhibited HIF-1[Formula: see text] expression in human HepG2 hepatic cells and Y1 adrenocortical cells. Further, we investigated the mechanisms of HIF-1[Formula: see text] expression in emodin-treated HepG2 cells. First, we found that HIF-1[Formula: see text] had normal stability in the presence of emodin. Thus, emodin did not decrease HIF-1[Formula: see text] by stimulating its degradation. Importantly, emodin decreased the activity of the signaling pathways that led to HIF-1[Formula: see text] biosynthesis. Interestingly, emodin increased HIF-1[Formula: see text] mRNA in HepG2 cells. This may be a result of feedback in response to the emodin-induced decrease in the protein of HIF-1[Formula: see text]. In conclusion, emodin decreases hepatic HIF-1[Formula: see text] by inhibiting its biosynthesis.

Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.

PubMed

Proper, Steven P; Saini, Yogesh; Greenwood, Krista K; Bramble, Lori A; Downing, Nathaniel J; Harkema, Jack R; Lapres, John J

2014-02-01

Hard metal lung disease (HMLD) is an occupational lung disease specific to inhalation of cobalt-containing particles whose mechanism is largely unknown. Cobalt is a known hypoxia mimic and stabilizer of the alpha subunits of hypoxia-inducible factors (HIFs). Previous work revealed that though HIF1α contrib utes to cobalt toxicity in vitro, loss of HIF1α in the alveolar epithelial cells does not provide in vivo protection from cobalt-induced lung inflammation. HIF1α and HIF2α show unique tissue expression profiles, and HIF2α is known to be the predominant HIF mRNA isoform in the adult lung. Thus, if HIF2α activation by cobalt contributes to pathophysiology of HMLD, we hypothesized that loss of HIF2α in lung epithelium would provide protection from cobalt-induced inflammation. Mice with HIF2α-deficiency in Club and alveolar type II epithelial cells (ATIIs) (HIF2α(Δ/Δ)) were exposed to cobalt (60 µg/day) or saline using a subacute occupational exposure model. Bronchoalveolar lavage cellularity, cytokines, qRT-PCR, and histopathology were analyzed. Results show that loss of HIF2α leads to enhanced eosinophilic inflammation and increased goblet cell metaplasia. Additionally, control mice demonstrated a mild recovery from cobalt-induced lung injury compared with HIF2α(Δ/Δ) mice, suggesting a role for epithelial HIF2α in repair mechanisms. The expression of important cytokines, such as interleukin (IL)-5 and IL-10, displayed significant differences following cobalt exposure when HIF2α(Δ/Δ) and control mice were compared. In summary, our data suggest that although loss of HIF2α does not afford protection from cobalt-induced lung inflammation, epithelial HIF2α signaling does play an important role in modulating the inflammatory and repair response in the lung.

Hypervelocity Impact Test Facility: A gun for hire

NASA Technical Reports Server (NTRS)

Johnson, Calvin R.; Rose, M. F.; Hill, D. C.; Best, S.; Chaloupka, T.; Crawford, G.; Crumpler, M.; Stephens, B.

1994-01-01

An affordable technique has been developed to duplicate the types of impacts observed on spacecraft, including the Shuttle, by use of a certified Hypervelocity Impact Facility (HIF) which propels particulates using capacitor driven electric gun techniques. The fully operational facility provides a flux of particles in the 10-100 micron diameter range with a velocity distribution covering the space debris and interplanetary dust particle environment. HIF measurements of particle size, composition, impact angle and velocity distribution indicate that such parameters can be controlled in a specified, tailored test designed for or by the user. Unique diagnostics enable researchers to fully describe the impact for evaluating the 'targets' under full power or load. Users regularly evaluate space hardware, including solar cells, coatings, and materials, exposing selected portions of space-qualified items to a wide range of impact events and environmental conditions. Benefits include corroboration of data obtained from impact events, flight simulation of designs, accelerated aging of systems, and development of manufacturing techniques.

Host lignin composition affects haustorium induction in the parasitic plants Phtheirospermum japonicum and Striga hermonthica.

PubMed

Cui, Songkui; Wada, Syogo; Tobimatsu, Yuki; Takeda, Yuri; Saucet, Simon B; Takano, Toshiyuki; Umezawa, Toshiaki; Shirasu, Ken; Yoshida, Satoko

2018-04-01

Parasitic plants in the family Orobanchaceae are destructive weeds of agriculture worldwide. The haustorium, an essential parasitic organ used by these plants to penetrate host tissues, is induced by host-derived phenolic compounds called haustorium-inducing factors (HIFs). The origin of HIFs remains unknown, although the structures of lignin monomers resemble that of HIFs. Lignin is a natural phenylpropanoid polymer, commonly found in secondary cell walls of vascular plants. We therefore investigated the possibility that HIFs are derived from host lignin. Various lignin-related phenolics, quinones and lignin polymers, together with nonhost and host plants that have different lignin compositions, were tested for their haustorium-inducing activity in two Orobanchaceae species, a facultative parasite, Phtheirospermum japonicum, and an obligate parasite, Striga hermonthica. Lignin-related compounds induced haustoria in P. japonicum and S. hermonthica with different specificities. High concentrations of lignin polymers induced haustorium formation. Treatment with laccase, a lignin degradation enzyme, promoted haustorium formation at low concentrations. The distinct lignin compositions of the host and nonhost plants affected haustorium induction, correlating with the response of the different parasitic plants to specific types of lignin-related compounds. Our study provides valuable insights into the important roles of lignin biosynthesis and degradation in the production of HIFs. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Three-dimensional particle simulation of heavy-ion fusion beams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedman, A.; Grote, D.P.; Haber, I.

1992-07-01

The beams in a heavy-ion-beam-driven inertial fusion (HIF) accelerator are collisionless, nonneutral plasmas, confined by applied magnetic and electric fields. These space-charge-dominated beams must be focused onto small (few mm) spots at the fusion target, and so preservation of a small emittance is crucial. The nonlinear beam self-fields can lead to emittance growth, and so a self-consistent field description is needed. To this end, a multidimensional particle simulation code, WARP (Friedman {ital et} {ital al}., Part. Accel. {bold 37}-{bold 38}, 131 (1992)), has been developed and is being used to study the transport of HIF beams. The code's three-dimensional (3-D)more » package combines features of an accelerator code and a particle-in-cell plasma simulation. Novel techniques allow it to follow beams through many accelerator elements over long distances and around bends. This paper first outlines the algorithms employed in WARP. A number of applications and corresponding results are then presented. These applications include studies of: beam drift-compression in a misaligned lattice of quadrupole focusing magnets; beam equilibria, and the approach to equilibrium; and the MBE-4 experiment ({ital AIP} {ital Conference} {ital Proceedings} 152 (AIP, New York, 1986), p. 145) recently concluded at Lawrence Berkeley Laboratory (LBL). Finally, 3-D simulations of bent-beam dynamics relevant to the planned Induction Linac Systems Experiments (ILSE) (Fessenden, Nucl. Instrum. Methods Plasma Res. A {bold 278}, 13 (1989)) at LBL are described. Axially cold beams are observed to exhibit little or no root-mean-square emittance growth at midpulse in transiting a (sharp) bend. Axially hot beams, in contrast, do exhibit some emittance growth.« less

Molecular characterization of hypoxia and hypoxia-inducible factor 1 alpha (HIF-1α) from Taiwan voles (Microtus kikuchii).

PubMed

Jiang, Yi-Fan; Chou, Chung-Hsi; Lin, En-Chung; Chiu, Chih-Hsien

2011-02-01

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that senses and adapts cells to hypoxic environmental conditions. HIF-1 is composed of an oxygen-regulated α subunit (HIF-1α) and a constitutively expressed β subunit (HIF-1β). Taiwan voles (Microtus kikuchii) are an endemic species in Taiwan, found only in mountainous areas greater than 2000m above sea level. In this study, the full-length HIF-1α cDNA was cloned and sequenced from liver tissues of Taiwan voles. We found that HIF-1α of Taiwan voles had high sequence similarity to HIF-1α of other species. Sequence alignment of HIF-1α functional domains indicated basic helix-loop-helix (bHLH), PER-ARNT-SIM (PAS) and C-terminal transactivation (TAD-C) domains were conserved among species, but sequence variations were found between the oxygen-dependent degradation domains (ODDD). To measure Taiwan vole HIF-1α responses to hypoxia, animals were challenged with cobalt chloride, and HIF-1α mRNA and protein expression in brain, lung, heart, liver, kidney, and muscle was assessed by quantitative RT-PCR and Western blot analysis. Upon induction of hypoxic stress with cobalt chloride, an increase in HIF-1α mRNA levels was detected in lung, heart, kidney, and muscle tissue. In contrast, protein expression levels showed greater variation between individual animals. These results suggest that the regulation of HIF-1α may be important to the Taiwan vole under cobalt chloride treatments. But more details regarding the evolutionary effect of environmental pressure on HIF-1α primary sequence, HIF-1α function and regulation in Taiwan voles remain to be identified. Copyright © 2010 Elsevier Inc. All rights reserved.

Characterization and functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in tilapia.

PubMed

Li, Hong Lian; Gu, Xiao Hui; Li, Bi Jun; Chen, Xiao; Lin, Hao Ran; Xia, Jun Hong

2017-01-01

Hypoxia is a major cause of fish morbidity and mortality in the aquatic environment. Hypoxia-inducible factors are very important modulators in the transcriptional response to hypoxic stress. In this study, we characterized and conducted functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in Nile tilapia (Oreochromis niloticus). By cloning and Sanger sequencing, we obtained the full length cDNA sequences for HIF1α (2686bp) and HIF1αn (1308bp), respectively. The CDS of HIF1α includes 15 exons encoding 768 amino acid residues and the CDS of HIF1αn contains 8 exons encoding 354 amino acid residues. The complete CDS sequences of HIF1α and HIF1αn cloned from tilapia shared very high homology with known genes from other fishes. HIF1α show differentiated expression in different tissues (brain, heart, gill, spleen, liver) and at different hypoxia exposure times (6h, 12h, 24h). HIF1αn expression level under hypoxia is generally increased (6h, 12h, 24h) and shows extremely highly upregulation in brain tissue under hypoxia. A functional determination site analysis in the protein sequences between fish and land animals identified 21 amino acid sites in HIF1α and 2 sites in HIF1αn as significantly associated sites (α = 0.05). Phylogenetic tree-based positive selection analysis suggested 22 sites in HIF1α as positively selected sites with a p-value of at least 95% for fish lineages compared to the land animals. Our study could be important for clarifying the mechanism of fish adaptation to aquatic hypoxia environment.

Characterization and functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in tilapia

PubMed Central

Li, Hong Lian; Gu, Xiao Hui; Li, Bi Jun; Chen, Xiao; Lin, Hao Ran; Xia, Jun Hong

2017-01-01

Hypoxia is a major cause of fish morbidity and mortality in the aquatic environment. Hypoxia-inducible factors are very important modulators in the transcriptional response to hypoxic stress. In this study, we characterized and conducted functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in Nile tilapia (Oreochromis niloticus). By cloning and Sanger sequencing, we obtained the full length cDNA sequences for HIF1α (2686bp) and HIF1αn (1308bp), respectively. The CDS of HIF1α includes 15 exons encoding 768 amino acid residues and the CDS of HIF1αn contains 8 exons encoding 354 amino acid residues. The complete CDS sequences of HIF1α and HIF1αn cloned from tilapia shared very high homology with known genes from other fishes. HIF1α show differentiated expression in different tissues (brain, heart, gill, spleen, liver) and at different hypoxia exposure times (6h, 12h, 24h). HIF1αn expression level under hypoxia is generally increased (6h, 12h, 24h) and shows extremely highly upregulation in brain tissue under hypoxia. A functional determination site analysis in the protein sequences between fish and land animals identified 21 amino acid sites in HIF1α and 2 sites in HIF1αn as significantly associated sites (α = 0.05). Phylogenetic tree-based positive selection analysis suggested 22 sites in HIF1α as positively selected sites with a p-value of at least 95% for fish lineages compared to the land animals. Our study could be important for clarifying the mechanism of fish adaptation to aquatic hypoxia environment. PMID:28278251

Hydroxyl-HIF2-alpha is potential therapeutic target for renal cell carcinomas

PubMed Central

Isono, Takahiro; Chano, Tokuhiro; Yoshida, Tetsuya; Kageyama, Susumu; Kawauchi, Akihiro; Suzaki, Masafumi; Yuasa, Takeshi

2016-01-01

Dormant cancer cells are deprivation-resistant, and cause a number of problems for therapeutic approaches for cancers. Renal cell carcinomas (RCCs) include deprivation-resistant cells that are resistant to various treatments. In this study, the specific characteristics of deprivation-resistant cells were transcriptionally identified by next generation sequencing. The hypoxia-inducible factors (HIF) transcription factor network was significantly enhanced in deprivation-resistant RCCs compared to the sensitive RCCs. Deprivation-resistant RCCs, that had lost Von Hippel-Lindau tumor suppressor expression, expressed hydroxyl-HIF2-alpha in the nucleus, but not sensitive-RCCs. Hydroxyl-HIF-alpha was also expressed in nuclei of RCC tissue samples. Knockdown for HIF2-alpha, but not HIF1-alpha, induced cell death related to a reduction in HIF-related gene expression in deprivation-resistant RCC cells. Chetomin, a nuclear HIF-inhibitor, induced marked level of cytotoxicity in deprivation-resistant cells, similar to the knockdown of HIF2-alpha. Therefore, hydroxyl-HIF2-alpha might be a potential therapeutic target for RCCs. PMID:27822416

HIF isoforms in the skin differentially regulate systemic arterial pressure

PubMed Central

Cowburn, Andrew S.; Takeda, Norihiko; Boutin, Adam T.; Kim, Jung-Whan; Sterling, Jane C.; Nakasaki, Manando; Southwood, Mark; Goldrath, Ananda W.; Jamora, Colin; Nizet, Victor; Chilvers, Edwin R.; Johnson, Randall S.

2013-01-01

Vascular flow through tissues is regulated via a number of homeostatic mechanisms. Localized control of tissue blood flow, or autoregulation, is a key factor in regulating tissue perfusion and oxygenation. We show here that the net balance between two hypoxia-inducible factor (HIF) transcription factor isoforms, HIF-1α and HIF-2α, is an essential mechanism regulating both local and systemic blood flow in the skin of mice. We also show that balance of HIF isoforms in keratinocyte-specific mutant mice affects thermal adaptation, exercise capacity, and systemic arterial pressure. The two primary HIF isoforms achieve these effects in opposing ways that are associated with HIF isoform regulation of nitric oxide production. We also show that a correlation exists between altered levels of HIF isoforms in the skin and the degree of idiopathic hypertension in human subjects. Thus, the balance between HIF-1α and HIF-2α expression in keratinocytes is a control element of both tissue perfusion and systemic arterial pressure, with potential implications in human hypertension. PMID:24101470

A HIF-LIMD1 negative feedback mechanism mitigates the pro-tumorigenic effects of hypoxia.

PubMed

Foxler, Daniel E; Bridge, Katherine S; Foster, John G; Grevitt, Paul; Curry, Sean; Shah, Kunal M; Davidson, Kathryn M; Nagano, Ai; Gadaleta, Emanuela; Rhys, Hefin I; Kennedy, Paul T; Hermida, Miguel A; Chang, Ting-Yu; Shaw, Peter E; Reynolds, Louise E; McKay, Tristan R; Wang, Hsei-Wei; Ribeiro, Paulo S; Plevin, Michael J; Lagos, Dimitris; Lemoine, Nicholas R; Rajan, Prabhakar; Graham, Trevor A; Chelala, Claude; Hodivala-Dilke, Kairbaan M; Spendlove, Ian; Sharp, Tyson V

2018-06-21

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-β subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1 -negative lung cancers. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Hypoxia induces hemorrhagic transformation in pituitary adenomas via the HIF-1α signaling pathway.

PubMed

Xiao, Zhengzheng; Liu, Qin; Zhao, Boxi; Wu, Jun; Lei, Ting

2011-12-01

The hypoxia inducible factor 1 α (HIF-1α) activity has been associated with various hemorrhagic events. The biological role of HIF-1α in the hemorrhagic transformation of pituitary adenomas remains unknown. We hypothesized that fast growing tumor cells tend to predispose themselves to sublethal hypoxia and activate the HIF-1α signaling pathway, leading to hemorrhagic transformation in pituitary adenomas. Here, we used apoplectic and non-apoplectic pituitary adenomas to determine the involvement of HIF-1α signaling in intratumoral hemorrhage. We employed HIF-1α overexpression/knockdown strategies to examine the association between HIF-1α signaling and hemorrhagic presentation in vitro and in vivo. In support of our hypothesis, compared with non-hemorrhagic pituitary adenomas, higher cellular proliferation was observed in hemorrhagic ones and it correlated with increased HIF-1α signaling. HIF-1α overexpression activated its downstream genes, vascular endothelial growth factor and the proapoptotic BNIP3, in MMQ pituitary adenoma cells and this up-regulation was attenuated by HIF-1 siRNA. In vivo studies using MMQ cell xenografts in nude mice showed that HIF-1α overexpression significantly promoted hemorrhagic transformation. Our study indicates that tumor hypoxia, following rapid tumor growth, may promote hemorrhagic transformation in pituitary adenomas via the HIF-1α signaling pathway.

Myeloid HIF-1 is protective in Helicobacter pylori-mediated gastritis.

PubMed

Matak, Pavle; Heinis, Mylène; Mathieu, Jacques R R; Corriden, Ross; Cuvellier, Sylvain; Delga, Stéphanie; Mounier, Rémi; Rouquette, Alexandre; Raymond, Josette; Lamarque, Dominique; Emile, Jean-François; Nizet, Victor; Touati, Eliette; Peyssonnaux, Carole

2015-04-01

Helicobacter pylori infection triggers chronic inflammation of the gastric mucosa that may progress to gastric cancer. The hypoxia-inducible factors (HIFs) are the central mediators of cellular adaptation to low oxygen levels (hypoxia), but they have emerged recently as major transcriptional regulators of immunity and inflammation. No studies have investigated whether H. pylori affects HIF signaling in immune cells and a potential role for HIF in H. pylori-mediated gastritis. HIF-1 and HIF-2 expression was examined in human H. pylori-positive gastritis biopsies. Subsequent experiments were performed in naive and polarized bone marrow-derived macrophages from wild-type (WT) and myeloid HIF-1α-null mice (HIF-1(Δmyel)). WT and HIF-1(Δmyel) mice were inoculated with H. pylori by oral gavage and sacrificed 6 mo postinfection. HIF-1 was specifically expressed in macrophages of human H. pylori-positive gastritis biopsies. Macrophage HIF-1 strongly contributed to the induction of proinflammatory genes (IL-6, IL-1β) and inducible NO synthase in response to H. pylori. HIF-2 expression and markers of M2 macrophage differentiation were decreased in response to H. pylori. HIF-1(Δmyel) mice inoculated with H. pylori for 6 mo presented with a similar bacterial colonization than WT mice but, surprisingly, a global increase of inflammation, leading to a worsening of the gastritis, measured by an increased epithelial cell proliferation. In conclusion, myeloid HIF-1 is protective in H. pylori-mediated gastritis, pointing to the complex counterbalancing roles of innate immune and inflammatory phenotypes in driving this pathology. Copyright © 2015 by The American Association of Immunologists, Inc.

Differential contribution of key metabolic substrates and cellular oxygen in HIF signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhdanov, Alexander V., E-mail: a.zhdanov@ucc.ie; Waters, Alicia H.C.; Golubeva, Anna V.

2015-01-01

Changes in availability and utilisation of O{sub 2} and metabolic substrates are common in ischemia and cancer. We examined effects of substrate deprivation on HIF signalling in PC12 cells exposed to different atmospheric O{sub 2}. Upon 2–4 h moderate hypoxia, HIF-α protein levels were dictated by the availability of glutamine and glucose, essential for deep cell deoxygenation and glycolytic ATP flux. Nuclear accumulation of HIF-1α dramatically decreased upon inhibition of glutaminolysis or glutamine deprivation. Elevation of HIF-2α levels was transcription-independent and associated with the activation of Akt and Erk1/2. Upon 2 h anoxia, HIF-2α levels strongly correlated with cellular ATP,more » produced exclusively via glycolysis. Without glucose, HIF signalling was suppressed, giving way to other regulators of cell adaptation to energy crisis, e.g. AMPK. Consequently, viability of cells deprived of O{sub 2} and glucose decreased upon inhibition of AMPK with dorsomorphin. The capacity of cells to accumulate HIF-2α decreased after 24 h glucose deprivation. This effect, associated with increased AMPKα phosphorylation, was sensitive to dorsomorphin. In chronically hypoxic cells, glutamine played no major role in HIF-2α accumulation, which became mainly glucose-dependent. Overall, the availability of O{sub 2} and metabolic substrates intricately regulates HIF signalling by affecting cell oxygenation, ATP levels and pathways involved in production of HIF-α. - Highlights: • Gln and Glc regulate HIF levels in hypoxic cells by maintaining low O{sub 2} and high ATP. • HIF-α levels under anoxia correlate with cellular ATP and critically depend on Glc. • Gln and Glc modulate activity of Akt, Erk and AMPK, regulating HIF production. • HIF signalling is differentially inhibited by prolonged Glc and Gln deprivation. • Unlike Glc, Gln plays no major role in HIF signalling in chronically hypoxic cells.« less

PTEN induces apoptosis and cavitation via HIF-2-dependent Bnip3 upregulation during epithelial lumen formation.

PubMed

Qi, Y; Liu, J; Saadat, S; Tian, X; Han, Y; Fong, G-H; Pandolfi, P P; Lee, L Y; Li, S

2015-05-01

The tumor suppressor phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 and antagonizes the prosurvival PI3K-Akt pathway. Targeted deletion of PTEN in mice led to early embryonic lethality. To elucidate its role in embryonic epithelial morphogenesis and the underlying mechanisms, we used embryonic stem cell-derived embryoid body (EB), an epithelial cyst structurally similar to the periimplantation embryo. PTEN is upregulated during EB morphogenesis in parallel with apoptosis of core cells, which mediates EB cavitation. Genetic ablation of PTEN causes Akt overactivation, apoptosis resistance and cavitation blockade. However, rescue experiments using mutant PTEN and pharmacological inhibition of Akt suggest that the phosphatase activity of PTEN and Akt are not involved in apoptosis-mediated cavitation. Instead, hypoxia-induced upregulation of Bnip3, a proapoptotic BH3-only protein, mediates PTEN-dependent apoptosis and cavitation. PTEN inactivation inhibits hypoxia- and reactive oxygen species-induced Bnip3 elevation. Overexpression of Bnip3 in PTEN-null EBs rescues apoptosis of the core cells. Mechanistically, suppression of Bnip3 following PTEN loss is likely due to reduction of hypoxia-inducible factor-2α (HIF-2α) because forced expression of an oxygen-stable HIF-2α mutant rescues Bnip3 expression and apoptosis. Lastly, we show that HIF-2α is upregulated by PTEN at both transcriptional and posttranscriptional levels. Ablation of prolyl hydroxylase domain-containing protein 2 (PHD2) in normal EBs or inhibition of PHD activities in PTEN-null EBs stabilizes HIF-2α and induces Bnip3 and caspase-3 activation. Altogether, these results suggest that PTEN is required for apoptosis-mediated cavitation during epithelial morphogenesis by regulating the expression of HIF-2α and Bnip3.

EF24, a novel curcumin analog, disrupts the microtubule cytoskeleton and inhibits HIF-1

PubMed Central

Thomas, Shala L.; Zhong, Diansheng; Zhou, Wei; Malik, Sanna; Liotta, Dennis; Snyder, James P.; Hamel, Ernest; Giannakakou, Paraskevi

2008-01-01

Curcumin, the yellow pigment of the spice turmeric, has emerged as a promising anticancer agent due to its antiproliferative and antiangiogenic properties. However, the molecular mechanism of action of this compound remains a subject of debate. In addition, curcumin’s low bioavailability and efficacy profile in vivo further hinders its clinical development. This study focuses on the mechanism of action of EF24, a novel curcumin analog with greater than curcumin biological activity and bioavailability, but no increased toxicity. Treatment of MDA-MB231 breast and PC3 prostate cancer cells with EF24 or curcumin led to inhibition of HIF-1α protein levels and, consequently, inhibition of HIF transcriptional activity. This drug-induced HIF inhibition occurred in a VHL-dependent but proteasome-independent manner. We found that, while curcumin inhibited HIF-1α gene transcription, EF24 exerted its activity by inhibiting HIF-1α posttranscriptionally. This result suggested that the two compounds are structurally similar but mechanistically distinct. Another cellular effect that further differentiated the two compounds was the ability of EF24, but not curcumin, to induce microtubule stabilization in cells. EF24 had no stabilizing effect on tubulin polymerization in an in vitro assay using purified bovine brain tubulin, suggesting that the EF24-induced cytoskeletal disruption in cells may be the result of upstream signaling events rather than EF24 direct binding to tubulin. In summary, our study identifies EF24 as a novel curcumin-related compound possessing a distinct mechanism of action, which we believe contributes to the potent anticancer activity of this agent and can be further exploited to investigate the therapeutic potential of EF24. PMID:18682687

EF24, a novel curcumin analog, disrupts the microtubule cytoskeleton and inhibits HIF-1.

PubMed

Thomas, Shala L; Zhong, Diansheng; Zhou, Wei; Malik, Sanna; Liotta, Dennis; Snyder, James P; Hamel, Ernest; Giannakakou, Paraskevi

2008-08-01

Curcumin, the yellow pigment of the spice turmeric, has emerged as a promising anticancer agent due to its antiproliferative and antiangiogenic properties. However, the molecular mechanism of action of this compound remains a subject of debate. In addition, curcumin's low bioavailability and efficacy profile in vivo further hinders its clinical development. This study focuses on the mechanism of action of EF24, a novel curcumin analog with greater than curcumin biological activity and bioavailability, but no increased toxicity. Treatment of MDA-MB231 breast and PC3 prostate cancer cells with EF24 or curcumin led to inhibition of HIF-1alpha protein levels and, consequently, inhibition of HIF transcriptional activity. This drug-induced HIF inhibition occurred in a VHL-dependent but proteasome-independent manner. We found that, while curcumin inhibited HIF-1alpha gene transcription, EF24 exerted its activity by inhibiting HIF-1alpha posttranscriptionally. This result suggested that the two compounds are structurally similar but mechanistically distinct. Another cellular effect that further differentiated the two compounds was the ability of EF24, but not curcumin, to induce microtubule stabilization in cells. EF24 had no stabilizing effect on tubulin polymerization in an in vitro assay using purified bovine brain tubulin, suggesting that the EF24-induced cytoskeletal disruption in cells may be the result of upstream signaling events rather than EF24 direct binding to tubulin. In summary, our study identifies EF24 as a novel curcumin-related compound possessing a distinct mechanism of action, which we believe contributes to the potent anticancer activity of this agent and can be further exploited to investigate the therapeutic potential of EF24.

The role of hypoxia-inducible factor-2 in digestive system cancers.

PubMed

Zhao, J; Du, F; Shen, G; Zheng, F; Xu, B

2015-01-15

Hypoxia is an all but ubiquitous phenomenon in cancers. Two known hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, primarily mediate the transcriptional response to hypoxia. Despite the high homology between HIF-1α and HIF-2α, emerging evidence suggests differences between both molecules in terms of transcriptional targets as well as impact on multiple physiological pathways and tumorigenesis. To date, much progress has been made toward understanding the roles of HIF-2α in digestive system cancers. Indeed, HIF-2α has been shown to regulate multiple aspects of digestive system cancers, including cell proliferation, angiogenesis and apoptosis, metabolism, metastasis and resistance to chemotherapy. These findings make HIF-2α a critical regulator of this malignant phenotype. Here we summarize the function of HIF-2 during cancer development as well as its contribution to tumorigenesis in digestive system malignancies.

HIF Oxygen Sensing Pathways in Lung Biology.

PubMed

Urrutia, Andrés A; Aragonés, Julián

2018-06-06

Cellular responses to oxygen fluctuations are largely mediated by hypoxia-inducible factors (HIFs). Upon inhalation, the first organ inspired oxygen comes into contact with is the lungs, but the understanding of the pulmonary HIF oxygen-sensing pathway is still limited. In this review we will focus on the role of HIF1α and HIF2α isoforms in lung responses to oxygen insufficiency. In particular, we will discuss novel findings regarding their role in the biology of smooth muscle cells and endothelial cells in the context of hypoxia-induced pulmonary vasoconstriction. Moreover, we will also discuss recent studies into HIF-dependent responses in the airway epithelium, which have been even less studied than the HIF-dependent vascular responses in the lungs. In summary, we will review the biological functions executed by HIF1 or HIF2 in the pulmonary vessels and epithelium to control lung responses to oxygen fluctuations as well as their pathological consequences in the hypoxic lung.

The role of hypoxia-inducible factor-2 in digestive system cancers

PubMed Central

Zhao, J; Du, F; Shen, G; Zheng, F; Xu, B

2015-01-01

Hypoxia is an all but ubiquitous phenomenon in cancers. Two known hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, primarily mediate the transcriptional response to hypoxia. Despite the high homology between HIF-1α and HIF-2α, emerging evidence suggests differences between both molecules in terms of transcriptional targets as well as impact on multiple physiological pathways and tumorigenesis. To date, much progress has been made toward understanding the roles of HIF-2α in digestive system cancers. Indeed, HIF-2α has been shown to regulate multiple aspects of digestive system cancers, including cell proliferation, angiogenesis and apoptosis, metabolism, metastasis and resistance to chemotherapy. These findings make HIF-2α a critical regulator of this malignant phenotype. Here we summarize the function of HIF-2 during cancer development as well as its contribution to tumorigenesis in digestive system malignancies. PMID:25590810

Induction of the nuclear factor HIF-1{alpha} in acetaminophen toxicity: Evidence for oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, Laura P.; Donahower, Brian; Burke, Angela S.

2006-04-28

Hypoxia inducible factor (HIF) controls the transcription of genes involved in angiogenesis, erythropoiesis, glycolysis, and cell survival. HIF-1{alpha} levels are a critical determinant of HIF activity. The induction of HIF-1{alpha} was examined in the livers of mice treated with a toxic dose of APAP (300 mg/kg IP) and sacrificed at 1, 2, 4, 8, and 12 h. HIF-1{alpha} was induced at 1-12 h and induction occurred prior to the onset of toxicity. Pre-treatment of mice with N-acetylcysteine (1200 mg/kg IP) prevented toxicity and HIF-1{alpha} induction. In further studies, hepatocyte suspensions were incubated with APAP (1 mM) in the presence ofmore » an oxygen atmosphere. HIF-1{alpha} was induced at 1 h, prior to the onset of toxicity. Inclusion of cyclosporine A (10 {mu}M), an inhibitor of mitochondrial permeability transition, oxidative stress, and toxicity, prevented the induction of HIF-1{alpha}. Thus, HIF-1{alpha} is induced before APAP toxicity and can occur under non-hypoxic conditions. The data suggest a role for oxidative stress in the induction of HIF-1{alpha} in APAP toxicity.« less

An oxygen-insensitive Hif-3α isoform inhibits Wnt signaling by destabilizing the nuclear β-catenin complex.

PubMed

Zhang, Peng; Bai, Yan; Lu, Ling; Li, Yun; Duan, Cunming

2016-01-14

Hypoxia-inducible factors (HIFs), while best known for their roles in the hypoxic response, have oxygen-independent roles in early development with poorly defined mechanisms. Here, we report a novel Hif-3α variant, Hif-3α2, in zebrafish. Hif-3α2 lacks the bHLH, PAS, PAC, and ODD domains, and is expressed in embryonic and adult tissues independently of oxygen availability. Hif-3α2 is a nuclear protein with significant hypoxia response element (HRE)-dependent transcriptional activity. Hif-3α2 overexpression not only decreases embryonic growth and developmental timing but also causes left-right asymmetry defects. Genetic deletion of Hif-3α2 by CRISPR/Cas9 genome editing increases, while Hif-3α2 overexpression decreases, Wnt/β-catenin signaling. This action is independent of its HRE-dependent transcriptional activity. Mechanistically, Hif-3α2 binds to β-catenin and destabilizes the nuclear β-catenin complex. This mechanism is distinct from GSK3β-mediated β-catenin degradation and is conserved in humans. These findings provide new insights into the oxygen-independent actions of HIFs and uncover a novel mechanism regulating Wnt/β-catenin signaling.

Mitochondrial targeting of HIF-1α inhibits hypoxia-induced apoptosis independently of its transcriptional activity.

PubMed

Li, Hong-Sheng; Zhou, Yan-Ni; Li, Lu; Li, Sheng-Fu; Long, Dan; Chen, Xue-Lu; Zhang, Jia-Bi; Li, You-Ping; Feng, Li

2018-04-25

The transcription factor hypoxia inducible factor-1α (HIF-1α) mediates adaptive responses to hypoxia by nuclear translocation and regulation of gene expression. Mitochondrial changes are critical for the adaptive response to hypoxia. However, the transcriptional and non-transcriptional mechanisms by which HIF-1α regulates mitochondria under hypoxia are poorly understood. Here, we examined the subcellular localization of HIF-1α in human cells and identified a small fraction of HIF-1α that translocated to the mitochondria after exposure to hypoxia or hypoxia-mimicking pharmacological agents. To probe the function of this HIF-1α population, we ectopically expressed a mitochondrial-targeted form of HIF-1α (mito-HIF-1α). Expression of mito-HIF-1α was sufficient to attenuate apoptosis induced by exposure to hypoxia or H 2 O 2 -induced oxidative stress. Moreover, mito-HIF-1α expression reduced the production of reactive oxygen species, the collapse of mitochondrial membrane potential, and the expression of mitochondrial DNA-encoded mRNA in response to hypoxia. However, these functions of mito-HIF-1α were independent of its conventional transcriptional activity. Finally, the livers of mice with CCl 4 -induced fibrosis showed a progressive increase in HIF-1α association with the mitochondria, indicating the clinical relevance of this finding. These data suggested that mitochondrial HIF-1α protects against apoptosis independently of its well-known role as a transcription factor. Copyright © 2018. Published by Elsevier Inc.

Effects of 2-methoxyestradiol on apoptosis and HIF-1α and HIF-2α expression in lung cancer cells under normoxia and hypoxia.

PubMed

Aquino-Gálvez, Arnoldo; González-Ávila, Georgina; Delgado-Tello, Javier; Castillejos-López, Manuel; Mendoza-Milla, Criselda; Zúñiga, Joaquín; Checa, Marco; Maldonado-Martínez, Héctor Aquiles; Trinidad-López, Axel; Cisneros, José; Torres-Espíndola, Luz María; Hernández-Jiménez, Claudia; Sommer, Bettina; Cabello-Gutiérrez, Carlos; Gutiérrez-González, Luis H

2016-01-01

Hypoxic tumor cells are known to be more resistant to conventional chemotherapy and radiation than normoxic cells. However, the effects of 2-methoxyestradiol (2-ME), an anti-angiogenic, antiproliferative and pro-apoptotic drug, on hypoxic lung cancer cells are unknown. The aim of the present study was to compare the effects of 2-ME on cell growth, apoptosis, hypoxia-inducible factor 1α (HIF-1α) and HIF-2α gene and protein expression in A549 cells under normoxic and hypoxic conditions. To establish the optimal 2-ME concentration with which to carry out the apoptosis assay and to examine mRNA and protein expression of HIFs, cell growth analysis was carried out through N-hexa-methylpararosaniline staining assays in A549 cell cultures treated with one of five different 2-ME concentrations at different times under normoxic or hypoxic growth conditions. The 2-ME concentration of 10 mM at 72 h was selected to perform all further experiments. Apoptotic cells were analyzed by flow cytometry. Western blotting was used to determine HIF-1α and HIF-2α protein expression in total cell extracts. Cellular localization of HIF-1α and HIF-2α was assessed by immunocytochemistry. HIF-1α and HIF-2α gene expression was determined by real-time PCR. A significant increase in the percentage of apoptosis was observed when cells were treated with 2-ME under a normoxic but not under hypoxic conditions (p=0.006). HIF-1α and HIF-2α protein expression levels were significantly decreased in cells cultured under hypoxic conditions and treated with 2-ME (p<0.001). Furthermore, 2-ME decreased the HIF-1α and HIF-2α nuclear staining in cells cultured under hypoxia. The HIF-1α and HIF-2α mRNA levels were significantly lower when cells were exposed to 2-ME under normoxia and hypoxia. Our results suggest that 2-ME could have beneficial results when used with conventional chemotherapy in an attempt to lower the invasive and metastatic processes during cancer development due to its effects on the gene expression and protein synthesis of HIFs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Wooyoung; Bazer, Fuller W.; Song, Gwonhwa, E-mail: ghsong@korea.ac.kr

The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially inmore » nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades. - Highlights: • HIF-1α expression is up-regulated in pTr cells under low oxygen concentrations. • EGF induces HIF-1α accumulation in pTr cells. • EGF-induced HIF-1α accumulation is blocked by de-novo translation inhibitor. • EGF-induced HIF-1α accumulation is mediated by AKT, ERK1/2 and mTOR pathways. • Oxygen deficiency and EGF has stimulatory effect on proliferation of pTr cells.« less

6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1alpha resulting in new vessel formation.

PubMed

Yoo, Y-G; Na, T-Y; Yang, W-K; Kim, H-J; Lee, I-K; Kong, G; Chung, J-H; Lee, M-O

2007-05-31

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. Previously, we reported that the orphan nuclear receptor Nur77 functions in stabilizing HIF-1alpha. Here, we demonstrate that 6-mercaptopurine (6-MP), an activator of the NR4A family members, enhances transcriptional activity of HIF-1. 6-MP enhanced the protein-level of HIF-1alpha as well as vascular endothelial growth factor (VEGF) in a dose- and time-dependent manner. The induction of HIF-1alpha was abolished by the transfection of either a dominant-negative Nur77 mutant or si-Nur77, indicating a critical role of Nur77 in the 6-MP action. The HIF-1alpha protein level remained up to 60 min in the presence of 6-MP when de novo protein synthesis was blocked by cycloheximide, suggesting that 6-MP induces stabilization of the HIF-1alpha protein. The fact that 6-MP decreased the association of HIF-1alpha with von Hippel-Lindau protein and the acetylation of HIF-1alpha, may explain how 6-MP induced stability of HIF-1alpha. Further, 6-MP induced the transactivation function of HIF-1alpha by recruiting co-activator cyclic-AMP-response-element-binding protein. Finally, 6-MP enhanced the expression of HIF-1alpha and VEGF, and the formation of capillary tubes in human umbilical vascular endothelial cells. Together, our results provide a new insight for 6-MP action in the stabilization of HIF-1alpha and imply a potential application of 6-MP in hypoxia-associated human vascular diseases.

HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geis, Theresa, E-mail: geis@biochem.uni-frankfurt.de; Döring, Claudia, E-mail: C.Doering@em.uni-frankfurt.de; Popp, Rüdiger, E-mail: popp@vrc.uni-frankfurt.de

Hypoxia promotes progression of hepatocellular carcinoma (HCC), not only affecting tumor cell proliferation and invasion, but also angiogenesis and thus, increasing the risk of metastasis. Hypoxia inducible factors (HIF)-1α and -2α cause adaptation of tumors to hypoxia, still with uncertainties towards the angiogenic switch. We created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids with embryonic bodies as an in vitro tumor model mimicking the cancer microenvironment. The naturally occuring oxygen and nutrient gradients within the cocultures allow us to question the role of distinct HIF isoforms in regulating HCC angiogenesis. Inmore » cocultures with a HIF-2α knockdown, angiogenesis was attenuated, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1) as a HIF-2α target gene in HepG2 cells. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis. Blocking plasmin, the downstream target of PAI-1, with aprotinin in HIF-2α knockdown (k/d) cells proved a cause–effect relation and restored angiogenesis, with no effect on control cocultures. Suggestively, HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. We conclude that the HIF-2α target gene PAI-1 favors the angiogenic switch in HCC. - Highlights: • HepG2 were cocultured with stem cells to mimic a cancer microenvironment in vitro. • A knockdown of HIF-2α reduces angiogenesis. • PAI-1 was identified as a HIF-2α target gene in HCC by microarray analysis. • HIF-2α induces the angiogenic switch via inhibition of plasmin.« less

Natural product-derived small molecule activators of hypoxia-inducible factor-1 (HIF-1).

PubMed

Nagle, Dale G; Zhou, Yu-Dong

2006-01-01

Hypoxia-inducible factor-1 (HIF-1) is a key mediator of oxygen homeostasis that was first identified as a transcription factor that is induced and activated by decreased oxygen tension. Upon activation, HIF-1 upregulates the transcription of genes that promote adaptation and survival under hypoxic conditions. HIF-1 is a heterodimer composed of an oxygen-regulated subunit known as HIF-1alpha and a constitutively expressed HIF-1beta subunit. In general, the availability and activity of the HIF-1alpha subunit determines the activity of HIF-1. Subsequent studies have revealed that HIF-1 is also activated by environmental and physiological stimuli that range from iron chelators to hormones. Preclinical studies suggest that HIF-1 activation may be a valuable therapeutic approach to treat tissue ischemia and other ischemia/hypoxia-related disorders. The focus of this review is natural product-derived small molecule HIF-1 activators. Natural products, relatively low molecular weight organic compounds produced by plants, animals, and microbes, have been and continue to be a major source of new drugs and molecular probes. The majority of known natural product-derived HIF-1 activators were discovered through the pharmacological evaluation of specifically selected individual compounds. On the other hand, the combination of natural products chemistry with appropriate high-throughput screening bioassays may yield novel natural product-derived HIF-1 activators. Potent natural product-derived HIF-1 activators that exhibit a low level of toxicity and side effects hold promise as new treatment options for diseases such as myocardial and peripheral ischemia, and as chemopreventative agents that could be used to reduce the level of ischemia/reperfusion injury following heart attack and stroke.

Effects of hypoxia-inducible factor-1α silencing on the proliferation of CBRH-7919 hepatoma cells

PubMed Central

Xu, Lin-Feng; Ni, Jia-Yan; Sun, Hong-Liang; Chen, Yao-Ting; Wu, Yu-Dan

2013-01-01

AIM: To study the effects of hypoxia-inducible factor-1α (HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells. METHODS: The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2. The HIF-1α-specific RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank. The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software. The small interfering RNA (siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000TM, and transfected into the hypoxic hepatoma cells. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assay were used to detect the expression levels of mRNA and protein. HIF-1α and vascular endothelial growth factor (VEGF) mRNA was determined using real time RT-PCR; the protein expression levels of AKT, p-AKT, p21 and cyclinD1 were determined using Western blotting. The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium (MTT) assay and the bromodeoxyuridine (BrdU) incorporation cell proliferation assay. RESULTS: Under induced hypoxia, the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2; the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L. Under hypoxia, the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia. HIF-1α-specific RNAi sequences were successfully transfected into hepatoma cells. The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF, along with the protein expression levels of p-AKT and cyclinD1; the protein expression of p21 was significantly increased, and there was no significant difference in the expression of AKT. The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group; in the siRNA transfection group, the amount of hepatoma cells in G1 phase was more than that in the control group. The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group. The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells. CONCLUSION: Hypoxia increases the expression of HIF-1α, and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells. PMID:23555163

Host - HIF- 1alpha Pathway And Hypoxia: In Vitro Studies And Mathematical Model

DTIC Science & Technology

2016-08-30

TERMS mathematical model, signaling pathways, hypoxia, immunohistochemistry, ELISA , inhalation chamber 16. SECURITY CLASSIFICATION OF: U 17...B. HIF-1α ELISA Procedure ........................................................................................27 Appendix C. HIF-1α Model...Quantifying Induction of HIF-1α Expression using ELISA .........................................15 Figure 10. Simulation Outputs from HIF-1α Kinetic

Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression.

PubMed

Liu, Juan; Zhang, Cen; Zhao, Yuhan; Yue, Xuetian; Wu, Hao; Huang, Shan; Chen, James; Tomsky, Kyle; Xie, Haiyang; Khella, Christen A; Gatza, Michael L; Xia, Dajing; Gao, Jimin; White, Eileen; Haffty, Bruce G; Hu, Wenwei; Feng, Zhaohui

2017-11-28

Mutations in E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Accumulating evidence suggests that Parkin is a tumor suppressor, but the underlying mechanism is poorly understood. Here we show that Parkin is an E3 ubiquitin ligase for hypoxia-inducible factor 1α (HIF-1α). Parkin interacts with HIF-1α and promotes HIF-1α degradation through ubiquitination, which in turn inhibits metastasis of breast cancer cells. Parkin downregulation in breast cancer cells promotes metastasis, which can be inhibited by targeting HIF-1α with RNA interference or the small-molecule inhibitor YC-1. We further identify lysine 477 (K477) of HIF-1α as a major ubiquitination site for Parkin. K477R HIF-1α mutation and specific cancer-associated Parkin mutations largely abolish the functions of Parkin to ubiquitinate HIF-1α and inhibit cancer metastasis. Importantly, Parkin expression is inversely correlated with HIF-1α expression and metastasis in breast cancer. Our results reveal an important mechanism for Parkin in tumor suppression and HIF-1α regulation.

The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha.

PubMed

Gort, E H; van Haaften, G; Verlaan, I; Groot, A J; Plasterk, R H A; Shvarts, A; Suijkerbuijk, K P M; van Laar, T; van der Wall, E; Raman, V; van Diest, P J; Tijsterman, M; Vooijs, M

2008-03-06

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that play a crucial role in oxygen homeostasis. Intratumoral hypoxia and genetic alterations lead to HIF activity, which is a hallmark of solid cancer and is associated with poor clinical outcome. HIF activity is regulated by an evolutionary conserved mechanism involving oxygen-dependent HIFalpha protein degradation. To identify novel components of the HIF pathway, we performed a genome-wide RNA interference screen in Caenorhabditis elegans, to suppress HIF-dependent phenotypes, like egg-laying defects and hypoxia survival. In addition to hif-1 (HIFalpha) and aha-1 (HIFbeta), we identified hlh-8, gska-3 and spe-8. The hlh-8 gene is homologous to the human oncogene TWIST1. We show that TWIST1 expression in human cancer cells is enhanced by hypoxia in a HIF-2alpha-dependent manner. Furthermore, intronic hypoxia response elements of TWIST1 are regulated by HIF-2alpha, but not HIF-1alpha. These results identify TWIST1 as a direct target gene of HIF-2alpha, which may provide insight into the acquired metastatic capacity of hypoxic tumors.

HIF1α and HIF2α: sibling rivalry in hypoxic tumour growth and progression.

PubMed

Keith, Brian; Johnson, Randall S; Simon, M Celeste

2011-12-15

Hypoxia-inducible factors (HIFs) are broadly expressed in human cancers, and HIF1α and HIF2α were previously suspected to promote tumour progression through largely overlapping functions. However, this relatively simple model has now been challenged in light of recent data from various approaches that reveal unique and sometimes opposing activities of these HIFα isoforms in both normal physiology and disease. These effects are mediated in part through the regulation of unique target genes, as well as through direct and indirect interactions with important oncoproteins and tumour suppressors, including MYC and p53. As HIF inhibitors are currently undergoing clinical evaluation as cancer therapeutics, a more thorough understanding of the unique roles performed by HIF1α and HIF2α in human neoplasia is warranted.

Yak response to high-altitude hypoxic stress by altering mRNA expression and DNA methylation of hypoxia-inducible factors.

PubMed

Xiong, Xianrong; Fu, Mei; Lan, Daoliang; Li, Jian; Zi, Xiangdong; Zhong, Jincheng

2015-01-01

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, which play crucial roles in tumor angiogenesis and mammalian development, and regulate the transcription of genes involved in oxygen homeostasis in response to hypoxia. However, information on HIF-1α and HIF-2α in yak (Bos grunniens) is scarce. The complete coding region of yak HIF-2α was cloned, its mRNA expression in several tissues were determined, and the expression levels were compared with those of closely related low-altitude cattle (Bos taurus), and the methylation status of promoter regions were analyzed to better understand the roles of HIF-1α and HIF-2α in domesticated yak. The yak HIF-2α cDNA was cloned and sequenced in the present work reveals the evolutionary conservation through multiple sequence alignment, although 15 bases changed, resulting in 8 amino acid substitutions in the translated proteins in cattle. The tissue-specific expression results showed that HIF-1α is ubiquitously expressed, whereas HIF-2α expression is limited to endothelial tissues (kidney, heart, lung, spleen, and liver) and blood in yak. Both HIF-1α and HIF-2α expressions were higher in yak tissues than in cattle. The HIF-1α expression level is much higher in yak than cattle in these organs, except for the lung (P < 0.05), but the HIF-2α gene is significantly different in the heart, spleen, and kidney (P < 0.05). Furthermore, the methylation levels in the 5' flanking regulatory regions of HIF-1α and HIF-2α in yak kidney were significantly decreased than cattle counterparts (P < 0.05). Identifying these genes and the comparison of different expressions facilitates the understanding of the biological high-altitude hypoxic stress response mechanism and may assist current medical research to understand hypoxia-related diseases.

Differential HIF and NOS responses to acute anemia: defining organ-specific hemoglobin thresholds for tissue hypoxia.

PubMed

Tsui, Albert K Y; Marsden, Philip A; Mazer, C David; Sled, John G; Lee, Keith M; Henkelman, R Mark; Cahill, Lindsay S; Zhou, Yu-Qing; Chan, Neville; Liu, Elaine; Hare, Gregory M T

2014-07-01

Tissue hypoxia likely contributes to anemia-induced organ injury and mortality. Severe anemia activates hypoxia-inducible factor (HIF) signaling by hypoxic- and neuronal nitric oxide (NO) synthase- (nNOS) dependent mechanisms. However, organ-specific hemoglobin (Hb) thresholds for increased HIF expression have not been defined. To assess organ-specific Hb thresholds for tissue hypoxia, HIF-α (oxygen-dependent degradation domain, ODD) luciferase mice were hemodiluted to mild, moderate, or severe anemia corresponding to Hb levels of 90, 70, and 50 g/l, respectively. HIF luciferase reporter activity, HIF protein, and HIF-dependent RNA levels were assessed. In the brain, HIF-1α was paradoxically decreased at mild anemia, returned to baseline at moderate anemia, and then increased at severe anemia. Brain HIF-2α remained unchanged at all Hb levels. Both kidney HIF-1α and HIF-2α increased earlier (Hb ∼70-90 g/l) in response to anemia. Liver also exhibited an early HIF-α response. Carotid blood flow was increased early (Hb ∼70, g/l), but renal blood flow remained relatively constant, only increased at Hb of 50 g/l. Anemia increased nNOS (brain and kidney) and endothelia NOS (eNOS) (kidney) levels. Whereas anemia-induced increases in brain HIFα were nNOS-dependent, our current data demonstrate that increased renal HIFα was nNOS independent. HIF-dependent RNA levels increased linearly (∼10-fold) in the brain. However, renal HIF-RNA responses (MCT4, EPO) increased exponentially (∼100-fold). Plasma EPO levels increased near Hb threshold of 90 g/l, suggesting that the EPO response is sensitive. Collectively, these observations suggest that each organ expresses a different threshold for cellular HIF/NOS hypoxia responses. This knowledge may help define the mechanism(s) by which the brain and kidney maintain oxygen homeostasis during anemia. Copyright © 2014 the American Physiological Society.

Integration of oxygen signaling at the consensus HRE.

PubMed

Wenger, Roland H; Stiehl, Daniel P; Camenisch, Gieri

2005-10-18

The hypoxia-inducible factor 1 (HIF-1) was initially identified as a transcription factor that regulated erythropoietin gene expression in response to a decrease in oxygen availability in kidney tissue. Subsequently, a family of oxygen-dependent protein hydroxylases was found to regulate the abundance and activity of three oxygen-sensitive HIFalpha subunits, which, as part of the HIF heterodimer, regulated the transcription of at least 70 different effector genes. In addition to responding to a decrease in tissue oxygenation, HIF is proactively induced, even under normoxic conditions, in response to stimuli that lead to cell growth, ultimately leading to higher oxygen consumption. The growing cell thus profits from an anticipatory increase in HIF-dependent target gene expression. Growth stimuli-activated signaling pathways that influence the abundance and activity of HIFs include pathways in which kinases are activated and pathways in which reactive oxygen species are liberated. These pathways signal to the HIF protein hydroxylases, as well as to HIF itself, by means of covalent or redox modifications and protein-protein interactions. The final point of integration of all of these pathways is the hypoxia-response element (HRE) of effector genes. Here, we provide comprehensive compilations of the known growth stimuli that promote increases in HIF abundance, of protein-protein interactions involving HIF, and of the known HIF effector genes. The consensus HRE derived from a comparison of the HREs of these HIF effectors will be useful for identification of novel HIF target genes, design of oxygen-regulated gene therapy, and prediction of effects of future drugs targeting the HIF system.

Hypoxia-inducible factors promote alveolar development and regeneration.

PubMed

Vadivel, Arul; Alphonse, Rajesh S; Etches, Nicholas; van Haaften, Timothy; Collins, Jennifer J P; O'Reilly, Megan; Eaton, Farah; Thébaud, Bernard

2014-01-01

Understanding how alveoli and the underlying capillary network develop and how these mechanisms are disrupted in disease states is critical for developing effective therapies for lung regeneration. Recent evidence suggests that lung angiogenesis promotes lung development and repair. Vascular endothelial growth factor (VEGF) preserves lung angiogenesis and alveolarization in experimental O2-induced arrested alveolar growth in newborn rats, but combined VEGF+angiopoietin 1 treatment is necessary to correct VEGF-induced vessel leakiness. Hypoxia-inducible factors (HIFs) are transcription factors that activate multiple O2-sensitive genes, including those encoding for angiogenic growth factors, but their role during postnatal lung growth is incompletely understood. By inducing the expression of a range of angiogenic factors in a coordinated fashion, HIF may orchestrate efficient and safe angiogenesis superior to VEGF. We hypothesized that HIF inhibition impairs alveolarization and that HIF activation regenerates irreversible O2-induced arrested alveolar growth. HIF inhibition by intratracheal dominant-negative adenovirus (dnHIF-1α)-mediated gene transfer or chetomin decreased lung HIF-1α, HIF-2α, and VEGF expression and led to air space enlargement and arrested lung vascular growth. In experimental O2-induced arrested alveolar growth in newborn rats, the characteristic features of air space enlargement and loss of lung capillaries were associated with decreased lung HIF-1α and HIF-2α expression. Intratracheal administration of Ad.HIF-1α restored HIF-1α, endothelial nitric oxide synthase, VEGF, VEGFR2, and Tie2 expression and preserved and rescued alveolar growth and lung capillary formation in this model. HIFs promote normal alveolar development and may be useful targets for alveolar regeneration.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leitner, M.; Bieniosek, F.; Kwan, J.

The Heavy Ion Fusion Science Virtual National Laboratory (HIFS-VNL), a collaboration between Lawrence Berkeley National Laboratory (LBNL), Lawrence Livermore National Laboratory (LLNL), and Princeton Plasma Physics Laboratory (PPPL), is currently constructing a new induction linear accelerator, called Neutralized Drift Compression eXperiment NDCX-II. The accelerator design makes effective use of existing components from LLNL's decommissioned Advanced Test Accelerator (ATA), especially induction cells and Blumlein voltage sources that have been transferred to LBNL. We have developed an aggressive acceleration 'schedule' that compresses the emitted ion pulse from 500 ns to 1 ns in just 15 meters. In the nominal design concept, 30more » nC of Li{sup +} are accelerated to 3.5 MeV and allowed to drift-compress to a peak current of about 30 A. That beam will be utilized for warm dense matter experiments investigating the interaction of ion beams with matter at high temperature and pressure. Construction of the accelerator will be complete within a period of approximately two and a half years and will provide a worldwide unique opportunity for ion-driven warm dense matter experiments as well as research related to novel beam manipulations for heavy ion fusion drivers.« less

Loss of HIF-1α in the notochord results in cell death and complete disappearance of the nucleus pulposus.

PubMed

Merceron, Christophe; Mangiavini, Laura; Robling, Alexander; Wilson, Tremika LeShan; Giaccia, Amato J; Shapiro, Irving M; Schipani, Ernestina; Risbud, Makarand V

2014-01-01

The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD degeneration in humans.

P08.14 In situ detection of hypoxia inducible factor 2 alpha in malignant gliomas

PubMed Central

Renfrow, J. J.; Strowd, R. E.; Huang, Y.; Herpai, D.; Mott, R. T.; Wong, T.; Lesser, G. J.; Debinski, W.

2017-01-01

Abstract Introduction: Direct intratumoral measurements confirm that necrosis and hypoxia are fundamental features of the pathobiology of glioblastoma (GBM). Tumor cells adapt to hypoxia through up regulation of hypoxia inducible factors (HIFs), a conserved family of transcription factors. Of the HIFs, HIF-2 alpha (HIF-2α) appears to be expressed in GBM tumor cells (not neural progenitors) and drives adaptation to prolonged (>24 hour) hypoxia. Targeting HIFs is an attractive cancer treatment strategy given that tumor hypoxia is a constant environmental cue driving genetic instability, cell migration, angiogenesis, and chemoradiation resistance. GBM, a tumor uniformly characterized by an intratumoral hypoxic environment, is a prime candidate for HIF therapeutic targeting but specific drugs were not available. PT2385 is a novel, first-in-class, HIF-2α inhibitor which recently entered clinical trials for renal carcinoma. PT2385 is orally administered and is small, lipophilic with a brain:plasma ratio of 0.9 in rats. To establish HIF-2α as a therapeutic target in GBM, we investigated in situ expression of HIF-2α protein in tissue samples from the Wake Forest Brain Tumor Center of Excellence Brain Tumor Bank. Methods: 22 formalin-fixed, paraffin-embedded glioma samples (grade II-IV) were analyzed for HIF-2α expression using immunohistochemistry. After rehydration, endogenous peroxidase was blocked, epitopes were retrieved using a microwaved Tris EDTA pH 9.0 solution, non-specific epitopes were blocked, and HIF-2α antibody (Santa Cruz, sc-13596) was added. A horseradish peroxidase conjugated anti-mouse antibody was then added followed by detection chromagen. An inverted bright field microscope captured images and representative samples were digitally scanned. Localization and quantification of HIF-2α was independently verified by a neuropathologist. Results: There was no detectable HIF-2α expression in the four Grade II and two Grade III gliomas studied. Of the 16 GBMs (Grade IV), HIF-2α was expressed in 13 (81%). HIF-2α was highly expressed in seven specimens (>10% cells positive), intermediate in six specimens (<10% of cells positive), and minimal to none in three specimens. Staining was specific to both tumor cells and occasionally monocytic cells (based on morphology). It was noted that HIF-2α was frequently present in perivascular and perinecrotic regions. Conclusions: HIF-2α expression is found in the majority of GBM specimens, but is absent in low-grade gliomas. Immunohistochemistry demonstrated a range of HIF-2α abundance along with regional staining patterns, clustered in perivascular and perinecrotic niches. HIF-2α appears to correlate with increasing malignancy grade. This is the first in situ description of HIF-2α in gliomas and further studies are in progress for preclinical in vitro and in vivo testing of PT2385, a first-in-class HIF-2α targeted agent, in GBM.

Beluga whale pVHL enhances HIF-2α activity via inducing HIF-2α proteasomal degradation under hypoxia

PubMed Central

Bi, Jianling; Hu, Bo; Wang, Jing; Liu, Xing; Zheng, Jinsong; Wang, Ding; Xiao, Wuhan

2017-01-01

Aquatic mammals, such as cetaceans experience various depths, with accordingly diverse oxygenation, thus, cetaceans have developed adaptations for hypoxia, but mechanisms underlying this tolerance to low oxygen are unclear. Here we analyzed VHL and HIF-2α, in the hypoxia signaling pathway. Variations in VHL are greater than HIF-2α between cetaceans and terrestrial mammals, and beluga whale VHL (BW-VHL) promotes HIF-2α degradation under hypoxia. BW-VHL catalyzes BW-HIF-2α to form K48-linked poly-ubiquitin chains mainly at the lysine 429 of BW-HIF-2α (K429) and induces BW-HIF-2α for proteasomal degradation. W100 within BW-VHL is a key site for BW-VHL functionally and BW-VHL enhances transcriptional activity of BW-HIF-2α under hypoxia. Our data therefore reveal that BW-VHL has a unique function that may contribute to hypoxic adaptation. PMID:28178687

Targeting HIF-2 α in clear cell renal cell carcinoma: A promising therapeutic strategy.

PubMed

Martínez-Sáez, Olga; Gajate Borau, Pablo; Alonso-Gordoa, Teresa; Molina-Cerrillo, Javier; Grande, Enrique

2017-03-01

The loss of the Von Hippel-Lindau tumor suppressor (VHL) is a key oncogenic event in the vast majority of patients with clear cell renal cell carcinoma (ccRCC). With the loss of the VHL protein (pVHL) function, the hypoxia inducible factor α (HIF-α) accumulates inside the tumor cell and dimerizes with HIF-β. The HIF-α/HIF-β complex transcriptionally activates hundreds of genes promoting the adaptation to hypoxia that is implicated in tumor development. There is growing evidence showing that HIF-2α subunit has a central role in ccRCC over HIF-1α. Thus, efforts have been made to specifically target this pathway. PT2385 and PT2399 are first-in-class, orally available, small molecule inhibitors of HIF-2 that selectively disrupt the heterodimerization of HIF-2α with HIF-1β. Preclinical and clinical data indicate that these new molecules are effective in blocking cancer cell growth, proliferation, and tumor angiogenesis characteristic in ccRCC. Treatment with HIF-2α specific antagonists, either alone or in combination with immunotherapy or other antiangiogenic agents have the potential to transform the therapeutic landscape in this tumor in the future. Herein, we summarize the molecular background behind the use of HIF-2α inhibitors in ccRCC and give an overview of the development of new agents in this setting. Copyright © 2017 Elsevier B.V. All rights reserved.

Steroid receptor coactivator-3 regulates glucose metabolism in bladder cancer cells through coactivation of hypoxia inducible factor 1α.

PubMed

Zhao, Wei; Chang, Cunjie; Cui, Yangyan; Zhao, Xiaozhi; Yang, Jun; Shen, Lan; Zhou, Ji; Hou, Zhibo; Zhang, Zhen; Ye, Changxiao; Hasenmayer, Donald; Perkins, Robert; Huang, Xiaojing; Yao, Xin; Yu, Like; Huang, Ruimin; Zhang, Dianzheng; Guo, Hongqian; Yan, Jun

2014-04-18

Cancer cell proliferation is a metabolically demanding process, requiring high glycolysis, which is known as "Warburg effect," to support anabolic growth. Steroid receptor coactivator-3 (SRC-3), a steroid receptor coactivator, is overexpressed and/or amplified in multiple cancer types, including non-steroid targeted cancers, such as urinary bladder cancer (UBC). However, whether SRC-3 regulates the metabolic reprogramming for cancer cell growth is unknown. Here, we reported that overexpression of SRC-3 accelerated UBC cell growth, accompanied by the increased expression of genes involved in glycolysis. Knockdown of SRC-3 reduced the UBC cell glycolytic rate under hypoxia, decreased tumor growth in nude mice, with reduction of proliferating cell nuclear antigen and lactate dehydrogenase expression levels. We further revealed that SRC-3 could interact with hypoxia inducible factor 1α (HIF1α), which is a key transcription factor required for glycolysis, and coactivate its transcriptional activity. SRC-3 was recruited to the promoters of HIF1α-target genes, such as glut1 and pgk1. The positive correlation of expression levels between SRC-3 and Glut1 proteins was demonstrated in human UBC patient samples. Inhibition of glycolysis through targeting HK2 or LDHA decelerated SRC-3 overexpression-induced cell growth. In summary, overexpression of SRC-3 promoted glycolysis in bladder cancer cells through HIF1α to facilitate tumorigenesis, which may be an intriguing drug target for bladder cancer therapy.

PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a)

PubMed Central

Emerling, Brooke M.; Weinberg, Frank; Liu, Juinn-Lin; Mak, Tak W.; Chandel, Navdeep S.

2008-01-01

The tumor suppressor PTEN is mutated or deleted in many tumors, causing the activation of the PI3K pathway. Here, we show that the loss of PTEN increases the transcriptional activity of hypoxia-inducible factor 1 (HIF-1) through the inactivation of Forkhead transcription factors (FOXO) in PTEN-null cells. Reintroduction of PTEN into the nucleus, overexpression of a nonphosphorylatable FOXO3a, which accumulates in the nucleus, or inhibition of nuclear export of FOXO3a by leptomycin B represses HIF-1 transcriptional activity in PTEN-null cells. HIF-1 transcriptional activity increases in PTEN-positive cells depleted of FOXO3a with siRNA. PTEN and FOXO3a regulate the transactivation domain of HIF-1α. Chromatin immunoprecipitation indicates that FOXO3a complexes with HIF-1α and p300 on the Glut-1 promoter, a HIF-1 target gene. Overexpression of p300 reverses FOXO3a-mediated repression of HIF-1 transcriptional activity. Coimmunoprecipitation and GAL4-HIF-1α transactivation assays reveal that FOXO3a interferes with p300-dependent HIF-1 transcriptional activity. Thus, FOXO3a negatively regulates HIF-1 transcriptional activity. PMID:18268343

Hypoxia and hypoxia-inducible factors in neuroblastoma.

PubMed

Påhlman, Sven; Mohlin, Sofie

2018-05-01

Hypoxia (i.e., low oxygen levels) is a known feature of aggressive tumors. Cells, including tumor cells, respond to conditions of insufficient oxygen by activating a transcriptional program mainly driven by hypoxia-inducible factors (HIF)-1 and HIF-2. Both HIF-1α and HIF-2α expression levels have been shown to correlate to patient outcome in various tumor forms and in neuroblastoma, a solid childhood tumor of the sympathetic nervous system, in particular, HIF-2α marks a subpopulation of immature neural crest-like perivascularly located cells and associates with aggressive disease and distant metastasis. It has for long been recognized that the HIF-α subunits are oxygen-dependently regulated at the post-translational level, via ubiquitination and proteasomal degradation. Evidence of oxygen-independent mechanisms of regulation, transcriptional control of EPAS1/HIF2A and possible cytoplasmic activities of HIF-2α has also emerged during recent years. In this review, we discuss these non-conventional actions of HIF-2α, its putative role as a therapeutic target and the constraints it carries, as well as the importance of HIF-2 activity in a vascularized setting, the so-called pseudo-hypoxic state.

Two structurally distinct chalcone dimers from Helichrysum zivojinii and their activities in cancer cell lines.

PubMed

Aljančić, Ivana S; Vučković, Ivan; Jadranin, Milka; Pešić, Milica; Dorđević, Iris; Podolski-Renić, Ana; Stojković, Sonja; Menković, Nebojša; Vajs, Vlatka E; Milosavljević, Slobodan M

2014-02-01

Dimers tomoroside A (1) and tomoroside B (2) of the co-occuring known chalcone monomer (3), along with the seven known flavonoid glucosides (4-10), were isolated from the aerial parts of Helichrysum zivojinii Černjavski & Soška. The structures of the isolated compounds were elucidated by spectroscopic techniques. Compound 1 inhibited topo IIα and hif-1α expression and stimulated doxorubicin anticancer effect, while 2 increased the expression of hif-1α, probably acting as antioxidant and redox status modulator. Notably, 2 synergized with Tipifarnib showing potential to improve the action of this new chemotherapeutic involved in the modulation of mitogene activated protein (MAP) kinase signaling pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

Hypoxia preconditioning protection of corneal stromal cells requires HIF1alpha but not VEGF.

PubMed

Xing, Dongmei; Bonanno, Joseph A

2009-05-18

Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1alpha (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor). Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1alpha, and HIF-1alpha siRNA to test if HIF-1alpha activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1alpha and VEGF in transcriptional and translational levels. During hypoxia (0.5% O2), 5 muM cadmium chloride completely inhibited HIF-1alpha expression and reversed the protection by hypoxia preconditioning. HIF-1alpha siRNA (15 nM) reduced HIF-1alpha expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1alpha dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning. VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning. However, the transcription and translation of VEGF were paradoxically increased by siHIF-1alpha, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1alpha. These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1alpha, but that VEGF is not a component of the protection.

Trichostatin A resistance is facilitated by HIF-1α acetylation in HeLa human cervical cancer cells under normoxic conditions

PubMed Central

Lee, Jae-Wook; Yang, Dong Hee; Park, Sojin; Han, Hae-Kyoung; Park, Jong-Wan; Kim, Bo Yeon; Um, Sung Hee; Moon, Eun-Yi

2018-01-01

Trichostatin A (TSA) is an anticancer drug that inhibits histone deacetylases (HDACs). Hypoxia-inducible factor 1 (HIF-1) participates in tumor angiogenesis by upregulating target genes, such as vascular endothelial growth factor (VEGF). In the present study, we investigated whether TSA treatment increases HIF-1α stabilization via acetylation under normoxic conditions, which would lead to VEGF upregulation and resistance to anticancer drugs. TSA enhanced total HIF-1α and VEGF-HRE reporter activity under normoxic conditions. When cells were transfected with GFP-HIF-1α, treatment with TSA increased the number of green fluorescence protein (GFP)-positive cells. TSA also enhanced the nuclear translocation of HIF-1α protein, as assessed by immunoblotting and as evidenced by increased nuclear localization of GFP-HIF-1α. An increase in the interaction between HIF-1α and the VEGF promoter, which was assessed by a chromatin immunoprecipitation (ChIP) assay, led to activation of the VEGF promoter. TSA acetylated HIF-1α at lysine (K) 674, which led to an increase in TSA-induced VEGF-HRE reporter activity. In addition, TSA-mediated cell death was reduced by the overexpression of HIF-1α but it was rescued by transfection with a HIF-1α mutant (K674R). These data demonstrate that HIF-1α may be stabilized and translocated into the nucleus for the activation of VEGF promoter by TSA-mediated acetylation at K674 under normoxic conditions. These findings suggest that HIF-1α acetylation may lead to resistance to anticancer therapeutics, such as HDAC inhibitors, including TSA. PMID:29416751

HIF-1α and HIF-2α induce angiogenesis and improve muscle energy recovery.

PubMed

Niemi, Henna; Honkonen, Krista; Korpisalo, Petra; Huusko, Jenni; Kansanen, Emilia; Merentie, Mari; Rissanen, Tuomas T; André, Helder; Pereira, Teresa; Poellinger, Lorenz; Alitalo, Kari; Ylä-Herttuala, Seppo

2014-10-01

Cardiovascular patients suffer from reduced blood flow leading to ischaemia and impaired tissue metabolism. Unfortunately, an increasing group of elderly patients cannot be treated with current revascularization methods. Thus, new treatment strategies are urgently needed. Hypoxia-inducible factors (HIFs) upregulate the expression of angiogenic mediators together with genes involved in energy metabolism and recovery of ischaemic tissues. Especially, HIF-2α is a novel factor, and only limited information is available about its therapeutic potential. Gene transfers with adenoviral HIF-1α and HIF-2α were performed into the mouse heart and rabbit ischaemic hindlimbs. Angiogenesis was evaluated by histology. Left ventricle function was analysed with echocardiography. Perfusion in rabbit skeletal muscles and energy recovery after electrical stimulation-induced exercise were measured with ultrasound and (31)P-magnetic resonance spectroscopy ((31)P-MRS), respectively. HIF-1α and HIF-2α gene transfers increased capillary size up to fivefold in myocardium and ischaemic skeletal muscles. Perfusion in skeletal muscles was increased by fourfold without oedema. Especially, AdHIF-1α enhanced the recovery of ischaemic muscles from electrical stimulation-induced energy depletion. Special characteristic of HIF-2α gene transfer was a strong capillary growth in muscle connective tissue and that HIF-2α gene transfer maintained left ventricle function. We conclude that both AdHIF-1α and AdHIF-2α gene transfers induced beneficial angiogenesis in vivo. Transient moderate increases in angiogenesis improved energy recovery after exercise in ischaemic muscles. This study shows for the first time that a moderate increase in angiogenesis is enough to improve tissue energy metabolism, which is potentially a very useful feature for cardiovascular gene therapy. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

Evidence for Adaptation to the Tibetan Plateau Inferred from Tibetan Loach Transcriptomes

PubMed Central

Wang, Ying; Yang, Liandong; Zhou, Kun; Zhang, Yanping; Song, Zhaobin; He, Shunping

2015-01-01

Abstract Triplophysa fishes are the primary component of the fish fauna on the Tibetan Plateau and are well adapted to the high-altitude environment. Despite the importance of Triplophysa fishes on the plateau, the genetic mechanisms of the adaptations of these fishes to this high-altitude environment remain poorly understood. In this study, we generated the transcriptome sequences for three Triplophysa fishes, that is, Triplophysa siluroides, Triplophysa scleroptera, and Triplophysa dalaica, and used these and the previously available transcriptome and genome sequences from fishes living at low altitudes to identify potential genetic mechanisms for the high-altitude adaptations in Triplophysa fishes. An analysis of 2,269 orthologous genes among cave fish (Astyanax mexicanus), zebrafish (Danio rerio), large-scale loach (Paramisgurnus dabryanus), and Triplophysa fishes revealed that each of the terminal branches of the Triplophysa fishes had a significantly higher ratio of nonsynonymous to synonymous substitutions than that of the branches of the fishes from low altitudes, which provided consistent evidence for genome-wide rapid evolution in the Triplophysa genus. Many of the GO (Gene Ontology) categories associated with energy metabolism and hypoxia response exhibited accelerated evolution in the Triplophysa fishes compared with the large-scale loach. The genes that exhibited signs of positive selection and rapid evolution in the Triplophysa fishes were also significantly enriched in energy metabolism and hypoxia response categories. Our analysis identified widespread Triplophysa-specific nonsynonymous mutations in the fast evolving genes and positively selected genes. Moreover, we detected significant evidence of positive selection in the HIF (hypoxia-inducible factor)-1A and HIF-2B genes in Triplophysa fishes and found that the Triplophysa-specific nonsynonymous mutations in the HIF-1A and HIF-2B genes were associated with functional changes. Overall, our study provides new insights into the adaptations and evolution of fishes in the high-altitude environment of the Tibetan Plateau and complements previous findings on the adaptations of mammals and birds to high altitudes. PMID:26454018

Arylsulfonamide KCN1 inhibits in vivo glioma growth and interferes with HIF signaling by disrupting HIF-1α interaction with co-factors p300/CBP

PubMed Central

Yin, Shaoman; Kaluz, Stefan; Devi, Narra S.; Jabbar, Adnan A.; de Noronha, Rita G.; Mun, Jiyoung; Zhang, Zhaobin; Boreddy, Purushotham R.; Wang, Wei; Wang, Zhibo; Abbruscato, Thomas; Chen, Zhengjia; Olson, Jeffrey J.; Zhang, Ruiwen; Goodman, Mark M.; Nicolaou, K.C.; Van Meir, Erwin G.

2012-01-01

Purpose The hypoxia inducible factor-1 (HIF-1) plays a critical role in tumor adaptation to hypoxia, and its elevated expression correlates with poor prognosis and treatment failure in cancer patients. In this study, we determined whether 3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide, KCN1, the lead inhibitor in a novel class of arylsulfonamide inhibitors of the HIF-1 pathway, had anti-tumorigenic properties in vivo and further defined its mechanism of action. Experimental Design We studied the inhibitory effect of systemic KCN1 delivery on the growth of human brain tumors in mice. To define mechanisms of KCN1 anti-HIF activities, we examined its influence on the assembly of a functional HIF1α/HIF1β/p300 transcription complex. Results KCN1 specifically inhibited HIF reporter gene activity in several glioma cell lines at the nanomolar level. KCN1 also downregulated transcription of endogenous HIF-1 target genes, such as VEGF, Glut-1 and carbonic anhydrase 9, in an HRE-dependent manner. KCN1 potently inhibited the growth of subcutaneous malignant glioma tumor xenografts with minimal adverse effects on the host. It also induced a temporary survival benefit in an intracranial model of glioma but had no effect in a model of melanoma metastasis to the brain. Mechanistically, KCN1 did not down-regulate levels of HIF-1α or other components of the HIF transcriptional complex; rather, it antagonized hypoxia-inducible transcription by disrupting the interaction of HIF-1α with transcriptional co-activators p300/CBP. Conclusions Our results suggest that the new HIF pathway inhibitor KCN1 has antitumor activity in mouse models, supporting its further translation for the treatment of human tumors displaying hypoxia or HIF overexpression. PMID:22923450

Requirement of Hsp105 in CoCl{sub 2}-induced HIF-1α accumulation and transcriptional activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mikami, Hiroki; Saito, Youhei, E-mail: ysaito@mb.kyoto-phu.ac.jp; Okamoto, Namiko

The mammalian stress protein Hsp105α protects cells from stress conditions. Several studies have indicated that Hsp105α is overexpressed in many types of solid tumors, which contain hypoxic microenvironments. However, the role of Hsp105α in hypoxic tumors remains largely unknown. We herein demonstrated the involvement of Hsp105α in HIF-1 functions induced by the hypoxia-mimetic agent CoCl{sub 2}. While Hsp105α is mainly localized in the cytoplasm under normal conditions, a treatment with CoCl{sub 2} induces the nuclear localization of Hsp105α, which correlated with HIF-1α expression levels. The overexpression of degradation-resistant HIF-1α enhances the nuclear localization of Hsp105α without the CoCl{sub 2} treatment.more » The CoCl{sub 2}-dependent transcriptional activation of HIF-1, which is measured using a reporter gene containing a HIF-responsive element, is reduced by the knockdown of Hsp105α. Furthermore, the CoCl{sub 2}-induced accumulation of HIF-1α is enhanced by heat shock, which results in the nuclear localization of Hsp105, and is suppressed by the knockdown of Hsp105. Hsp105 associates with HIF-1α in CoCl{sub 2}-treated cells. These results suggest that Hsp105α plays an important role in the functions of HIF-1 under hypoxic conditions, in which Hsp105α enhances the accumulation and transcriptional activity of HIF-1 through the HIF-1α-mediated nuclear localization of Hsp105α. - Highlights: • Hsp105α is required for the CoCl{sub 2}-induced transcriptional activation and accumulation of HIF-1. • Hsp105α localizes to the nucleus and interacts with HIF-1α in CoCl{sub 2}-treated cells. • Hsp105 enhances the CoCl{sub 2}-induced accumulation of HIF-1α under heat shock conditions.« less

Prolonged fasting activates hypoxia inducible factor -1α, -2α and -3α in a tissue-specific manner in northern elephant seal pups

PubMed Central

Soñanez-Organis, José G.; Vázquez-Medina, José P.; Crocker, Daniel E.; Ortiz, Rudy M.

2013-01-01

Hypoxia inducible factors (HIFs) are important regulators of energy homeostasis and cellular adaptation to low oxygen conditions. Northern elephant seals are naturally adapted to prolonged periods (1–2 months) of food deprivation (fasting) that result in metabolic changes that may activate HIF-1. However, the effects of prolonged fasting on HIFs are not well defined. We obtained the full-length cDNAs of HIF-1α and HIF-2α, and partial cDNA of HIF-3α in northern elephant seal pups. We also measured mRNA and nuclear protein content of HIF-1α, -2α, -3α in muscle and adipose during prolonged fasting (1, 3, 5 & 7 wks), along with mRNA expression of HIF-mediated genes, LDH and VEGF. HIF-1α, -2α and -3α are 2595, 2852 and 1842 bp and encode proteins of 823, 864 and 586 amino acid residues with conserved domains needed for their function (bHLH and PAS) and regulation (ODD and TAD). HIF-1α and -2α mRNA expression increased 3- to 5-fold after 7 weeks of fasting in adipose and muscle, whereas HIF-3α increased 5-fold after 7 weeks of fasting in adipose. HIF-2α protein expression was detected in nuclear fractions from adipose and muscle, increasing approximately 2-fold, respectively with fasting. Expression of VEGF increased 3-fold after 7 weeks in adipose and muscle, whereas LDH mRNA expression increased 12-fold after 7 weeks in adipose. While the 3 HIFα genes are expressed in muscle and adipose, only HIF-2α protein was detectable in the nucleus suggesting that HIF-2α may contribute more significantly in the up-regulation of genes involved in the metabolic adaption during fasting in the elephant seal. PMID:23707926

VHL and HIF-1α: gene variations and prognosis in early-stage clear cell renal cell carcinoma.

PubMed

Lessi, Francesca; Mazzanti, Chiara Maria; Tomei, Sara; Di Cristofano, Claudio; Minervini, Andrea; Menicagli, Michele; Apollo, Alessandro; Masieri, Lorenzo; Collecchi, Paola; Minervini, Riccardo; Carini, Marco; Bevilacqua, Generoso

2014-03-01

Von Hipple-Lindau gene (VHL) inactivation represents the most frequent abnormality in clear cell renal cell carcinoma (ccRCC). Hypoxia-inducible factor-1α (HIF-1α) expression is regulated by O2 level. In normal O2 conditions, VHL binds HIF-1α and allows HIF-1α proteasomal degradation. A single-nucleotide polymorphism (SNP) has been found located in the oxygen-dependent degradation domain at codon 582 (C1772T, rs11549465, Pro582Ser). In hypoxia, VHL/HIF-1α interaction is abolished and HIF-1α activates target genes in the nucleus. This study analyzes the impact of genetic alterations and protein expression of VHL and the C1772T SNP of HIF-1α gene (HIF-1α) on prognosis in early-stage ccRCC (pT1a, pT1b, and pT2). Mutational analysis of the entire VHL sequence and the genotyping of HIF-1α C1772T SNP were performed together with VHL promoter methylation analysis and loss of heterozygosis (LOH) analysis at (3p25) locus. Data obtained were correlated with VHL and HIF-1α protein expression and with tumor-specific survival (TSS). VHL mutations, methylation status, and LOH were detected in 51, 11, and 12% of cases, respectively. Our results support the association between biallelic alterations and/or VHL silencing with a worse TSS. Moreover, we found a significant association between the HIF-1α C1772C genotype and a worse TSS. The same association was found when testing the presence of HIF-1α protein in the nucleus. Our results highlight the role of VHL/HIF-1α pathway in RCC and support the molecular heterogeneity of early-stage ccRCC. More important, we show the involvement of HIF-1α C1772T SNP in ccRCC progression.

Prolonged fasting activates hypoxia inducible factors-1α, -2α and -3α in a tissue-specific manner in northern elephant seal pups.

PubMed

Soñanez-Organis, José G; Vázquez-Medina, José P; Crocker, Daniel E; Ortiz, Rudy M

2013-09-10

Hypoxia inducible factors (HIFs) are important regulators of energy homeostasis and cellular adaptation to low oxygen conditions. Northern elephant seals are naturally adapted to prolonged periods (1-2 months) of food deprivation (fasting) which result in metabolic changes that may activate HIF-1. However, the effects of prolonged fasting on HIFs are not well defined. We obtained the full-length cDNAs of HIF-1α and HIF-2α, and partial cDNA of HIF-3α in northern elephant seal pups. We also measured mRNA and nuclear protein content of HIF-1α, -2α, -3α in muscle and adipose during prolonged fasting (1, 3, 5 & 7 weeks), along with mRNA expression of HIF-mediated genes, LDH and VEGF. HIF-1α, -2α and -3α are 2595, 2852 and 1842 bp and encode proteins of 823, 864 and 586 amino acid residues with conserved domains needed for their function (bHLH and PAS) and regulation (ODD and TAD). HIF-1α and -2α mRNA expression increased 3- to 5-fold after 7 weeks of fasting in adipose and muscle, whereas HIF-3α increased 5-fold after 7 weeks of fasting in adipose. HIF-2α protein expression was detected in nuclear fractions from adipose and muscle, increasing approximately 2-fold, respectively with fasting. Expression of VEGF increased 3-fold after 7 weeks in adipose and muscle, whereas LDH mRNA expression increased 12-fold after 7 weeks in adipose. While the 3 HIFα genes are expressed in muscle and adipose, only HIF-2α protein was detectable in the nucleus suggesting that HIF-2α may contribute more significantly in the up-regulation of genes involved in the metabolic adaptation during fasting in the elephant seal. Copyright © 2013 Elsevier B.V. All rights reserved.

Endothelial Hypoxia-Inducible Factor-1α Promotes Atherosclerosis and Monocyte Recruitment by Upregulating MicroRNA-19a.

PubMed

Akhtar, Shamima; Hartmann, Petra; Karshovska, Ela; Rinderknecht, Fatuma-Ayaan; Subramanian, Pallavi; Gremse, Felix; Grommes, Jochen; Jacobs, Michael; Kiessling, Fabian; Weber, Christian; Steffens, Sabine; Schober, Andreas

2015-12-01

Chemokines mediate monocyte adhesion to dysfunctional endothelial cells (ECs) and promote arterial inflammation during atherosclerosis. Hypoxia-inducible factor (HIF)-1α is expressed in various cell types of atherosclerotic lesions and is associated with lesional inflammation. However, the impact of endothelial HIF-1α in atherosclerosis is unclear. HIF-1α was detectable in the nucleus of ECs covering murine and human atherosclerotic lesions. To study the role of endothelial HIF-1α in atherosclerosis, deletion of the Hif1a gene was induced in ECs from apolipoprotein E knockout mice (EC-Hif1a(-/-)) by Tamoxifen injection. The formation of atherosclerotic lesions, the lesional macrophage accumulation, and the expression of CXCL1 in ECs were reduced after partial carotid ligation in EC-Hif1a(-/-) compared with control mice. Moreover, the lesion area and the lesional macrophage accumulation were decreased in the aortas of EC-Hif1a(-/-) mice compared with control mice during diet-induced atherosclerosis. In vitro, mildly oxidized low-density lipoprotein or lysophosphatidic acid 20:4 increased endothelial CXCL1 expression and monocyte adhesion by inducing HIF-1α expression. Moreover, endothelial Hif1a deficiency resulted in downregulation of miR-19a in atherosclerotic arteries determined by microRNA profiling. In vitro, HIF-1α-induced miR-19a expression mediated the upregulation of CXCL1 in mildly oxidized low-density lipoprotein-stimulated ECs. These results indicate that hyperlipidemia upregulates HIF-1α expression in ECs by mildly oxidized low-density lipoprotein-derived unsaturated lysophosphatidic acid. Endothelial HIF-1α promoted atherosclerosis by triggering miR-19a-mediated CXCL1 expression and monocyte adhesion, indicating that inhibition of the endothelial HIF-1α/miR-19a pathway may be a therapeutic option against atherosclerosis. © 2015 American Heart Association, Inc.

Sphingosine-1-phosphate: a novel nonhypoxic activator of hypoxia-inducible factor-1 in vascular cells.

PubMed

Michaud, Maude D; Robitaille, Geneviève A; Gratton, Jean-Philippe; Richard, Darren E

2009-06-01

Sphingosine-1-phosphate (S1P) is a potent bioactive phospholipid responsible for a variety of vascular cell responses. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of genes essential for adaptation to low oxygen. S1P and HIF-1 are both important mediators of vascular cell responses such as migation, proliferation, and survival. Studies have shown that nonhypoxic stimuli can activate HIF-1 in oxygenated conditions. Here, we attempt to determine whether S1P can modulate the vascular activation of HIF-1. We show that in vascular endothelial and smooth muscle cells, activation of the S1P type-2 receptor by S1P strongly increases HIF-1 alpha protein levels, the active subunit of HIF-1. This is achieved through pVHL-independent stabilization of HIF-1 alpha. We demonstrate that the HIF-1 nuclear complex, formed on S1P stimulation, is transcriptionally active and specifically binds to a hypoxia-responsive elements. Moreover, S1P activates the expression of genes known to be closely regulated by HIF-1. Our results identify S1P as a novel and potent nonhypoxic activator of HIF-1. We believe that understanding the role played by HIF-1 in S1P gene regulation will have a strong impact on different aspects of vascular biology.

Erythrocytosis and Pulmonary Hypertension in a Mouse Model of Human HIF2A Gain of Function Mutation*

PubMed Central

Tan, Qiulin; Kerestes, Heddy; Percy, Melanie J.; Pietrofesa, Ralph; Chen, Li; Khurana, Tejvir S.; Christofidou-Solomidou, Melpo; Lappin, Terence R. J.; Lee, Frank S.

2013-01-01

The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-α, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-α to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2α, one of two main Hif-α isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2α as a cause of erythrocytosis, highlight the importance of this HIF-α isoform in erythropoiesis, and point to physiologic consequences of HIF-2α dysregulation. PMID:23640890

Targeted inactivation of fh1 causes proliferative renal cyst development and activation of the hypoxia pathway.

PubMed

Pollard, Patrick J; Spencer-Dene, Bradley; Shukla, Deepa; Howarth, Kimberley; Nye, Emma; El-Bahrawy, Mona; Deheragoda, Maesha; Joannou, Maria; McDonald, Stuart; Martin, Alison; Igarashi, Peter; Varsani-Brown, Sunita; Rosewell, Ian; Poulsom, Richard; Maxwell, Patrick; Stamp, Gordon W; Tomlinson, Ian P M

2007-04-01

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.

Targeting HIF-2α as therapy for advanced cancers.

PubMed

Murugesan, Thanabal; Rajajeyabalachandran, Gurukumari; Kumar, Swetha; Nagaraju, Shruthi; Kumar, Sooriya

2018-05-14

Hypoxia-inducible factors (HIF-1α, -2α -3α, and -β) are key factors that control hypoxia-induced carcinogenic pathways. HIF-1α is predominantly involved in the early stages of cancer, whereas HIF-2α is actively involved in the later stages; in addition, chronic (prolonged) rather than acute (short) hypoxia is a feature of metastasis and chemoresistance that occur during the later stages of cancer. Oncometabolites, onco-miRNAs, glucose deprivation, pseudohypoxia, cytokine/chemokine secretion, and some unique upstream proteins are involved in the signaling switch from HIF-1α to HIF-2α; thus, understanding this signaling switch is critical for the treatment of advanced cancer. In this review, we highlight data relating to HIF-2α rather than HIF-1α signaling in cancer pathways and discuss prospective drugs that target this important factor. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sequence and functional characterization of hypoxia-inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus

PubMed Central

Townley, Ian K.; Karchner, Sibel I.; Skripnikova, Elena; Wiese, Thomas E.; Hahn, Mark E.

2017-01-01

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α—HIF2αb—a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations. PMID:28039194

Sequence and functional characterization of hypoxia-inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus.

PubMed

Townley, Ian K; Karchner, Sibel I; Skripnikova, Elena; Wiese, Thomas E; Hahn, Mark E; Rees, Bernard B

2017-03-01

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α-HIF2αb-a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations. Copyright © 2017 the American Physiological Society.

Overcoming cisplatin resistance of ovarian cancer cells by targeting HIF-1-regulated cancer metabolism

PubMed Central

Ai, Zhihong; Lu, Yang; Qiu, Songbo; Fan, Zhen

2016-01-01

Cisplatin is currently one of the most effective chemotherapeutic drugs used for treating ovarian cancer; however, resistance to cisplatin is common. In this study, we explored an experimental strategy for overcoming cisplatin resistance of human ovarian cancer from the new perspective of cancer cell metabolism. By using two pairs of genetically matched cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines, we tested the hypothesis that downregulating hypoxia-inducible factor-1 (HIF-1), which regulates metabolic enzymes involved in glycolysis, is a promising strategy for overcoming cisplatin resistance of human ovarian cancer cells. We found that cisplatin downregulated the level of the regulatable α subunit of HIF-1, HIF-1α, in cisplatin-sensitive ovarian cancer cells through enhancing HIF-1α degradation but did not downregulate HIF-1α in their cisplatin-resistant counterparts. Overexpression of a degradation-resistant HIF-1α (HIF-1α ΔODD) reduced cisplatin-induced apoptosis in cisplatin-sensitive cells, whereas genetic knockdown of HIF-1α or pharmacological promotion of HIF-1α degradation enhanced response to cisplatin in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cells. We further demonstrated that knockdown of HIF-1α improved the response of cisplatin-resistant ovarian cancer cells to cisplatin by redirecting the aerobic glycolysis in the resistant cancer cells towards mitochondrial oxidative phosphorylation, leading to cell death through overproduction of reactive oxygen species. Our findings suggest that the HIF-1α-regulated cancer metabolism pathway could be a novel target for overcoming cisplatin resistance in ovarian cancer. PMID:26801746

SUPPRESSION OF HIF-1α TRANSCRIPTIONAL ACTIVITY BY THE HIF PROLYL HYDROXYLASE EGLN1*

PubMed Central

To, Kenneth K. W.; Huang, L. Eric

2005-01-01

The cellular response to hypoxia is, at least in part, mediated by the transcriptional regulation of hypoxia-responsive genes involved in balancing the intracellular ATP production and consumption. Recent evidence suggests that the transcription factor, HIF-1α, functions as a master regulator of oxygen homeostasis by controlling a broad range of cellular events in hypoxia. In normoxia, HIF-1α is targeted for destruction via prolyl hydroxylation, an oxygen-dependent modification that signals for recognition by the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor (VHL). Three HIF prolyl hydroxylases (EGLN1, EGLN2, and EGLN3) have been identified in mammals, among which, EGLN1 and EGLN3, are hypoxia-inducible at their mRNA levels in a HIF-1α-dependent manner. In this study, we demonstrate that apart from promoting HIF-1α proteolysis in normoxia, EGLN1 specifically represses HIF-1α transcriptional activity in hypoxia. Ectopic expression of EGLN1 inhibited HIF-1α transcriptional activity without altering its protein levels in a VHL-deficient cell line, indicating a discrete activity of EGLN1 in transcriptional repression. Conversely, silencing of EGLN1 expression augmented HIF-1α transcriptional activity and its target gene expression in hypoxia. Hence, we propose that the accumulated EGLN1 in hypoxia acts as a negative-feedback mechanism to modulate HIF-1α target gene expression. Our finding also provides new insight into the pharmacological manipulation of the HIF prolyl hydroxylase for ischemic diseases. PMID:16157596

The involvement of hypoxia-inducible transcription factor-1-dependent pathway in nickel carcinogenesis.

PubMed

Salnikow, Konstantin; Davidson, Todd; Zhang, Qunwei; Chen, Lung Chi; Su, Weichen; Costa, Max

2003-07-01

Nickel is a potent environmental pollutant in industrial countries. Because nickel compounds are carcinogenic, exposure to nickel represents a serious hazard to human health. The understanding of how nickel exerts its toxic and carcinogenic effects at a molecular level may be important in risk assessment, as well as in the treatment and prevention of occupational diseases. Previously, using human and rodent cells in vitro, we showed that hypoxia-inducible signaling pathway was activated by carcinogenic nickel compounds. Acute exposure to nickel resulted in the accumulation of hypoxia-inducible transcription factor (HIF)-1, which strongly activated hypoxia-inducible genes, including the recently discovered tumor marker NDRG1 (Cap43). To further identify HIF-1-dependent nickel-inducible genes and to understand the role of the HIF-dependent signaling pathway in nickel-induced transformation, we used the Affymetrix GeneChip to compare the gene expression profiles in wild-type cells or in cells from HIF-1 alpha knockout mouse embryos exposed to nickel chloride. As expected, when we examined 12,000 genes for expression changes, we found that genes coding for glycolytic enzymes and glucose transporters, known to be regulated by HIF-1 transcription factor, were induced by nickel only in HIF-1 alpha-proficient cells. In addition, we found a number of other hypoxia-inducible genes up-regulated by nickel in a HIF-dependent manner including BCL-2-binding protein Nip3, EGLN1, hypoxia-inducible gene 1 (HIG1), and prolyl 4-hydroxylase. Additionally, we found a number of genes induced by nickel in a HIF-independent manner, suggesting that Ni activated other signaling pathways besides HIF-1. Finally, we found that in HIF-1 alpha knockout cells, nickel strongly induced the expression of the whole group of genes that were not expressed in the presence of HIF-1. Because the majority of modulated genes were induced or suppressed by nickel in a HIF-1-dependent manner, we elucidated the role of HIF-1 transcription factor in cell transformation. In HIF-1 alpha-proficient cells, nickel exposure increased soft agar growth, whereas it decreased soft agar growth in HIF-1 alpha-deficient cells. We hypothesize that the induction of HIF-1 transcription factor by nickel may be important during the nickel-induced carcinogenic process.

Expression and clinical significance of the HIF-1a/ET-2 signaling pathway during the development and treatment of polycystic ovary syndrome.

PubMed

Wang, Fan; Zhang, Zhenghong; Wang, Zhaokai; Xiao, Kaizhuan; Wang, Qing; Su, Jingqian; Wang, Zhengchao

2015-04-01

Polycystic ovary syndrome (PCOS) is a major health problem in reproductive-aged women worldwide, but the precise pathogenesis of PCOS remains unclear. Our previous study revealed that hypoxia-inducible factor (HIF)-1a mediated endothelin (ET)-2 signaling plays an important role in ovulation in rats. Therefore, the present study used a PCOS rat model to test the hypotheses that HIF-1a signaling is expressed and inhibited in ovaries during PCOS formation and that the HIF-1a/ET-2 signaling pathway is a target of dimethyldiguanide (DMBG) in the clinical treatment of PCOS. First, the development of a PCOS model and the effect of DMBG treatment were examined through ovarian histology and serum hormone levels, which were consistent with previous reports. Second, HIF-1a and ET-2 expression were detected by immunohistochemistry and western blot. The results showed decreased HIF-1a/ET-2 expression in the ovaries of PCOS rats, whereas DMBG treatment reversed the protein decreases and improved the PCOS symptoms. Third, to understand the molecular mechanism, HIF-1a/ET-2 mRNA expression was also examined. Interestingly, HIF-1a mRNA increased in the ovaries of PCOS rats, while ET-2 mRNA decreased, indicating that HIF-1a protein degradation may be involved in POCS development and treatment. Finally, HIF prolyl hydroxylase (PHD) activity was examined to further clarify the contribution of HIF-1a signaling to the development and treatment of PCOS. The results suggested that the inhibition of HIF-1a/ET-2 signaling may be caused by increased PHD activity in PCOS. DMBG-treated PCOS may further activate HIF-1a signaling at least partly through inhibiting PHD activity. Taken together, these results indicate that HIF-1a signaling is inhibited in a PCOS rat model through increasing PHD activity. DMBG treatment improved PCOS by rescuing this pathway, suggesting that HIF-1a signaling plays an important role in the development and treatment of PCOS. This HIF-1a-mediated ET-2 signaling pathway may be an important mechanism regulating PCOS formation and treatment in mammalian ovaries in vivo and should be a new clinical target for PCOS prevention and treatment in the future.

Hypoxia-induced HIF1α targets in melanocytes reveal a molecular profile associated with poor melanoma prognosis

PubMed Central

Loftus, Stacie K.; Baxter, Laura L.; Cronin, Julia C.; Fufa, Temesgen D.; Pavan, William J.

2017-01-01

Summary Hypoxia and HIF1α signaling direct tissue-specific gene responses regulating tumor progression, invasion and metastasis. By integrating HIF1α knockdown and hypoxia-induced gene expression changes, this study identifies a melanocyte-specific, HIF1α-dependent/hypoxia-responsive gene expression signature. Integration of these gene expression changes with HIF1α ChIP-Seq analysis identifies 81 HIF1α direct target genes in melanocytes. The expression levels for ten of the HIF1α direct targets – GAPDH, PKM, PPAT, DARS, DTWD1, SEH1L, ZNF292, RLF, AGTRAP, and GPC6 – are significantly correlated with reduced time of Disease Free Status (DFS) in melanoma by logistic regression (P-value =0.0013) and ROC curve analysis (AUC= 0.826, P-value<0.0001). This HIF1α-regulated profile defines a melanocyte-specific response under hypoxia, and demonstrates the role of HIF1α as an invasive cell state gatekeeper in regulating cellular metabolism, chromatin and transcriptional regulation, vascularization and invasion. PMID:28168807

Erythrocytosis associated with a novel missense mutation in the HIF2A gene

PubMed Central

van Wijk, Richard; Sutherland, Scott; Van Wesel, Annet C.W.; Huizinga, Eric G.; Percy, Melanie J.; Bierings, Marc; Lee, Frank S.

2010-01-01

The ERYTHROPOIETIN (EPO) gene is regulated by the transcription factor Hypoxia Inducible Factor-α (HIF-α). In this pathway, Prolyl Hydroxylase Domain protein 2 (PHD2) hydroxylates two prolyl residues in HIF-α, which in turn promotes HIF-α degradation by the von Hippel Lindau (VHL) protein. Evidence that HIF-2α is the important isoform for EPO regulation in humans comes from the recent observation that mutations in the HIF2A gene are associated with cases of erythrocytosis. We report here a new erythrocytosis-associated mutation, p.Asp539Glu, in the HIF2A gene. Similar to all reported cases, the affected residue is in close vicinity and C-terminal to the primary hydroxylation site in HIF-2α, Pro531. This mutation, however, is notable in producing a rather subtle amino acid substitution. Nonetheless, we find that this mutation compromises binding of HIF-2α to both PHD2 and VHL, and we propose that this mutation is the cause of erythrocytosis in this individual. PMID:20007141

Mitochondrial deficiency impairs hypoxic induction of HIF-1 transcriptional activity and retards tumor growth

PubMed Central

Koido, Masaru; Haga, Naomi; Furuno, Aki; Tsukahara, Satomi; Sakurai, Junko; Tani, Yuri; Sato, Shigeo; Tomida, Akihiro

2017-01-01

Mitochondria can be involved in regulating cellular stress response to hypoxia and tumor growth, but little is known about that mechanistic relationship. Here, we show that mitochondrial deficiency severely retards tumor xenograft growth with impairing hypoxic induction of HIF-1 transcriptional activity. Using mtDNA-deficient ρ0 cells, we found that HIF-1 pathway activation was comparable in slow-growing ρ0 xenografts and rapid-growing parental xenografts. Interestingly, we found that ex vivo ρ0 cells derived from ρ0 xenografts exhibited slightly increased HIF-1α expression and modest HIF-1 pathway activation regardless of oxygen concentration. Surprisingly, ρ0 cells, as well as parental cells treated with oxidative phosphorylation inhibitors, were unable to boost HIF-1 transcriptional activity during hypoxia, although HIF-1α protein levels were ordinarily increased in these cells under hypoxic conditions. These findings indicate that mitochondrial deficiency causes loss of hypoxia-induced HIF-1 transcriptional activity and thereby might lead to a constitutive HIF-1 pathway activation as a cellular adaptation mechanism in tumor microenvironment. PMID:28060746

HIF-1α activates hypoxia-induced BCL-9 expression in human colorectal cancer cells

PubMed Central

Chen, Tian-Rui; Wei, Hai-feng; Song, Dian-Wen; Liu, Tie-Long; Yang, Xing-Hai; Fu, Chuan-Gang; Hu, Zhi-qian; Zhou, Wang; Yan, Wang-Jun; Xiao, Jian-Ru

2017-01-01

B-cell CLL/lymphoma 9 protein (BCL-9), a multi-functional co-factor in Wnt signaling, induced carcinogenesis as well as promoting tumor progression, metastasis and chemo-resistance in colorectal cancer (CRC). However, the mechanisms for increased BCL-9 expression in CRC were not well understood. Here, we report that hypoxia, a hallmark of solid tumors, induced BCL-9 mRNA expression in human CRC cells. Analysis of BCL-9 promoter revealed two functional hypoxia-responsive elements (HRE-B and HRE-C) that can be specifically bound with and be transactivated by hypoxia inducible factors (HIF) -1α but not HIF-2α. Consistently, ectopic expression of HIF-1α but not HIF-2α transcriptionally induced BCL-9 expression levels in cells. Knockdown of endogenous HIF-1α but not HIF-2α by siRNA largely abolished the induction of HIF by hypoxia. Furthermore, there was a strong association of HIF-1α expression with BCL-9 expression in human CRC specimens. In summary, results from this study demonstrated that hypoxia induced BCL-9 expression in human CRC cells mainly through HIF-1α, which could be an important underlying mechanism for increased BCL-9 expression in CRC. PMID:27121066

The yeast genome may harbor hypoxia response elements (HRE).

PubMed

Ferreira, Túlio César; Hertzberg, Libi; Gassmann, Max; Campos, Elida Geralda

2007-01-01

The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor activated when cells are submitted to hypoxia. The heterodimer is composed of two subunits, HIF-1alpha and the constitutively expressed HIF-1beta. During normoxia, HIF-1alpha is degraded by the 26S proteasome, but hypoxia causes HIF-1alpha to be stabilized, enter the nucleus and bind to HIF-1beta, thus forming the active complex. The complex then binds to the regulatory sequences of various genes involved in physiological and pathological processes. The specific regulatory sequence recognized by HIF-1 is the hypoxia response element (HRE) that has the consensus sequence 5'BRCGTGVBBB3'. Although the basic transcriptional regulation machinery is conserved between yeast and mammals, Saccharomyces cerevisiae does not express HIF-1 subunits. However, we hypothesized that baker's yeast has a protein analogous to HIF-1 which participates in the response to changes in oxygen levels by binding to HRE sequences. In this study we screened the yeast genome for HREs using probabilistic motif search tools. We described 24 yeast genes containing motifs with high probability of being HREs (p-value<0.1) and classified them according to biological function. Our results show that S. cerevisiae may harbor HREs and indicate that a transcription factor analogous to HIF-1 may exist in this organism.

Development of Inhibitors Targeting Hypoxia-Inducible Factor 1 and 2 for Cancer Therapy

PubMed Central

Yu, Tianchi

2017-01-01

Hypoxia is frequently observed in solid tumors and also one of the major obstacles for effective cancer therapies. Cancer cells take advantage of their ability to adapt hypoxia to initiate a special transcriptional program that renders them more aggressive biological behaviors. Hypoxia-inducible factors (HIFs) are the key factors that control hypoxia-inducible pathways by regulating the expression of a vast array of genes involved in cancer progression and treatment resistance. HIFs, mainly HIF-1 and -2, have become potential targets for developing novel cancer therapeutics. This article reviews the updated information in tumor HIF pathways, particularly recent advances in the development of HIF inhibitors. These inhibitors interfere with mRNA expression, protein synthesis, protein degradation and dimerization, DNA binding and transcriptional activity of HIF-1 and -2, or both. Despite efforts in the past two decades, no agents directly inhibiting HIFs have been approved for treating cancer patients. By analyzing results of the published reports, we put the perspectives at the end of the article. The therapeutic efficacy of HIF inhibitors may be improved if more efforts are devoted on developing agents that are able to simultaneously target HIF-1 and -2, increasing the penetrating capacity of HIF inhibitors, and selecting suitable patient subpopulations for clinical trials. PMID:28332352

PHGDH as a Key Enzyme for Serine Biosynthesis in HIF2α-Targeting Therapy for Renal Cell Carcinoma.

PubMed

Yoshino, Hirofumi; Nohata, Nijiro; Miyamoto, Kazutaka; Yonemori, Masaya; Sakaguchi, Takashi; Sugita, Satoshi; Itesako, Toshihiko; Kofuji, Satoshi; Nakagawa, Masayuki; Dahiya, Rajvir; Enokida, Hideki

2017-11-15

Continuous activation of hypoxia-inducible factor (HIF) is important for progression of renal cell carcinoma (RCC) and acquired resistance to antiangiogenic multikinase and mTOR inhibitors. Recently, HIF2α antagonists PT2385 and PT2399 were developed and are being evaluated in a phase I clinical trial for advanced or metastatic clear cell RCC (ccRCC). However, resistance to HIF2α antagonists would be expected to develop. In this study, we identified signals activated by HIF2α deficiency as candidate mediators of resistance to the HIF2α antagonists. We established sunitinib-resistant tumor cells in vivo and created HIF2α-deficient variants of these cells using CRISPR/Cas9 technology. Mechanistic investigations revealed that a regulator of the serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), was upregulated commonly in HIF2α-deficient tumor cells along with the serine biosynthesis pathway itself. Accordingly, treatment with a PHGDH inhibitor reduced the growth of HIF2α-deficient tumor cells in vivo and in vitro by inducing apoptosis. Our findings identify the serine biosynthesis pathway as a source of candidate therapeutic targets to eradicate advanced or metastatic ccRCC resistant to HIF2α antagonists. Cancer Res; 77(22); 6321-9. ©2017 AACR . ©2017 American Association for Cancer Research.

Regulation of Carotid Body Oxygen Sensing by Hypoxia-Inducible Factors

PubMed Central

Prabhakar, Nanduri R.; Semenza, Gregg L.

2015-01-01

Oxygen (O2) sensing by the carotid body and its chemosensory reflex is critical for homeostatic regulation of breathing and blood pressure. Carotid body responses to hypoxia are not uniform but instead exhibit remarkable inter-individual variations. The molecular mechanisms underlying variations in carotid body O2 sensing are not known. Hypoxia-inducible factor-1 (HIF-1) and HIF-2 mediate transcriptional responses to hypoxia. This article reviews the emerging evidence that proper expression of the HIF-α isoforms is a key molecular determinant for carotid body O2 sensing. HIF-1α deficiency leads to a blunted carotid body hypoxic response, which is due to increased abundance of HIF-2α, elevated anti-oxidant enzyme activity, and a reduced intracellular redox state. Conversely, HIF-2α deficiency results in augmented carotid body sensitivity to hypoxia, which is due to increased abundance of HIF-1α, elevated pro-oxidant enzyme activity, and an oxidized intracellular redox state. Double heterozygous mice with equally reduced HIF-1α and HIF-2α showed no abnormality in redox state or carotid body O2 sensing. Thus, mutual antagonism between HIF-α isoforms determines the redox state and thereby establishes the set point for hypoxic sensing by the carotid body. PMID:26265380

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ronghai; Zhang, Ping, E-mail: zpskx001@163.com; Li, Jinhang

The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent ofmore » HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.« less

Tet1 facilitates hypoxia tolerance by stabilizing the HIF-α proteins independent of its methylcytosine dioxygenase activity.

PubMed

Wang, Jing; Zhang, Dawei; Du, Juan; Zhou, Chi; Li, Zhi; Liu, Xing; Ouyang, Gang; Xiao, Wuhan

2017-12-15

Because of the requirement of oxygen (O2) to produce energy, aerobic organisms developed mechanisms to protect themselves against a shortage of oxygen in both acute status and chronic status. To date, how organisms tolerate acute hypoxia and the underlying mechanisms remain largely unknown. Here, we identify that Tet1, one member of the ten-eleven translocation (TET) family of methylcytosine dioxygenases, is required for hypoxia tolerance in zebrafish and mice. Tet1-null zebrafish and mice are more sensitive to hypoxic conditions compared with their wild-type siblings. We demonstrate that Tet1 stabilizes hypoxia-inducible factor α (HIF-α) and enhances HIF-α transcription activity independent of its enzymatic activity. In addition, we show that Tet1 modulates HIF-2α and HIF-1α through different mechanisms. Tet1 competes with prolyl hydroxylase protein 2 (PHD2) to bind to HIF-2α, resulting in a reduction of HIF-2α hydroxylation by PHD2. For HIF-1α, however, Tet1 has no effect on HIF-1α hydroxylation, but rather it appears to stabilize the C-terminus of HIF-1α by affecting lysine site modification. Furthermore, we found that Tet1 enhances rather than prevents poly-ubiquitination on HIF-α. Our results reveal a previously unrecognized function of Tet1 independent of its methylcytosine dioxygenase activity in hypoxia signaling. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis

PubMed Central

Liu, Xian-De; Yao, Jun; Tripathi, Durga Nand; Ding, Zhiyong; Xu, Yi; Sun, Mianen; Zhang, Jiangwei; Bai, Shanshan; German, Peter; Hoang, Anh; Zhou, Lijun; Jonasch, Darius; Zhang, Xuesong; Conti, Claudio J.; Efstathiou, Eleni; Tannir, Nizar M; Eissa, N. Tony; Mills, Gordon B.; Walker, Cheryl Lyn; Jonasch, Eric

2014-01-01

Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin-proteasome system (UPS). Here we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy-lysosome system components. Inhibition of autophagy increases HIF2α, while induction of autophagy decreases HIF2α. The E3 ligase von Hippel Lindau (VHL) and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, while proteasome inhibition induces autophagy and increases the HIF2α-p62 interaction. Importantly, clear cell renal cell carcinoma (ccRCC) is frequently associated with mono-allelic loss and/or mutation of autophagy related gene ATG7, and low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy plays an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism. PMID:24998849

Iron and oxygen sensing: a tale of 2 interacting elements?

PubMed

Simpson, Robert J; McKie, Andrew T

2015-02-01

Iron and oxygen metabolism are intimately linked with one another. A change in the level of either metabolite results in activation of common pathways. At the heart of these responses lies a group of iron and oxygen dependent enzymes called prolyl hydroxylases. Prolyl hydroxylases (PHDs) require both iron and oxygen for optimal activity and their biological activity is to carry out the critical post-translational modification of the addition of a hydroxyl group to specific proline residues within Hypoxia Inducible Factor (HIFs)-well known transcription factors originally thought to regulate responses to hypoxia but which are now known to regulate key iron metabolism proteins too. The addition of the hydroxyl group ultimately leads to the unbiquitylation and destruction of HIFs, thus PHDs control appropriate HIF transcriptional responses depending on cellular oxygen or iron levels. There are two major HIFs; HIF1α and HIF2α. In terms of responses to iron HIF2α is of major importance in key tissues such as the intestine where several iron transporters (Ferroportin, Dcytb) contain HREs within their promoters which bind HIF2α. Furthermore the recent discovery that HIF2α contains a 5' iron responsive element (IRE) has underlined the importance of HIF2α as a major player in iron metabolism. This review brings together recent findings with regard to the HIF2α/IRP network as well as other aspects of iron sensing in cells and tissues.

Hepatic Stellate Cells Orchestrate Clearance of Necrotic Cells in a HIF-1α-dependent Manner by Modulating Macrophage Phenotype in Mice

PubMed Central

Rockwell, Cheryl E.; Roth, Katherine J.; Chow, Aaron; O'Brien, Kate M; Albee, Ryan; Kelly, Kara; Towery, Keara; Luyendyk, James P.; Copple, Bryan L.

2014-01-01

Hypoxia-inducible factor-1α (HIF-1α) is activated in hepatic stellate cells (HSCs) by hypoxia, and regulates genes important for tissue repair. Whether HIF-1α is activated in HSCs after acute injury and contributes to liver regeneration, however, is not known. To investigate this, mice were generated with reduced levels of HIF-1α in HSCs by crossing HIF-1α floxed mice with mice that express Cre recombinase under control of the glial fibrillary acidic protein (GFAP) promoter (i.e., HIF-1α-GFAP Cre+ mice). These mice and control mice (i.e., HIF-1α-GFAP Cre- mice) were treated with a single dose of carbon tetrachloride, and liver injury and repair were assessed. After carbon tetrachloride, HIF-1α was activated in HSCs. Although liver injury was not different between the two strains of mice, during resolution of injury, clearance of necrotic cells was decreased in HIF-1α-GFAP Cre+ mice. In these mice, the persistence of necrotic cells stimulated a fibrotic response characterized by extensive collagen deposition. Hepatic accumulation of macrophages, which clear necrotic cells from the liver after carbon tetrachloride, was not affected by HIF-1α deletion in HSCs. Conversion of macrophages to M1-like, pro-inflammatory macrophages, which have increased phagocytic activity, however, was reduced in HIF-1α-GFAP Cre+ mice as indicated by a decrease in pro-inflammatory cytokines, and a decrease in the percentage of Gr1hi macrophages. Collectively, these studies have identified a novel function for HSCs and HIF-1α in orchestrating the clearance of necrotic cells from the liver, and demonstrated a key role for HSCs in modulating macrophage phenotype during acute liver injury. PMID:24639359

A novel long non-coding RNA, hypoxia-inducible factor-2α promoter upstream transcript, functions as an inhibitor of osteosarcoma stem cells in vitro.

PubMed

Wang, Yongcheng; Yao, Jie; Meng, Haoye; Yu, Zhiguo; Wang, Zhigang; Yuan, Xueling; Chen, Hong; Wang, Aiyuan

2015-04-01

Long non‑coding RNAs (lncRNAs) have recently been identified as novel modulators of malignant tumors. However, the function of lncRNAs in cancer stem cells (CSCs) remains to be elucidated. The present study aimed to investigate the regulating role of a novel lncRNA, hypoxia‑inducible factor‑2α (HIF‑2α) promoter upstream transcript (HIF2PUT), in osteosarcoma stem cells. The expression levels of HIF2PUT were assessed by quantitative polymerase chain reaction in 17 osteosarcoma tissue specimens, and the correlation between the expression of HIF2PUT and its host transcript‑HIF‑2α was determined. In functional experiments, HIF2PUT expression was knocked down by small interfering RNAs, or overexpressed by transfection with pcDNA‑HIF2PUT, in order to evaluate the effects of HIF2PUT on cell proliferation, migration, expression rate of osteosarcoma stem cell marker CD133, and stem sphere‑forming ability in MG63 cells. HIF2PUT expression levels were positively correlated with HIF‑2α in osteosarcoma tissues. Overexpression of HIF2PUT markedly inhibited cell proliferation and migration, decreased the percentage of CD133 expressing cells, and impaired the osteosarcoma stem sphere‑forming ability of the MG63 cells. Whereas, knockdown of HIF2PUT expression had the opposite effect. Furthermore, altering the expression of HIF2PUT resulted in a concomitant change to HIF‑2α mRNA expression. These results indicate that the lncRNA HIF2PUT may be a novel regulatory factor of osteosarcoma stem cells, which may exert its function partly by controlling HIF‑2α expression. Further studies regarding HIF2PUT may provide a novel therapeutic target of osteosarcoma in the future.

Inositol Polyphosphate Multikinase Inhibits Angiogenesis via Inositol Pentakisphosphate-Induced HIF-1α Degradation.

PubMed

Fu, Chenglai; Tyagi, Richa; Chin, Alfred C; Rojas, Tomas; Li, Ruo-Jing; Guha, Prasun; Bernstein, Isaac A; Rao, Feng; Xu, Risheng; Cha, Jiyoung Y; Xu, Jing; Snowman, Adele M; Semenza, Gregg L; Snyder, Solomon H

2018-02-02

Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored. We have investigated the role of IPMK in regulating angiogenesis. Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1α is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1α protein and thus VEGF. HIF-1α is constitutively ubiquitinated by pVHL (von Hippel-Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1α is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1α and pVHL. HIF-1α prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1α prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1α. Deletion of IPMK in mouse brain increases HIF-1α/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood-brain barrier and enhances brain blood vessel permeability. IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1α hydroxylation and thus pVHL-dependent degradation of HIF-1α. © 2017 American Heart Association, Inc.

Mesoporous bioactive glass nanolayer-functionalized 3D-printed scaffolds for accelerating osteogenesis and angiogenesis

NASA Astrophysics Data System (ADS)

Zhang, Yali; Xia, Lunguo; Zhai, Dong; Shi, Mengchao; Luo, Yongxiang; Feng, Chun; Fang, Bing; Yin, Jingbo; Chang, Jiang; Wu, Chengtie

2015-11-01

The hierarchical microstructure, surface and interface of biomaterials are important factors influencing their bioactivity. Porous bioceramic scaffolds have been widely used for bone tissue engineering by optimizing their chemical composition and large-pore structure. However, the surface and interface of struts in bioceramic scaffolds are often ignored. The aim of this study is to incorporate hierarchical pores and bioactive components into the bioceramic scaffolds by constructing nanopores and bioactive elements on the struts of scaffolds and further improve their bone-forming activity. Mesoporous bioactive glass (MBG) modified β-tricalcium phosphate (MBG-β-TCP) scaffolds with a hierarchical pore structure and a functional strut surface (~100 nm of MBG nanolayer) were successfully prepared via 3D printing and spin coating. The compressive strength and apatite-mineralization ability of MBG-β-TCP scaffolds were significantly enhanced as compared to β-TCP scaffolds without the MBG nanolayer. The attachment, viability, alkaline phosphatase (ALP) activity, osteogenic gene expression (Runx2, BMP2, OPN and Col I) and protein expression (OPN, Col I, VEGF, HIF-1α) of rabbit bone marrow stromal cells (rBMSCs) as well as the attachment, viability and angiogenic gene expression (VEGF and HIF-1α) of human umbilical vein endothelial cells (HUVECs) in MBG-β-TCP scaffolds were significantly upregulated compared with conventional bioactive glass (BG)-modified β-TCP (BG-β-TCP) and pure β-TCP scaffolds. Furthermore, MBG-β-TCP scaffolds significantly enhanced the formation of new bone in vivo as compared to BG-β-TCP and β-TCP scaffolds. The results suggest that application of the MBG nanolayer to modify 3D-printed bioceramic scaffolds offers a new strategy to construct hierarchically porous scaffolds with significantly improved physicochemical and biological properties, such as mechanical properties, osteogenesis, angiogenesis and protein expression for bone tissue engineering applications, in which the incorporation of nanostructures and bioactive components into the scaffold struts synergistically play a key role in the improved bone formation.

Hypoxia-Independent Downregulation of Hypoxia-Inducible Factor 1 Targets by Androgen Deprivation Therapy in Prostate Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ragnum, Harald Bull; Røe, Kathrine; Division of Medicine, Department of Oncology, Akershus University Hospital, Lørenskog

2013-11-15

Purpose: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. Methods and Materials: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvantmore » ADT was assessed by immunohistochemistry. Results: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). Conclusions: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to tumor regression. HIF1α expression is probably not a useful hypoxia biomarker during ADT in prostate cancer.« less

Hypoxia in cartilage: HIF-1alpha is essential for chondrocyte growth arrest and survival.

PubMed

Schipani, E; Ryan, H E; Didrickson, S; Kobayashi, T; Knight, M; Johnson, R S

2001-11-01

Breakdown or absence of vascular oxygen delivery is a hallmark of many common human diseases, including cancer, myocardial infarction, and stroke. The chief mediator of hypoxic response in mammalian tissues is the transcription factor hypoxia-inducible factor 1 (HIF-1), and its oxygen-sensitive component HIF-1alpha. A key question surrounding HIF-1alpha and the hypoxic response is the role of this transcription factor in cells removed from a functional vascular bed; in this regard there is evidence indicating that it can act as either a survival factor or induce growth arrest and apoptosis. To study more closely how HIF-1alpha functions in hypoxia in vivo, we used tissue-specific targeting to delete HIF-1alpha in an avascular tissue: the cartilaginous growth plate of developing bone. We show here the first evidence that the developmental growth plate in mammals is hypoxic, and that this hypoxia occurs in its interior rather than at its periphery. As a result of this developmental hypoxia, cells that lack HIF-1alpha in the interior of the growth plate die. This is coupled to decreased expression of the CDK inhibitor p57, and increased levels of BrdU incorporation in HIF-1alpha null growth plates, indicating defects in HIF-1alpha-regulated growth arrest occurs in these animals. Furthermore, we find that VEGF expression in the growth plate is regulated through both HIF-1alpha-dependent and -independent mechanisms. In particular, we provide evidence that VEGF expression is up-regulated in a HIF-1alpha-independent manner in chondrocytes surrounding areas of cell death, and this in turn induces ectopic angiogenesis. Altogether, our findings have important implications for the role of hypoxic response and HIF-1alpha in development, and in cell survival in tissues challenged by interruption of vascular flow; they also illustrate the complexities of HIF-1alpha response in vivo, and they provide new insights into mechanisms of growth plate development.

Increased size of solid organs in patients with Chuvash polycythemia and in mice with altered expression of HIF-1α and HIF-2α

PubMed Central

Yoon, Donghoon; Okhotin, David V.; Kim, Bumjun; Okhotina, Yulia; Okhotin, Daniel J.; Miasnikova, Galina Y.; Sergueeva, Adelina I.; Polyakova, Lydia A.; Maslow, Alexei; Lee, Yonggu; Semenza, Gregg L.; Prchal, Josef T.

2010-01-01

Chuvash polycythemia, the first hereditary disease associated with dysregulated oxygen-sensing to be recognized, is characterized by a homozygous germ-line loss-of-function mutation of the VHL gene (VHLR200W) resulting in elevated hypoxia inducible factor (HIF)-1α and HIF-2α levels, increased red cell mass and propensity to thrombosis. Organ volume is determined by the size and number of cells, and the underlying molecular control mechanisms are not fully elucidated. Work from several groups has demonstrated that the proliferation of cells is regulated in opposite directions by HIF-1α and HIF-2α. HIF-1α inhibits cell proliferation by displacing MYC from the promoter of the gene encoding the cyclin-dependent kinase inhibitor, p21Cip1, thereby inducing its expression. In contrast, HIF-2α promotes MYC activity and cell proliferation. Here we report that the volumes of liver, spleen, and kidneys relative to body mass were larger in 30 individuals with Chuvash polycythemia than in 30 matched Chuvash controls. In Hif1a+/− mice, which are heterozygous for a null (knockout) allele at the locus encoding HIF-1α, hepatic HIF-2α mRNA was increased (2-fold) and the mass of the liver was increased, compared with wild-type littermates, without significant difference in cell volume. Hepatic p21Cip1 mRNA levels were 9.5-fold lower in Hif1a+/− mice compared with wild-type littermates. These data suggest that, in addition to increased red cell mass, the sizes of liver, spleen, and kidneys are increased in Chuvash polycythemia. At least in the liver, this phenotype may result from increased HIF-2α and decreased p21Cip1 levels leading to increased hepatocyte proliferation. PMID:20140661

Activation of Hif1α by the Prolylhydroxylase Inhibitor Dimethyoxalyglycine Decreases Radiosensitivity

PubMed Central

Ayrapetov, Marina K.; Zhu, Kaya; Parmar, Kalindi; D'Andrea, Alan D.; Price, Brendan D.

2011-01-01

Hypoxia inducible factor 1α (Hif1α) is a stress responsive transcription factor, which regulates the expression of genes required for adaption to hypoxia. Hif1α is normally hydroxylated by an oxygen-dependent prolylhydroxylase, leading to degradation and clearance of Hif1α from the cell. Under hypoxic conditions, the activity of the prolylhydroxylase is reduced and Hif1α accumulates. Hif1α is also constitutively expressed in tumor cells, where it is associated with resistance to ionizing radiation. Activation of the Hif1α transcriptional regulatory pathway may therefore function to protect normal cells from DNA damage caused by ionizing radiation. Here, we utilized the prolylhydroxylase inhibitor dimethyloxalylglycine (DMOG) to elevate Hif1α levels in mouse embryonic fibroblasts (MEFs) to determine if DMOG could function as a radioprotector. The results demonstrate that DMOG increased Hif1α protein levels and decreased the sensitivity of MEFs to ionizing radiation. Further, the ability of DMOG to function as a radioprotector required Hif1α, indicating a key role for Hif1α's transcriptional activity. DMOG also induced the Hif1α -dependent accumulation of several DNA damage response proteins, including CHD4 and MTA3 (sub-units of the NuRD deacetylase complex) and the Suv39h1 histone H3 methyltransferase. Depletion of Suv39h1, but not CHD4 or MTA3, reduced the ability of DMOG to protect cells from radiation damage, implicating increased histone H3 methylation in the radioprotection of cells. Finally, treatment of mice with DMOG prior to total body irradiation resulted in significant radioprotection of the mice, demonstrating the utility of DMOG and related prolylhydroxylase inhibitors to protect whole organisms from ionizing radiation. Activation of Hif1α through prolylhydroxylase inhibition therefore identifies a new pathway for the development of novel radiation protectors. PMID:22016813

Metabolic Consequences of High-Fat Diet Are Attenuated by Suppression of HIF-1α

PubMed Central

Shin, Mi-Kyung; Drager, Luciano F.; Yao, Qiaoling; Bevans-Fonti, Shannon; Yoo, Doo-Young; Jun, Jonathan C.; Aja, Susan; Bhanot, Sanjay; Polotsky, Vsevolod Y.

2012-01-01

Obesity is associated with tissue hypoxia and the up-regulation of hypoxia inducible factor 1 alpha (HIF-1α). Prior studies in transgenic mice have shown that HIF-1α plays a role in the metabolic dysfunction associated with obesity. Therefore, we hypothesized that, after the development of diet-induced obesity (DIO), metabolic function could be improved by administration of HIF-1α antisense oligonucleotides (ASO). DIO mice were treated with HIF-1α ASO or with control ASO for 8 weeks and compared with an untreated group. We found that HIF-1α ASO markedly suppressed Hif-1α gene expression in adipose tissue and the liver. HIF-1α ASO administration induced weight loss. Final body weight was 41.6±1.4 g in the HIF-1α ASO group vs 46.7±0.9 g in the control ASO group and 47.9±0.8 g in untreated mice (p<0.001). HIF-1α ASO increased energy expenditure (13.3±0.6 vs 12±0.1 and 11.9±0.4 kcal/kg/hr, respectively, p<0.001) and decreased the respiratory exchange ratio (0.71±0.01 vs 0.75±0.01 and 0.76±0.01, respectively, p<0.001), which suggested switching metabolism to fat oxidation. In contrast, HIF-1a ASO had no effect on food intake or activity. HIF-1α ASO treatment decreased fasting blood glucose (195.5±8.4 mg/dl vs 239±7.8 mg/dl in the control ASO group and 222±8.2 mg/dl in untreated mice, p<0.01), plasma insulin, hepatic glucose output, and liver fat content. These findings demonstrate that the metabolic consequences of DIO are attenuated by HIF-1α ASO treatment. PMID:23049707

Design and Synthesis of Novel Small-molecule Inhibitors of the Hypoxia Inducible Factor Pathway

PubMed Central

Mooring, Suazette Reid; Jin, Hui; Devi, Narra S.; Jabbar, Adnan A.; Kaluz, Stefan; Liu, Yuan; Van Meir, Erwin G.; Wang, Binghe

2012-01-01

Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels, but interfere with the HIF-1α/HIF-1β/p300/CBP complex formation by interacting with p300 and CBP. PMID:22032632

Protein arginine methyltransferase 5 is an essential component of the hypoxia-inducible factor 1 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Ji-Hong; Choi, Yong-Joon; Cho, Chung-Hyun

2012-02-10

Highlights: Black-Right-Pointing-Pointer HIF-1{alpha} is expressed PRMT5-dependently in hypoxic cancer cells. Black-Right-Pointing-Pointer The HIF-1 regulation of hypoxia-induced genes is attenuated in PRMT5-knocked-down cells. Black-Right-Pointing-Pointer The de novo synthesis of HIF-1{alpha} depends on PRMT5. Black-Right-Pointing-Pointer PRMT5 is involved in the HIF-1{alpha} translation initiated by 5 Prime UTR of HIF-1{alpha} mRNA. -- Abstract: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that transfers one or two methyl groups to the arginine residues of histones or non-histone proteins, and that plays critical roles in cellular processes as diverse as receptor signaling and gene expression. Furthermore, PRMT5 is highly expressed in tumors, where it maymore » be associated with tumor growth. Although much research has been conducted on PRMT5, little is known regarding its role in adaption to hypoxia. As hypoxia-inducible factor 1 (HIF-1) is a key player in hypoxic response, we examined the possible involvement of PRMT5 in the HIF-1 signaling pathway. Of the siRNAs targeting PRMT1-8, only PRMT5 siRNA attenuated the hypoxic induction of HIF-1{alpha} in A549 cells, and this result was reproducible in all three cancer cell lines examined. PRMT5 knock-down also repressed the promoter activities and the transcript levels of HIF-1-governed genes. Mechanistically, de novo synthesis of HIF-1{alpha} protein was reduced in PRMT5-knocked-down A549 cells, and this was rescued by PRMT5 restoration. In contrast, HIF-1{alpha} transcription, RNA processing, and protein stability were unaffected by PRMT5 knock-down. Furthermore, PRMT5 was found to be essential for the HIF-1{alpha} translation initiated by the 5 Prime UTR of HIF-1{alpha} mRNA. Given our results and previous reports, we believe that PRMT5 probably promotes tumor growth by stimulating cell proliferation and by participating in the construction of a tumor-favorable microenvironment via HIF-1 activation.« less

HIF-2alpha: Achilles' heel of pseudohypoxic subtype paraganglioma and other related conditions.

PubMed

Tella, Sri Harsha; Taïeb, David; Pacak, Karel

2017-11-01

Paragangliomas (PGLs) belong to the most hereditary endocrine tumours. The existence of mutated HIF2A in these tumours, the role of oncometabolites on HIFs stabilisation and a recent concept proposing how hereditary PGLs converge on the hypoxia-signalling pathway, brought solid evidence of the existence of PGL hypoxiom. Hypoxia-inducible factor 2alpha (HIF-2α) antagonists -PT2385, and PT2399 have been shown to have promising results in the management of clear cell renal cell carcinoma by targeting the HIF-2α pathway in recent and ongoing clinical trials (PT2799). The main aim of this perspective is to address the possibility of HIF-2α antagonists in the management of tumours, beyond clear cell renal cell carcinoma, where the dysfunctional hypoxia-signalling pathway, especially HIF-2α, referred here as the Achilles' heel, plays a unique role in tumorigenesis and other disorders. These tumours or disorders include PGLs, somatostatinomas, hemangioblastomas, gastrointestinal stromal tumours, pituitary tumours, leiomyomas/leiomyosarcomas, polycythaemia and retinal abnormalities. We hope that HIF-2α antagonists are likely to emerge as a potential effective treatment of choice for HIF-2α-related tumours and disorders. Copyright © 2017. Published by Elsevier Ltd.

Parkin modulates expression of HIF-1α and HIF-3α during hypoxia in gliobastoma-derived cell lines in vitro.

PubMed

Maugeri, Grazia; D'Amico, Agata Grazia; Reitano, Rita; Saccone, Salvatore; Federico, Concetta; Cavallaro, Sebastiano; D'Agata, Velia

2016-06-01

Mutation of the Parkin gene causes an autosomal recessive juvenile-onset form of Parkinson's disease. However, recently, it has been also linked to a wide variety of malignancies, including glioblastoma multiforme (GBM). In this pathology, Parkin exhibits a tumor suppressor role by mitigating the proliferation rate in both in vitro and in vivo models. However, Parkin involvement in the hypoxic process has not as yet been investigated. GBM is the most common and aggressive primary brain tumor in adults and is characterized by hypoxic areas. The low oxygen supply causes the expression of hypoxia-inducible factors (HIFs) leading to an accumulation of pro-angiogenic factors and tumoral invasiveness. We assess the relationship between Parkin and two HIFs expressed during hypoxic conditions, namely HIF-1α and HIF-3α. Our data show that Parkin is downregulated under hypoxia and that it interferes with HIF expression based on cellular oxygen tension. These results suggest a role for the involvement of Parkin in GBM, although further studies will be needed to understand the mechanism by which it modulates HIF-1α and HIF-3α expression.

Expression of HIF-1α and Markers of Angiogenesis Are Not Significantly Different in Triple Negative Breast Cancer Compared to Other Breast Cancer Molecular Subtypes: Implications for Future Therapy.

PubMed

Yehia, Lamis; Boulos, Fouad; Jabbour, Mark; Mahfoud, Ziyad; Fakhruddin, Najla; El-Sabban, Marwan

2015-01-01

Triple negative breast cancer lacks estrogen, progesterone and epidermal growth factor receptors rendering it refractory to available targetedtherapies. TNBC is associated with central fibrosis and necrosis, both indicators of tumor hypoxia. Hypoxia inducible factor 1α is up-regulated under hypoxia and its expression is associated with induction of angiogenesis resulting in proliferation, aggressive tumor phenotype and metastasis. In this study we evaluate the potential use of HIF-1α as aTNBC-specific marker. 62 TNBC, 64 HER2+, and 64 hormone-receptors positive breast cancer cases were evaluated for central fibrosis and necrosis, HIF-1α, HIF-1β, VEGFR3, CD31 expression and microvessel density. RNA extraction from paraffin-embedded samples, followed by quantitative real-time polymerase chain reaction (qRT-PCR) evaluation of HIF-1α and VEGF transcripts was performed on 54 cases (18 from each subtype). HIF-1α protein was expressed in 35.5% TNBC, 45.3% HER2+and 25.0% ER+/PR+ (p = 0.055; χ2 test). PCRanalysis of subgroup of breast cancers, 84.2% expressed HIF-1α protein and its transcripts, while only 66.7% expressed VEGF transcripts simultaneously with the HIF-1α protein and its transcripts. Central fibrosis and necrosis was highest in TNBC (p = 0.015; χ2 test), while MVD was comparable among all groups (p = 0.928; χ2 test). VEGFR3 was highest in TNBC expressing HIF-1α. HIF-1β protein was expressed in 32.0% of HIF-1α(+), and in (44.3%) of HIF-1α(-) breast cancer cases (p = 0.033; χ2 test). Moreover, HIF-1α expression in cases with central fibrosis and necrosis was highest in the HER2+ followed by the TNBC (p = 0.156; χ2 test). A proportion of TNBC express HIF-1α but not in a significantly different manner from other breast cancer subtypes. The potential of anti-HIF-1α targeted therapy is therefore not a candidate for exclusive use in TNBC, but should be considered in all breast cancers, especially in the setting of clinically aggressive or refractory disease.

The copper dependent-lysyl oxidases contribute to the pathogenesis of pulmonary emphysema in chronic obstructive pulmonary disease patients.

PubMed

Besiktepe, Neziha; Kayalar, Ozgecan; Ersen, Ezel; Oztay, Fusun

2017-12-01

Abnormalities in the elastic fiber biology are seen in pulmonary emphysema (PE). The copper-dependent lysyl oxidases regulate the production and accumulation of elastic fibers in the connective tissue. This study focused on the relationship between lysyl oxidase (LOX), LOX-like protein 1 (LOXL1), and LOXL2 and PE pathogenesis. Lung samples with or without PE from patients with chronic obstructive lung disease (n=35) were used. Protein levels of elastin, LOX, LOXL1, LOXL2, hypoxia inducible factor 1-alpha (HIF-1α), copper metabolism domain containing-1 (COMMD1), and phosphatase and tensin homolog (PTEN) were assayed using microscopic and biochemical methods The emphysematous areas were characterized by enlargement of the alveoli, destruction of the alveolar structure, accumulation of macrophages in the alveolar lumens, and showed increased HIF-1α immunoreactivity. Additionally, the emphysematous areas had significantly lower elastin, LOX, LOXL1, LOXL2, HIF-1α, COMMD1, and PTEN protein levels than the non-emphysematous areas. We suppose that the reductions in the HIF-1α levels led to decreases in the protein levels of active LOX, LOXL1, and LOXL2. These decreases might cause abnormalities in the elastic fiber biology. HIF-1α activation induced by decreased COMMD1 and protease activation induced by decreased PTEN might contribute to the development of PE. Finally, methods aimed at increasing the protein levels of LOXs, COMMD1 and PTEN might be effective for treating PE. Copyright © 2017 Elsevier GmbH. All rights reserved.

Hydrostatic pressure influences HIF-2 alpha expression in chondrocytes.

PubMed

Inoue, Hiroaki; Arai, Yuji; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Tsuchida, Shinji; Matsuki, Tomohiro; Ueshima, Keiichirou; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

2015-01-05

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.

Paradoxical Regulation of Hypoxia Inducible Factor-1α (HIF-1α) by Histone Deacetylase Inhibitor in Diffuse Large B-Cell Lymphoma

PubMed Central

Bhalla, Savita; Evens, Andrew M.; Prachand, Sheila; Schumacker, Paul T.; Gordon, Leo I.

2013-01-01

Hypoxia inducible factor (HIF) is important in cancer, as it regulates various oncogenic genes as well as genes involved in cell survival, proliferation, and migration. Elevated HIF-1 protein promotes a more aggressive tumor phenotype, and greater HIF-1 expression has been demonstrated to correlate with poorer prognosis, increased risk of metastasis and increased mortality. Recent reports suggest that HIF-1 activates autophagy, a lysosomal degradation pathway which may promote tumor cell survival. We show here that HIF-1α expression is constitutively active in multiple diffuse large B cell lymphoma (DLBCL) cell lines under normoxia and it is regulated by the PI3K/AKT pathway. PCI-24781, a pan histone deacetylase inhibitor (HDACI), enhanced accumulation of HIF-1α and induced autophagy initially, while extended incubation with the drug resulted in inhibition of HIF-1α. We tested the hypothesis that PCI-24781- induced autophagy is mediated by HIF-1α and that inhibition of HIF-1α in these cells results in attenuation of autophagy and decreased survival. We also provide evidence that autophagy serves as a survival pathway in DLBCL cells treated with PCI-24781 which suggests that the use of autophagy inhibitors such as chloroquine or 3-methyl adenine in combination with PCI-24781 may enhance apoptosis in lymphoma cells. PMID:24312289

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression

PubMed Central

Harashima, Nanae; Takenaga, Keizo; Akimoto, Miho; Harada, Mamoru

2017-01-01

Cancer cells develop resistance to therapy by adapting to hypoxic microenvironments, and hypoxia-inducible factors (HIFs) play crucial roles in this process. We investigated the roles of HIF-1α and HIF-2α in cancer cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) using human pancreatic cancer cell lines. siRNA-mediated knockdown of HIF-2α, but not HIF-1α, increased susceptibility of two pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL in vitro under normoxic and hypoxic conditions. The enhanced sensitivity to TRAIL was also observed in vivo. This in vitro increased TRAIL sensitivity was observed in other three pancreatic cancer cell lines. An array assay of apoptosis-related proteins showed that knockdown of HIF-2α decreased survivin expression. Additionally, survivin promoter activity was decreased in HIF-2α knockdown Panc-1 cells and HIF-2α bound to the hypoxia-responsive element in the survivin promoter region. Conversely, forced expression of the survivin gene in HIF-2α shRNA-expressing Panc-1 cells increased resistance to TRAIL. In a xenograft mouse model, the survivin suppressant YM155 sensitized Panc-1 cells to TRAIL. Collectively, our results indicate that HIF-2α dictates the susceptibility of human pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL by regulating survivin expression transcriptionally, and that survivin could be a promising target to augment the therapeutic efficacy of death receptor-targeting anti-cancer therapy. PMID:28476028

HIF-1α coordinates lymphangiogenesis during wound healing and in response to inflammation

PubMed Central

Zampell, Jamie C.; Yan, Alan; Avraham, Tomer; Daluvoy, Sanjay; Weitman, Evan S.; Mehrara, Babak J.

2012-01-01

This study aimed to investigate the mechanisms that coordinate lymphangiogenesis. Using mouse models of lymphatic regeneration and inflammatory lymphangiogenesis, we explored the hypothesis that hypoxia inducible factor-α (HIF-1α) is a central regulator of lymphangiogenesis. We show that HIF-1α inhibition by small molecule inhibitors (YC-1 and 2-methyoxyestradiol) results in delayed lymphatic repair, decreased local vascular endothelial growth factor-C (VEGF-C) expression, reduced numbers of VEGF-C+ cells, and reductions in inflammatory lymphangiogenesis. Using transgenic HIF-1α/luciferase mice to image HIF-1α expression in real time in addition to Western blot analysis and pimonidazole staining for cellular hypoxia, we demonstrate that hypoxia stabilizes HIF-1α during initial stages of wound repair (1–2 wk); whereas inflammation secondary to gradients of lymphatic fluid stasis stabilizes HIF-1α thereafter (3–6 wk). In addition, we show that CD4+ cell-mediated inflammation is necessary for this response and regulates HIF-1α expression by macrophages, as CD4-deficient or CD4-depleted mice demonstrate 2-fold reductions in HIF-1α expression as compared to wild-types. In summary, we show that HIF-1α is a critical coordinator of lymphangiogenesis by regulating the expression of lymphangiogenic cytokines as part of an early response mechanism to hypoxia, inflammation, and lymphatic fluid stasis.—Zampell, J. C., Yan, A., Avraham, T., Daluvoy, S., Weitman, E. S., Mehrara, B. J. HIF-1α coordinates lymphangiogenesis during wound healing and in response to inflammation. PMID:22067482

BAG3 and HIF-1 α coexpression detected by immunohistochemistry correlated with prognosis in hepatocellular carcinoma after liver transplantation.

PubMed

Xiao, Heng; Tong, Rongliang; Cheng, Shaobing; Lv, Zhen; Ding, Chaofeng; Du, Chengli; Xie, Haiyang; Zhou, Lin; Wu, Jian; Zheng, Shusen

2014-01-01

The objective is to determine the effects of BAG3 and HIF-1 α expression on the prognosis of HCC patients after liver transplantation. Samples from 31 patients with HCC receiving liver transplantation were collected for this study. The immunohistochemistry was used to detect the expression of BAG3 and HIF-1 α of HCC samples. According to the immunohistochemistry results, BAG3 and HIF-1 α staining were significantly associated with tumor TNM stage (P = 0.004, P = 0.012). A significant association between high BAG3/HIF-1 α levels and a shorter overall survival was detected, so as the combined BAG3 and HIF-1 α analysis. The results suggested that the expression level of BAG3 and HIF-1 α is efficient prognostic parameters in patients with HCC after liver transplantation.

Changes in the Anatomic and Microscopic Structure and the Expression of HIF-1α and VEGF of the Yak Heart with Aging and Hypoxia

PubMed Central

He, Yanyu; Yu, Sijiu; Hu, Junwei; Cui, Yan; Liu, Penggang

2016-01-01

The study aimed to identify the changes of anatomic and microscopic structure and the expression and localization of hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in the myocardium and coronary artery of the yak heart adapted to chronic hypoxia with aging. Thirty-two yaks (1 day, 6 months, 1 year, 2 years, and 5 year old) were included, and immunoelectronmicroscopy, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used. Right ventricular hypertrophy was not present in yaks with aging. There was no intima thickening phenomenon in the coronary artery. The ultrastructure of myofibrils, mitochondria, and collagen fibers and the diameter and quantity of collagen changed significantly with aging. The enzymatic activity of complexes I, II, and V increased with age. Immunogold labeling showed the localization of HIF-1α protein in the cytoplasm and nuclei of endothelial cells and cytoplasm of cardiac muscle cells, and VEGF protein in the nuclei and perinuclei areas of smooth muscle cells of coronary artery, and in the cytoplasm and nuclei of endothelial cells. ELISA results showed that HIF-1α secretion significantly increased in the myocardium and coronary artery from an age of 1 day to 2 years of yaks and decreased in old yaks. However, VEGF protein always increased with aging. The findings of this study suggest that 6 months is a key age of yak before which there are some adaptive changes to deal with low-oxygen environment, and there is a maturation of the yak heart from the age of 6 months to 2 years. PMID:26914488

[Transcription factors NF-kB, HIF-1, HIF-2, growth factor VEGF, VEGFR2 and carboanhydrase IX mRNA and protein level in the development of kidney cancer metastasis].

PubMed

Spirina, L V; Usynin, Y A; Yurmazov, Z A; Slonimskaya, E M; Kolegova, E S; Kondakova, I V

2017-01-01

Here, we have investigated the participation of nuclear factors NF-kB, HIF-1 and HIF-2, VEGF, VEGFR2, and carboanhydrase IX in clear-cell renal cancer. We have determined the expression and protein level of transcription factors, VEGF, VEGFR2, and carboanhydrase IX in tumor and normal tissues of 30 patients with kidney cancer. The Real-Time PCR and ELISA were used in the study. The low levels of HIF-1 mRNA expression associated with high levels of HIF-1 protein were also associated with metastasis. The expression levels of VEGF, VEGFR2, and their protein levels are increased in primary tumors of patients with disseminated kidney cancer compared to nonmetastatic cancer. No correlation was revealed between the content of mRNA and encoded proteins in the kidney cancer tissues. The changes in the ratios of mRNA levels and the respective proteins (HIF-1α, HIF-2, NF-kB, VEGF, VEGFR2, and carboanhydrase IX) may contribute to kidney-cancer metastasis.

Activation of intestinal hypoxia-inducible factor 2α during obesity contributes to hepatic steatosis.

PubMed

Xie, Cen; Yagai, Tomoki; Luo, Yuhong; Liang, Xianyi; Chen, Tao; Wang, Qiong; Sun, Dongxue; Zhao, Jie; Ramakrishnan, Sadeesh K; Sun, Lulu; Jiang, Chunmei; Xue, Xiang; Tian, Yuan; Krausz, Kristopher W; Patterson, Andrew D; Shah, Yatrik M; Wu, Yue; Jiang, Changtao; Gonzalez, Frank J

2017-11-01

Nonalcoholic fatty liver disease is becoming the most common chronic liver disease in Western countries, and limited therapeutic options are available. Here we uncovered a role for intestinal hypoxia-inducible factor (HIF) in hepatic steatosis. Human-intestine biopsies from individuals with or without obesity revealed that intestinal HIF-2α signaling was positively correlated with body-mass index and hepatic toxicity. The causality of this correlation was verified in mice with an intestine-specific disruption of Hif2a, in which high-fat-diet-induced hepatic steatosis and obesity were substantially lower as compared to control mice. PT2385, a HIF-2α-specific inhibitor, had preventive and therapeutic effects on metabolic disorders that were dependent on intestine HIF-2α. Intestine HIF-2α inhibition markedly reduced intestine and serum ceramide levels. Mechanistically, intestine HIF-2α regulates ceramide metabolism mainly from the salvage pathway, by positively regulating the expression of Neu3, the gene encoding neuraminidase 3. These results suggest that intestinal HIF-2α could be a viable target for hepatic steatosis therapy.

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

PubMed

Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

2005-08-01

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

The hypoxia factor Hif-1α controls neural crest chemotaxis and epithelial to mesenchymal transition

PubMed Central

Barriga, Elias H.; Maxwell, Patrick H.

2013-01-01

One of the most important mechanisms that promotes metastasis is the stabilization of Hif-1 (hypoxia-inducible transcription factor 1). We decided to test whether Hif-1α also was required for early embryonic development. We focused our attention on the development of the neural crest, a highly migratory embryonic cell population whose behavior has been likened to cancer metastasis. Inhibition of Hif-1α by antisense morpholinos in Xenopus laevis or zebrafish embryos led to complete inhibition of neural crest migration. We show that Hif-1α controls the expression of Twist, which in turn represses E-cadherin during epithelial to mesenchymal transition (EMT) of neural crest cells. Thus, Hif-1α allows cells to initiate migration by promoting the release of cell–cell adhesions. Additionally, Hif-1α controls chemotaxis toward the chemokine SDF-1 by regulating expression of its receptor Cxcr4. Our results point to Hif-1α as a novel and key regulator that integrates EMT and chemotaxis during migration of neural crest cells. PMID:23712262

The expression and significance of HIF-1alpha and GLUT-3 in glioma.

PubMed

Liu, Yang; Li, Yun-ming; Tian, Rui-feng; Liu, Wei-ping; Fei, Zhou; Long, Qian-fa; Wang, Xiao-an; Zhang, Xiang

2009-12-22

HIF-1alpha plays an indispensable role in tumor formation and histogenesis. Target genes involved in glucose transport are acutely transactivated by HIF-1alpha. GLUT-3 protein is the rate-limiting factor related to glucose transport, which is classified as brain-type glucose transporter. This study was the initial one aiming to probe into the co-expression and clinical significance of HIF-1alpha and GLUT-3 in glioma. One hundred and twenty cases of glioma tissues and ten human normal cerebral tissues decompressed in glioma excision were examined using immunohistochemistry and Western blot. The expression of HIF-1alpha and GLUT-3 increased gradually with the increase of pathological grade of glioma, respectively. There was significant difference in the expression of HIF-1alpha and GLUT-3 in every two groups, respectively. There was a positive correlation between HIF-1alpha and GLUT-3. In conclusion, the expression of HIF-1alpha and GLUT-3 in glioma was correlated significantly with tumors' pathological grade, which can be taken as a pair of useful markers for predicting the biological behavior of glioma.

HIF-1 and ventilatory acclimatization to chronic hypoxia

PubMed Central

Powell, Frank L.; Fu, Zhenxing

2008-01-01

Ventilatory acclimatization to hypoxia (VAH) is a time-dependent increase in ventilation and ventilatory O2-sensitivity that involves plasticity in carotid body chemoreceptors and CNS respiratory centers. Hypoxia inducible factor-1α (HIF-1α) controls the expression of several genes that increase physiological O2 supply. Studies using transgenic mice show HIF-1α expression in the carotid bodies and CNS with chronic sustained and intermittent hypoxia is important for VAH. Other O2-sensitive transcription factors such as HIF-2α may be important for VAH by reducing metabolic O2 demands also. Specific gene targets of HIF-1α shown to be involved in VAH include erythropoietin, endothelin-1, neuronal nitric oxide synthase and tyrosine hydroxylase. Other HIF-1α targets that may be involved in VAH include vascular endothelial growth factor, heme oxygenase 1 and cytoglobin. Interactions between these multiple pathways and feedback control of HIF-1α expression from some of the targets support a complex and powerful role for HIF-1α in neural plasticity of physiological control circuits with chronic hypoxia. PMID:18708172

PHGDH as a key enzyme for serine biosynthesis in HIF2α-targeting therapy for renal cell carcinoma

PubMed Central

Yoshino, Hirofumi; Nohata, Nijiro; Miyamoto, Kazutaka; Yonemori, Masaya; Sakaguchi, Takashi; Sugita, Satoshi; Itesako, Toshihiko; Kofuji, Satoshi; Nakagawa, Masayuki; Dahiya, Rajvir; Enokida, Hideki

2018-01-01

Continuous activation of hypoxia-inducible factor (HIF) is important for progression of renal cell carcinoma (RCC) and acquired resistance to anti-angiogenic multi-kinase and mTOR inhibitors. Recently, HIF2α antagonists PT2385 and PT2399 were developed and are being evaluated in a Phase I clinical trial for advanced or metastatic clear cell RCC (ccRCC). However, resistance to HIF2α antagonists would be expected to develop. In this study, we identified signals activated by HIF2α deficiency as candidate mediators of resistance to the multi-kinase inhibitor sunitinib. We established sunitinib-resistant tumor cells in vivo and created HIF2α-deficient variants of these cells using CRISPR/Cas9 technology. Mechanistic investigations revealed that a regulator of the serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), was upregulated commonly in HIF2α-deficient tumor cells along with the serine biosynthesis pathway itself. Accordingly, treatment with a PHGDH inhibitor reduced the growth of HIF2α-deficient tumor cells in vivo and in vitro by inducing apoptosis. Our findings identify the serine biosynthesis pathway as a source of candidate therapeutic targets to eradicate advanced or metastatic ccRCC resistant to HIF2α antagonists. PMID:28951458

The therapeutic potential of HIF-2 antagonism in renal cell carcinoma

PubMed Central

2017-01-01

Hypoxia, the insufficient delivery of oxygen for the demand of a tissue, contributes to the development of an aggressive phenotype, resistance to radiation therapy and chemotherapy, and is predictive of a poor outcome in numerous tumor types. Adaptation to hypoxia is mediated by hypoxia-inducible factors (HIFs), including HIF-1α and HIF-2α, which regulate genes promoting angiogenesis, increased tumor growth or metastasis. In kidney cancer, HIF-2α is believed to be the most important driver for development and progression of clear cell renal cell carcinoma (ccRCC), highlighting the therapeutic potential of HIF-2 antagonists in this disease. Recent studies show that HIF-2α can be targeted by selective, and orally active new class of inhibitors. In conjunction with the restricted expression of HIF-2α in normal adult physiology, these studies suggest that such therapeutic approach might be favorable for patients with lower toxicity than current anti-angiogenic drugs like sunitinib. However, the differential sensitivity to these HIF-2α antagonists along with the potential mechanisms of resistance reported in these studies advocate for the identification of biomarkers to determine which patients are more likely to benefit from these therapies as well as paving the way for second generation inhibitors or complementary inhibitory approaches. PMID:28217462

Hypoxia-Inducible Factor and Its Role in the Management of Anemia in Chronic Kidney Disease

PubMed Central

Kaplan, Joshua M.; Sharma, Neeraj

2018-01-01

Hypoxia-inducible factor (HIF) plays a crucial role in the response to hypoxia at the cellular, tissue, and organism level. New agents under development to pharmacologically manipulate HIF may provide new and exciting possibilities in the treatment of anemia of chronic kidney disease (CKD) as well as in multiple other disease states involving ischemia–reperfusion injury. This article provides an overview of recent studies describing current standards of care for patients with anemia in CKD and associated clinical issues, and those supporting the clinical potential for targeting HIF stabilization with HIF prolyl-hydroxylase inhibitors (HIF-PHI) in these patients. Additionally, articles reporting the clinical potential for HIF-PHIs in ‘other’ putative therapeutic areas, the tissue and intracellular distribution of HIF- and prolyl-hydroxylase domain (PHD) isoforms, and HIF isoforms targeted by the different PHDs, were identified. There is increasing uncertainty regarding the optimal treatment for anemia of CKD with poorer outcomes associated with treatment to higher hemoglobin targets, and the increasing use of iron and consequent risk of iron imbalance. Attainment and maintenance of more physiologic erythropoietin levels associated with HIF stabilization may improve the management of patients resistant to treatment with erythropoiesis-stimulating agents and improve outcomes at higher hemoglobin targets. PMID:29382128

Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Yun-Jeong; Cho, Hyun-Ji; Magae, Junji

2013-12-15

Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α throughmore » an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action. - Highlights: • Inhibitory effect of ascofuranone on HIF-1α expression is EGF-specific regulation. • Ascofuranone decreases HIF-1α protein synthesis through Akt/mTOR pathways. • Ascofuranone suppresses EGF-induced VEGF production and tumor angiogenesis.« less

Hispidulin inhibits proliferation and enhances chemosensitivity of gallbladder cancer cells by targeting HIF-1α

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Hui; Xie, Jing; Peng, Jianjun, E-mail: jianjunpeng@126.com

2015-03-15

Gallbladder cancer (GBC) is an aggressive malignancy of the bile duct, which is associated with a low (5-year) survival and poor prognosis. The transcription factor HIF-1α is implicated in the angiogenesis, cell survival, epithelial mesenchymal transition (EMT) and invasiveness of GBC. In this study, we have investigated the role of HIF-1α in the pathobilogy of GBC and effect of hispidulin on the molecular events controlled by this transcription factor. We observed that hispidulin caused induction of apoptosis, blockade of growth and cell cycle progression in GBC cells. Our results have demonstrated for the first time that hispidulin-exerted anti-tumor effect involvedmore » the suppression of HIF-1α signaling. Hispidulin was found to repress the expression of HIF-1α protein dose-dependently without affecting the HIF-1α mRNA expression. In addition, the inhibition of HIF-1α protein synthesis was revealed to be mediated through the activation of AMPK signaling. Hispidulin also sensitized the tumor cells to Gemcitabine and 5-Fluoroucil by down-regulating HIF-1α/P-gp signaling. Given the low cost and exceedingly safe profile, hispidulin appears to be a promising and novel chemosensitizer for GBC treatment. - Highlights: • Hispidulin inhibits proliferation of gallbladder cancer cells by targeting HIF-1α. • Hispidulin regulates HIF-1α via activating AMPK signaling. • Hispidulin sensitized the GBC cells to chemotherapeutics by down-regulating P-gp.« less

Menadione and ethacrynic acid inhibit the hypoxia-inducible factor (HIF) pathway by disrupting HIF-1α interaction with p300.

PubMed

Na, Yu-Ran; Han, Ki-Cheol; Park, Hyunsung; Yang, Eun Gyeong

2013-05-17

Hypoxia is a general characteristic of most solid malignancies and intimately related to neoplastic diseases and cancer progression. Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor (HIF)-1α that elicits transcriptional activity through recruitment of the CREB binding protein (CBP)/p300 coactivator. Targeted blockade of HIF-1α binding to CBP/p300 would thus constitute a novel approach for cancer treatment by suppressing tumor angiogenesis and metastasis. Here, we identified inhibitors against the interaction between HIF-1α and p300 by a fluorescence polarization-based assay employing a fluorescently-labeled peptide containing the C-terminal activation domain of HIF-1α. Two small molecule inhibitors, menadione (MD) and ethacrynic acid (EA), were found to decrease expression of luciferase under the control of hypoxia-responsive elements in hypoxic cells as well as to efficiently block the interaction between the full-length HIF-1α and p300. While these compounds did not alter the expression level of HIF-1α, they down-regulated expression of a HIF-1α target vascular endothelial growth factor (VEGF) gene. Considering hypoxia-induced VEGF expression leading to highly aggressive tumor growth, MD and EA may provide new scaffolds for development of tumor therapeutic reagents as well as tools for a better understanding of HIF-1α-mediated hypoxic regulation. Copyright © 2013 Elsevier Inc. All rights reserved.

Hydrostatic Pressure Influences HIF-2 Alpha Expression in Chondrocytes

PubMed Central

Inoue, Hiroaki; Arai, Yuji; Kishida, Tsunao; Terauchi, Ryu; Honjo, Kuniaki; Nakagawa, Shuji; Tsuchida, Shinji; Matsuki, Tomohiro; Ueshima, Keiichirou; Fujiwara, Hiroyoshi; Mazda, Osam; Kubo, Toshikazu

2015-01-01

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α. PMID:25569085

(1)H NMR metabolomics analysis of renal cell carcinoma cells: Effect of VHL inactivation on metabolism.

PubMed

Cuperlovic-Culf, Miroslava; Cormier, Kevin; Touaibia, Mohamed; Reyjal, Julie; Robichaud, Sarah; Belbraouet, Mehdi; Turcotte, Sandra

2016-05-15

Von Hippel-Lindau (VHL) is an onco-suppressor involved in oxygen and energy-dependent promotion of protein ubiquitination and proteosomal degradation. Loss of function mutations of VHL (VHL-cells) result in organ specific cancers with the best studied example in renal cell carcinomas. VHL has a well-established role in deactivation of hypoxia-inducible factor (HIF-1) and in regulation of PI3K/AKT/mTOR activity. Cell culture metabolomics analysis was utilized to determined effect of VHL and HIF-1α or HIF-2α on metabolism of renal cell carcinomas (RCC). RCC cells were stably transfected with VHL or shRNA designed to silence HIF-1α or HIF-2α genes. Obtained metabolic data was analysed qualitatively, searching for overall effects on metabolism as well as quantitatively, using methods developed in our group in order to determine specific metabolic changes. Analysis of the effect of VHL and HIF silencing on cellular metabolic footprints and fingerprints provided information about the metabolic pathways affected by VHL through HIF function as well as independently of HIF. Through correlation network analysis as well as statistical analysis of significant metabolic changes we have determined effects of VHL and HIF on energy production, amino acid metabolism, choline metabolism as well as cell regulation and signaling. VHL was shown to influence cellular metabolism through its effect on HIF proteins as well as by affecting activity of other factors. © 2015 UICC.

Anemia in conventional hemodialysis: Finding the optimal treatment balance.

PubMed

Hasegawa, Takeshi; Koiwa, Fumihiko; Akizawa, Tadao

2018-06-17

Renal anemia is a serious and common complication in hemodialysis (HD) patients. The introduction of erythropoiesis-stimulating agents (ESAs) has dramatically improved hemoglobin levels and outcomes. Several interventional studies reported that excessive correction of anemia and the massive use of ESA can trigger cardiovascular disease (CVD), and consequently may worsen the prognosis of patients undergoing HD. Therefore, it has been widely recognized that large doses of ESA should be used with caution. An effective use of iron preparations is required to yield the optimal effect of ESA. It is well-known that iron utilization is inhibited under pathological conditions, such as chronic inflammation, resulting in ESA resistance. It is postulated that a new class of therapeutic agents for renal anemia, hypoxia inducible factor prolyl hydroxylase (HIF-PH) inhibitors, will have beneficial treatment effects in patients on HD. HIF is induced by hypoxia and promotes erythropoietin production. In the absence of a hypoxic state, HIF is decomposed by the HIF catabolic enzyme. HIF-PH inhibitors inhibit this degrading enzyme and stimulate endogenous erythropoietin production via HIF induction. Additionally, HIF-PH inhibitors promote effective utilization of iron and raise erythropoietin to physiological concentrations. Accordingly, HIF-PH inhibitors improve anemia and iron metabolism. It appears that this effect persists irrespective of chronic inflammatory conditions. HIF-PH inhibitors do not overshoot erythropoietin above physiological concentrations like ESAs. Therefore, it is hypothesized that HIF-PH inhibitors would not increase the risk of CVD in patients undergoing HD. © 2018 Wiley Periodicals, Inc.

The epigenetic regulation of HIF-1α by SIRT1 in MPP{sup +} treated SH-SY5Y cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Su-Yan; Guo, Yan-Jie; Feng, Ya

Both silent information regulator 1 (SIRT1) and hypoxia inducible factor 1 (HIF-1) have been found to play important roles in the pathophysiology of Parkinson's disease (PD). However, their mechanisms and their relationship still require further study. In the present study, we focused on the change and relationship of SIRT1 and HIF-1α in PD. PD cell models were established by using methyl-4-phenylpyridinium (MPP{sup +}), which induced inhibition of cell proliferation, cell cycle arrest and apoptosis. We found that the expression of HIF-1α and its target genes VEGFA and LDHA increased and that SIRT1 expression was inhibited in MPP{sup +} treated cells.more » With further analysis, we found that the acetylation of H3K14 combined with the HIF-1α promoter was dramatically increased in cells treated with MPP{sup +}, which resulted in the transcriptional activation of HIF-1α. Moreover, the acetylation of H3K14 and the expression of HIF-1α increased when SIRT1 was knocked down, suggesting that SIRT1 was involved in the epigenetic regulation of HIF-1α. At last, phenformin, another mitochondrial complex1 inhibitor, was used to testify that the increased HIF-1a was not due to off target effects of MPP{sup +}. Therefore, our results support a link between PD and SIRT1/HIF-1α signaling, which may serve as a clue for understanding PD.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doi, Nobutaka; New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd.; Ogawa, Ryohei, E-mail: ogawa@med.u-toyama.ac.jp

The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutuallymore » complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.« less

β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer

PubMed Central

Luo, Yong; Li, Mingchuan; Zuo, Xuemei; Basourakos, Spyridon P.; Zhang, Jiao; Zhao, Jiahui; Han, Yili; Lin, Yunhua; Wang, Yongxing; Jiang, Yongguang; Lan, Ling

2018-01-01

Hypoxia-inducible factor-1α (HIF-1α) is known to play crucial roles in tumor radioresistance; however, the molecular mechanisms responsible for the promotion of tumor radioresistance by HIF-1α remain unclear. β-catenin is known to be involved in the metastatic potential of prostate cancer (PCa). In this study, to investigate the role of HIF-1α and β-catenin in the radioresistance of PCa, two PCa cell lines, LNCaP and C4-2B, were grouped as follows: Negative control (no treatment), HIF-1α overexpression group (transfected with HIF-1α overexpression plasmid) and β-catenin silenced group (transfected with HIF-1α plasmids and β-catenin-shRNA). Cell proliferation, cell cycle, cell invasion and radiosensitivity were examined under normal or hypoxic conditions. In addition, radiosensitivity was examined in two mouse PCa models (the LNCaP orthotopic BALB/c-nu mice model and the C4-2B subcutaneous SCID mice model). Our results revealed that in both the LNCaP and C4-2B cells, transfection with HIF-1α overexpression plasmid led to an enhanced β-catenin nuclear translocation, while β-catenin silencing inhibited β-catenin nuclear translocation. The enhanced β-catenin nuclear translocation induced by HIF-1α overexpression resulted in an enhanced cell proliferation and cell invasion, an altered cell cycle distribution, decreased apoptosis, and improved non-homologous end joining (NHEJ) repair under normal and irradiation conditions. Similar results were observed in the animal models. HIF-1α overexpression enhanced β-catenin nuclear translocation, which led to the activation of the β-catenin/NHEJ signaling pathway and increased cell proliferation, cell invasion and DNA repair. These results thus suggest that HIF-1α overexpression promotes the radioresistance of PCa cells. PMID:29658569

ERRα augments HIF-1 signalling by directly interacting with HIF-1α in normoxic and hypoxic prostate cancer cells.

PubMed

Zou, Chang; Yu, Shan; Xu, Zhenyu; Wu, Dinglan; Ng, Chi-Fai; Yao, Xiaoqiang; Yew, David T; Vanacker, Jean-Marc; Chan, Franky L

2014-05-01

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up-regulation of hypoxia-inducible factor 1 (HIF-1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen-related receptor-α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα-over-expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF-1α protein expression and HIF-1 signalling. These effects could also be observed in ERRα-over-expressing cells grown under normoxia, suggesting that ERRα could function to pre-adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF-1α via its AF-2 domain. A ubiquitination assay showed that this ERRα-HIF-1α interaction could inhibit ubiquitination of HIF-1α and thus reduce its degradation. Such ERRα-HIF-1α interaction could be attenuated by XCT790, an ERRα-specific inverse agonist, resulting in reduced HIF-1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF-1α, suggesting ERRα as a potential therapeutic target for cancer treatment. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Targeting Heat Shock Protein 90 Overrides the Resistance of Lung Cancer Cells by Blocking Radiation-induced Stabilization of Hypoxia-inducible Factor 1α

PubMed Central

Kim, Woo-Young; Oh, Seung Hyun; Woo, Jong-Kyu; Hong, Waun Ki; Lee, Ho-Young

2008-01-01

Hypoxia-inducible factor-1 (HIF-1) has been suggested to play a major role in tumor radioresistance. However, the mechanisms through which irradiation regulates HIF-1α expression remain unclear. The purpose of this study was to investigate the mechanisms that mediate HIF-1 activation and thus radioresistance. Here we show that irradiation induces survival and angiogenic activity in a subset of radioresistant lung cancer cell lines by elevating HIF-1α protein expression. Radiation induced HIF-1α protein expression mainly through two distinct pathways, including an increase in de novo protein synthesis via activation of PI3K/Akt/mTOR and stabilization of HIF-1α protein via augmenting the interaction between heat shock protein 90 (Hsp90) and HIF-1α protein. While the PI3K/Akt/mTOR pathway was activated by irradiation in all the lung cancer cells examined, the HSP90-HIF-1α interaction was enhanced in the resistant cells only. Inhibition of Hsp90 function by 17-AAG or deguelin, a novel natural inhibitor of HSP90, suppressed increases in HIF-1α/Hsp90 interaction and HIF-1α expression in radioresistant cells. Furthermore, combined treatment of radiation with deguelin significantly decreased the survival and angiogenic potential of radioresistant lung cancer cells in vitro. We finally determined in vivo that systemic administration of deguelin resulted in profound inhibition of tumor growth and angiogenesis when combined with radiation. These results provide a strong rationale to target Hsp90 as a means to block radiation-induced HIF-1α and thus to circumvent radioresistance in lung cancer cells. PMID:19176399

HIF-2α-induced chemokines stimulate motility of fibroblast-like synoviocytes and chondrocytes into the cartilage-pannus interface in experimental rheumatoid arthritis mouse models.

PubMed

Huh, Yun Hyun; Lee, Gyuseok; Lee, Keun-Bae; Koh, Jeong-Tae; Chun, Jang-Soo; Ryu, Je-Hwang

2015-10-29

Pannus formation and resulting cartilage destruction during rheumatoid arthritis (RA) depends on the migration of synoviocytes to cartilage tissue. Here, we focused on the role of hypoxia-inducible factor (HIF)-2α-induced chemokines by chondrocytes in the regulation of fibroblast-like synoviocyte (FLS) migration into the cartilage-pannus interface and cartilage erosion. Collagen-induced arthritis (CIA), K/BxN serum transfer, and tumor necrosis factor-α transgenic mice were used as experimental RA models. Expression patterns of HIF-2α and chemokines were determined via immunostaining, Western blotting and RT-PCR. FLS motility was evaluated using transwell migration and invasion assays. The specific role of HIF-2α was determined via local deletion of HIF-2α in joint tissues or using conditional knockout (KO) mice. Cartilage destruction, synovitis and pannus formation were assessed via histological analysis. HIF-2α and various chemokines were markedly upregulated in degenerating cartilage and pannus of RA joints. HIF-2α induced chemokine expression by chondrocytes in both primary culture and cartilage tissue. HIF-2α -induced chemokines by chondrocytes regulated the migration and invasion of FLS. Local deletion of HIF-2α in joint tissues inhibited pannus formation adjacent to cartilage tissue and cartilage destruction caused by K/BxN serum transfer. Furthermore, conditional knockout of HIF-2α in cartilage blocked pannus formation in adjacent cartilage but not bone tissue, along with inhibition of cartilage erosion caused by K/BxN serum transfer. Our findings suggest that chemokines induced by IL-1β or HIF-2α in chondrocytes regulate pannus expansion by stimulating FLS migration and invasion, leading to cartilage erosion during RA pathogenesis.

HIF-2α Expression Regulates Sprout Formation into 3D Fibrin Matrices in Prolonged Hypoxia in Human Microvascular Endothelial Cells.

PubMed

Nauta, Tessa D; Duyndam, Monique C A; Weijers, Ester M; van Hinsbergh, Victor M W; Koolwijk, Pieter

2016-01-01

During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis. We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling. Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction. Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.

Polycythemia and paraganglioma with a novel somatic HIF2A mutation in a male.

PubMed

Toyoda, Hidemi; Hirayama, Jyunya; Sugimoto, Yuka; Uchida, Keiichi; Ohishi, Kohshi; Hirayama, Masahiro; Komada, Yoshihiro

2014-06-01

Recently, a new syndrome of paraganglioma, somatostatinoma, and polycythemia has been discovered (known as Pacak-Zhuang syndrome). This new syndrome, with somatic HIF2A gain-of-function mutations, has never been reported in male patients. We describe a male patient with Pacak-Zhuang syndrome who carries a newly discovered HIF2A mutation. Congenital polycythemias have diverse etiologies, including germline mutations in the oxygen-sensing pathway. These include von Hippel-Lindau (Chuvash polycythemia), prolyl hydroxylase domain-containing protein-2, and hypoxia-inducible factor-2α (HIF-2α). Somatic gain-of-function mutations in the gene encoding HIF-2α were reported in patients with paraganglioma and polycythemia and have been found exclusively in female patients. Through sequencing of the HIF2A using DNA from paraganglioma in 15-year-old male patient, we identified a novel mutation of HIF2A: a heterozygous C to A substitution at base 1589 in exon 12 of HIF2A. The mutation was not found in germline DNA from leukocytes. The C1589A mutations resulted in substitution of alanine 530 in the HIF-2α protein with glutamic acid. This mutation is undoubtedly associated with increased HIF-2α activity and increased protein half-life, because it affects the vicinity of the prolyl hydroxylase target residue, proline 531. To our knowledge, this is the first report describing Pacak-Zhuang syndrome with somatic gain-of-function mutation in HIF2A in a male patient. Congenital polycythemia of unknown origin should raise suspicion for the novel disorder Pacak-Zhuang syndrome, even in male patients. Copyright © 2014 by the American Academy of Pediatrics.

Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

PubMed

Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

2017-09-29

Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK cell lysis and, hence, their potential in vivo lifespan. Our results further support the use of HIF-1 alpha-expressing dental MSCs for cell therapy in tissue injury and immune disorders.

Hydrogen Sulfide Inhibits Hypoxia- But Not Anoxia-Induced Hypoxia-Inducible Factor 1 Activation in a von Hippel-Lindau- and Mitochondria-Dependent Manner

PubMed Central

Kai, Shinichi; Tanaka, Tomoharu; Daijo, Hiroki; Harada, Hiroshi; Kishimoto, Shun; Suzuki, Kengo; Takabuchi, Satoshi; Takenaga, Keizo; Fukuda, Kazuhiko

2012-01-01

Abstract Aims: In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H2S) is an endogenously synthesized gaseous molecule that acts as an important signaling molecule in the living body. Transcription factor hypoxia-inducible factor 1 (HIF-1) is known to respond to intracellular reduced oxygen (O2) availability, which is regulated by an elaborate balance between O2 supply and demand. However, the effect of H2S on HIF-1 activity under hypoxic conditions is largely unknown in mammalian cells. In this study, we tried to elucidate the effect of H2S on hypoxia-induced HIF-1 activation adopting cultured cells and mice. Results: The H2S donors sodium hydrosulfide and sodium sulfide in pharmacological concentrations reversibly reduced cellular O2 consumption and inhibited hypoxia- but not anoxia-induced HIF-1α protein accumulation and expression of genes downstream of HIF-1 in established cell lines. H2S did not affect HIF-1 activation induced by the HIF-α hydroxylases inhibitors desferrioxamine or CoCl2. Experimental evidence adopting von Hippel-Lindau (VHL)- or mitochondria-deficient cells indicated that H2S did not affect neosynthesis of HIF-1α protein but destabilized HIF-1α in a VHL- and mitochondria-dependent manner. We also demonstrate that exogenously administered H2S inhibited HIF-1–dependent gene expression in mice. Innovation: For the first time, we show that H2S modulates intracellular O2 homeostasis and regulates activation of HIF-1 and the subsequent gene expression induced by hypoxia by using an in vitro system with established cell lines and an in vivo system in mice. Conclusions: We demonstrate that H2S inhibits hypoxia-induced HIF-1 activation in a VHL- and mitochondria-dependent manner. Antioxid. Redox Signal. 16, 203–216. PMID:22004513

Hypoxia-independent upregulation of placental hypoxia inducible factor-1α gene expression contributes to the pathogenesis of preeclampsia.

PubMed

Iriyama, Takayuki; Wang, Wei; Parchim, Nicholas F; Song, Anren; Blackwell, Sean C; Sibai, Baha M; Kellems, Rodney E; Xia, Yang

2015-06-01

Accumulation of hypoxia inducible factor-1α (HIF-1α) is commonly an acute and beneficial response to hypoxia, whereas chronically elevated HIF-1α is associated with multiple disease conditions, including preeclampsia, a serious hypertensive disease of pregnancy. However, the molecular basis underlying the persistent elevation of placental HIF-1α in preeclampsia and its role in the pathogenesis of preeclampsia are poorly understood. Here we report that Hif-1α mRNA and HIF-1α protein were elevated in the placentas of pregnant mice infused with angiotensin II type I receptor agonistic autoantibody, a pathogenic factor in preeclampsia. Knockdown of placental Hif-1α mRNA by specific siRNA significantly attenuated hallmark features of preeclampsia induced by angiotensin II type I receptor agonistic autoantibody in pregnant mice, including hypertension, proteinuria, kidney damage, impaired placental vasculature, and elevated maternal circulating soluble fms-like tyrosine kinase-1 levels. Next, we discovered that Hif-1α mRNA levels and HIF-1α protein levels were induced in an independent preeclampsia model with infusion of the inflammatory cytokine tumor necrosis factor superfamily member 14 (LIGHT). SiRNA knockdown experiments also demonstrated that elevated HIF-1α contributed to LIGHT-induced preeclampsia features. Translational studies with human placentas showed that angiotensin II type I receptor agonistic autoantibody or LIGHT is capable of inducing HIF-1α in a hypoxia-independent manner. Moreover, increased HIF-1α was found to be responsible for angiotensin II type I receptor agonistic autoantibody or LIGHT-induced elevation of Flt-1 gene expression and production of soluble fms-like tyrosine kinase-1 in human villous explants. Overall, we demonstrated that hypoxia-independent stimulation of HIF-1α gene expression in the placenta is a common pathogenic mechanism promoting disease progression. Our findings reveal new insight to preeclampsia and highlight novel therapeutic possibilities for the disease. © 2015 American Heart Association, Inc.

Investigation of hypoxia-inducible factor-1α in hippocampal sclerosis: a postmortem study.

PubMed

Feast, Alexandra; Martinian, Lillian; Liu, Joan; Catarino, Claudia B; Thom, Maria; Sisodiya, Sanjay M

2012-08-01

Hypoxia-inducible factor-1α (HIF-1α) is involved in critical aspects of cell survival in response to hypoxia and regulates vascular endothelial growth factor (VEGF) expression. Previous experimental and human studies in epilepsy show up-regulation of VEGF following seizures, although expression of HIF-1α as its potential regulator has not been explored. We used a postmortem (PM) series from patients with epilepsy and hippocampal sclerosis (HS) to investigate patterns of expression of HIF-1α and VEGF and their potential contribution to neuroprotection. In 33 PMs (17 cases with unilateral HS, 3 with bilateral HS, 3 with No-HS, and 10 controls), we quantified neuronal immunolabeling for HIF-1α and VEGF in hippocampal subfields. HIF-1α- and VEGF-immunopositive hippocampal neurones were observed in HS, No-HS, and also in control cases; there was no significant difference in overall labeling between epilepsy cases and controls. In positive cases, HIF-1α and VEGF neuronal labeling localized primarily in CA1, CA4, and CA3 subfields in all groups; significantly more positive neurons were seen in the entorhinal cortex in epilepsy cases (p < 0.05). Labeling lateralized to the side of sclerosis in unilateral HS cases, with significant differences between hemispheres (p < 0.05). There was a trend for high HIF-1α labeling scores in patients with Dravet syndrome without HS and sudden unexpected death in epilepsy (SUDEP) cases, and lower scores with long seizure-free periods prior to death. Hippocampal HIF-1α and VEGF labeling showed a significant correlation. There was neuronal colocalization of HIF-1α and VEGF. Regional expression patterns are in keeping with seizure-related activation of HIF-1α and VEGF. The prominent expression in non-HS cases could support an overall neuroprotective effect. Correlation between HIF-1α and VEGF neuronal immunolabeling supports HIF-1α-mediated induction of VEGF in epilepsy. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner

PubMed Central

Lohanatha, Ferenz L.; Hahne, Martin; Strehl, Cindy; Fangradt, Monique; Tran, Cam Loan; Schönbeck, Kerstin; Hoff, Paula; Ode, Andrea; Perka, Carsten; Duda, Georg N.; Buttgereit, Frank

2012-01-01

Background Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages. Methodology/Principal Findings Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis. Conclusions/Significance Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches. PMID:23029528

Nuclear translocation of HIF-1α induced by influenza A (H1N1) infection is critical to the production of proinflammatory cytokines.

PubMed

Guo, Xinkun; Zhu, Zhaoqin; Zhang, Wanju; Meng, Xiaoxiao; Zhu, Yong; Han, Peng; Zhou, Xiaohui; Hu, Yunwen; Wang, Ruilan

2017-05-24

Infection with the influenza A (H1N1) virus is a major challenge for public health because it can cause severe morbidity and even mortality in humans. The over-secretion of inflammatory cytokines (cytokine storm) is considered to be a key contributor to the severe pneumonia caused by H1N1 infection. It has been reported that hypoxia-inducible factor 1-alpha (HIF-1α) is associated with the production of proinflammatory molecules, but whether HIF-1α participates in the acute inflammatory responses against H1N1 infection is still unclear. To investigate the role of HIF-1α in H1N1 infection, the expression and nuclear translocation of HIF-1α in A549 and THP-1 cell lines infected with H1N1 virus were observed. The results showed that without altering the intracellular mRNA or protein expression of HIF-1α, H1N1 infection only induced nuclear translocation of HIF-1α under normal oxygen concentrations. The use of 2-methoxyestradiol (2ME2), a HIF-1α inhibitor that blocks HIF-1α nuclear accumulation, in H1N1-infected cells decreased the mRNA and protein expression of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 and increased the levels of IL-10. In contrast, H1N1-infected cells under hypoxic conditions had increased HIF-1α nuclear accumulation, increased expression of TNF-α and IL-6 and decreased levels of IL-10. In conclusion, our data implied that in vitro H1N1 infection induced nuclear translocation of HIF-1α without altering the expression of HIF-1α, which may promote the secretion of proinflammatory cytokines during H1N1 infection.

Functional cooperation between HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR.

PubMed

Meng, Shuyan; Wang, Guorui; Lu, Yang; Fan, Zhen

2018-07-01

Hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are important transcription factors regulating expression of genes involved in cell survival. HIF-1α and c-Jun are key components of HIF-1 and AP-1, respectively, and are regulated by epidermal growth factor receptor (EGFR)-mediated cell signaling and tumor microenvironmental cues. The roles of HIF-1α and c-Jun in development of resistance to EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC) with activating mutation of EGFR have not been explored. In this study, we investigated the roles of HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR. Changes in HIF-1α protein and in total and phosphorylated c-Jun levels in relation to changes in total and phosphorylated EGFR levels before and after gefitinib treatment were measured using Western blot analysis in NSCLC cells sensitive or resistant to gefitinib. The impact of overexpression of a constitutively expressed HIF-1α (HIF-1α/ΔODD) or a constitutively active c-Jun upstream regulator (SEK1 S220E/T224D mutant) on cell response to gefitinib was also examined. The effect of pharmacological inhibition of SEK1-JNK-c-Jun pathway on cell response to gefitinib was evaluated. Downregulation of HIF-1α and total and phosphorylated c-Jun levels correlated with cell inhibitory response to gefitinib better than decrease in phosphorylated EGFR did in NSCLC cells with intrinsic or acquired resistance to gefitinib. Overexpression of HIF-1α/ΔODD or SEK1 S220E/T224D mutant conferred resistance to gefitinib. There exists a positive feed-forward regulation loop between HIF-1 and c-Jun. The JNK inhibitor SP600125 sensitized gefitinib-resistant NSCLC cells to gefitinib. HIF-1α and c-Jun functionally cooperate in development of resistance to gefitinib in NSCLC cells. The translational value of inhibiting HIF-1α/c-Jun cooperation in overcoming resistance to EGFR TKI treatment of NSCLC cells with activating mutation of EGFR deserves further investigation. Copyright © 2018 Elsevier B.V. All rights reserved.

Cold Inducible RNA Binding Protein Is Involved in Chronic Hypoxia Induced Neuron Apoptosis by Down-Regulating HIF-1α Expression and Regulated By microRNA-23a.

PubMed

Chen, Xiaoming; Liu, Xinqin; Li, Bin; Zhang, Qian; Wang, Jiye; Zhang, Wenbin; Luo, Wenjing; Chen, Jingyuan

2017-01-01

Background: Neuron apoptosis mediated by hypoxia inducible factor 1α (HIF-1α) in hippocampus is one of the most important factors accounting for the chronic hypobaric hypoxia induced cognitive impairment. As a neuroprotective molecule that is up-regulated in response to various environmental stress, CIRBP was reported to crosstalk with HIF-1α under cellular stress. However, its function under chronic hypobaric hypoxia remains unknown. Objective: In this study, we tried to identify the role of CIRBP in HIF-1α mediated neuron apoptosis under chronic hypobaric hypoxia and find a possible method to maintain its potential neuroprotective in long-term high altitude environmental exposure. Methods: We established a chronic hypobaric hypoxia rat model as well as a tissue culture model where SH-SY5Y cells were exposed to 1% hypoxia. Based on these models, we measured the expressions of HIF-1α and CIRBP under hypoxia exposure and examined the apoptosis of neurons by TUNEL immunofluorescence staining and western blot analysis of apoptosis related proteins. In addition, by establishing HIF-1α shRNA and pEGFP-CIRBP plasmid transfected cells, we confirmed the role of HIF-1α in chronic hypoxia induced neuron apoptosis and identified the influence of CIRBP over-expression upon HIF-1α and neuron apoptosis in the process of exposure. Furthermore, we measured the expression of the reported hypoxia related miRNAs in both models and the influence of miRNAs' over-expression/knock-down upon CIRBP in the process of HIF-1α mediated neuron apoptosis. Results: HIF-1α expression as well as neuron apoptosis was significantly elevated by chronic hypobaric hypoxia both in vivo and in vitro . CIRBP was induced in the early stage of exposure (3d/7d); however as the exposure was prolonged (21d), CIRBP level of the hypoxia group became significantly lower than that of control. In addition, HIF-1α knockdown significantly decreased neuron apoptosis under hypoxia, suggesting HIF-1α may be pro-apoptotic in the process of exposure. CIRBP over-expression significantly suppressed HIF-1α up-regulation in hypoxia and inhibited HIF-1α mediated neuron apoptosis. Interestingly, miR-23a was also induced by hypoxia exposure and showed the same changing tendency with CIRBP (increasing in 3d/7d, decreasing in 21d). In addition, over-expressing miR-23a up-regulated CIRBP, down-regulated HIF-1α and attenuated neuron apoptosis. Conclusion: Cold inducible RNA binding protein is involved in chronic hypoxia induced neuron apoptosis by down-regulating HIF-1α expression, and MiR-23a may be an important tool to maintain CIRBP level and function.

CYP2E1 Potentiates Ethanol-induction of Hypoxia and HIF-1α in vivo

PubMed Central

Wang, Xiaodong; Wu, Defeng; Yang, Lili; Gan, Lixia; Cederbaum, Arthur I

2013-01-01

Ethanol induces hypoxia and elevates HIF-1α in the liver. CYP2E1 plays a role in the mechanisms by which ethanol generates oxidative stress, fatty liver and liver injury. The current study evaluated whether CYP2E1 contributes to ethanol-induced hypoxia and activation of HIF-1α in vivo and whether HIF-1α protects against or promotes CYP2E1-dependent toxicity in vitro. Wild type (WT), CYP2E1-knockin (KI) and CYP2E1 knockout (KO) mice were fed ethanol chronically; pair fed controls received isocaloric dextrose. Ethanol produced liver injury in the KI mice to a much greater extent than in the WT and KO mice. Protein levels of HIF-1α and downstream targets of HIF-1α activation were elevated in the ethanol-fed KI mice compared to the WT and KO mice. Levels of HIF prolylhydroxlase 2 which promotes HIF-1α degradation were decreased in the ethanol-fed KI mice in association with the increases in HIF-1α. Hypoxia occurred in the ethanol-fed CYP2E1 KI mice as shown by an increased area of staining using the hypoxia-specific marker pimonidazole. Hypoxia was lower in the ethanol-fed WT mice and lowest in the ethanol fed KO mice and all the dextrose-fed mice. In situ double staining showed that pimonidazole and CYP2E1 were co-localized to the same area of injury in the hepatic centrilobule. Increased protein levels of HIF-1α were also found after acute ethanol treatment of KI mice. Treatment of HepG2 E47 cells which express CYP2E1 with ethanol plus arachidonic (AA) acid or ethanol plus buthionine sulfoximine (BSO) which depletes GSH caused loss of cell viability to greater extent than in HepG2 C34 cells which do not express CYP2E1. These treatments elevated protein levels of HIF-1α to a greater extent in E47 cells than C34 cells. 2-Methoxyestradiol, an inhibitor of HIF-1α, blunted the toxic effects of ethanol plus AA and ethanol plus BSO in the E47 cells in association with inhibition of HIF-1α. The HIF-1α inhibitor also blocked the elevated oxidative stress produced by ethanol/AA or ethanol/BSO in the E47 cells. These results suggest that CYP2E1 plays a role in ethanol-induced hypoxia, oxidative stress and activation of HIF-1α and that HIF-1α contributes to CYP2E1-dependent ethanol-induced toxicity. Blocking HIF-1α activation and actions may have therapeutic implications for protection against ethanol/CYP2E1-induced oxidative stress, steatosis and liver injury. PMID:23669278

Transcriptome analysis of the plateau fish (Triplophysa dalaica): Implications for adaptation to hypoxia in fishes.

PubMed

Wang, Ying; Yang, Liandong; Wu, Bo; Song, Zhaobin; He, Shunping

2015-07-10

Triplophysa dalaica, endemic species of Qinghai-Tibetan Plateau, is informative for understanding the genetic basis of adaptation to hypoxic conditions of high altitude habitats. Here, a comprehensive gene repertoire for this plateau fish was generated using the Illumina deep paired-end high-throughput sequencing technology. De novo assembly yielded 145, 256 unigenes with an average length of 1632 bp. Blast searches against GenBank non-redundant database annotated 74,594 (51.4%) unigenes encoding for 30,047 gene descriptions in T. dalaica. Functional annotation and classification of assembled sequences were performed using Gene Ontology (GO), clusters of euKaryotic Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. After comparison with other fish transcriptomes, including silver carp (Hypophthalmichthys molitrix) and mud loach (Misgurnus anguillicaudatus), 2621 high-quality orthologous gene alignments were constructed among these species. 61 (2.3%) of the genes were identified as having undergone positive selection in the T. dalaica lineage. Within the positively selected genes, 13 genes were involved in hypoxia response, of which 11 were listed in HypoxiaDB. Furthermore, duplicated hif-α (hif-1αA/B and hif-2αA/B), EGLN1 and PPARA candidate genes involved in adaptation to hypoxia were identified in T. dalaica transcriptome. Branch-site model in PAML validated that hif-1αB and hif-2αA genes have undergone positive selection in T.dalaica. Finally, 37,501 simple sequence repeats (SSRs) and 19,497 high-quality single nucleotide polymorphisms (SNPs) were identified in T. dalaica. The identified SSR and SNP markers will facilitate the genetic structure, population geography and ecological studies of Triplophysa fishes. Copyright © 2015 Elsevier B.V. All rights reserved.

Response of skeletal muscle mitochondria to hypoxia.

PubMed

Hoppeler, Hans; Vogt, Michael; Weibel, Ewald R; Flück, Martin

2003-01-01

This review explores the current concepts relating the structural and functional modifications of skeletal muscle mitochondria to the molecular mechanisms activated when organisms are exposed to a hypoxic environment. In contrast to earlier assumptions it is now established that permanent or long-term exposure to severe environmental hypoxia decreases the mitochondrial content of muscle fibres. Oxidative muscle metabolism is shifted towards a higher reliance on carbohydrates as a fuel, and intramyocellular lipid substrate stores are reduced. Moreover, in muscle cells of mountaineers returning from the Himalayas, we find accumulations of lipofuscin, believed to be a mitochondrial degradation product. Low mitochondrial contents are also observed in high-altitude natives such as Sherpas. In these subjects high-altitude performance seems to be improved by better coupling between ATP demand and supply pathways as well as better metabolite homeostasis. The hypoxia-inducible factor 1 (HIF-1) has been identified as a master regulator for the expression of genes involved in the hypoxia response, such as genes coding for glucose transporters, glycolytic enzymes and vascular endothelial growth factor (VEGF). HIF-1 achieves this by binding to hypoxia response elements in the promoter regions of these genes, whereby the increase of HIF-1 in hypoxia is the consequence of a reduced degradation of its dominant subunit HIF-1a. A further mechanism that seems implicated in the hypoxia response of muscle mitochondria is related to the formation of reactive oxygen species (ROS) in mitochondria during oxidative phosphorylation. How exactly ROS interfere with HIF-1a as well as MAP kinase and other signalling pathways is debated. The current evidence suggests that mitochondria themselves could be important players in oxygen sensing.

SIRT1 and HIF1α signaling in metabolism and immune responses.

PubMed

Yu, Qing; Dong, Lin; Li, Yan; Liu, Gaungwei

2018-04-01

SIRT1 and HIF1α are regarded as two key metabolic sensors in cellular metabolism pathways and play vital roles in influencing immune responses. SIRT1 and HIF1α regulate immune responses in metabolism-dependent and -independent ways. Here, we summarized the recent knowledge of SIRT1 and HIF1α signaling in metabolism and immune responses. HIF1α is a direct target of SIRT1. Sometimes, SIRT1 and HIF1α cooperate or act separately to mediate immune responses. In innate immune responses, SIRT1 can regulate the glycolytic activity of myeloid-derived suppressor cells (MDSCs) and influence MDSC functional differentiation. SIRT1 can regulate monocyte function through NF-κB and PGC-1, accompanying an increased NAD + level. The SIRT1-HIF1α axis bridges the innate immune signal to an adaptive immune response by directing cytokine production of dendritic cells in a metabolism-independent manner, promoting the differentiation of CD4 + T cells. For adaptive immune cells, SIRT1 can mediate the differentiation of inflammatory T cell subsets in a NAD + -dependent manner. HIF1α can stimulate some glycolysis-associated genes and regulate the ATP and ROS generations. In addition, SIRT1-and HIF1α-associated metabolism inhibits the activity of mTOR, thus negatively regulating the differentiation and function of Th9 cells. As immune cells are crucial in controlling immune-associated diseases, SIRT1-and HIF1α associated-metabolism is closely linked to immune-associated diseases, including infection, tumors, allergic airway inflammation, and autoimmune diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Drug resistance and cancer stem cells: the shared but distinct roles of hypoxia-inducible factors HIF1α and HIF2α.

PubMed

Schöning, Jennifer Petra; Monteiro, Michael; Gu, Wenyi

2017-02-01

Chemotherapy resistance is a major contributor to poor treatment responses and tumour relapse, the development of which has been strongly linked to the action of cancer stem cells (CSCs). Mounting evidence suggests that CSCs are reliant on low oxygen conditions and hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) to maintain their stem cell features. Research in the last decade has begun to clarify the functional differences between the two HIFα subtypes (HIFαs). Here, we review and discuss these differences in relation to CSC-associated drug resistance. Both HIFαs contribute to CSC survival but play different roles -HIF1α being more responsible for survival functions and HIF2α for stemness traits such as self-renewal - and are sensitive to different degrees of hypoxia. Failure to account for physiologically relevant oxygen concentrations in many studies may influence the current understanding of the roles of HIFαs. We also discuss how hypoxia and HIFαs contribute to CSC drug resistance via promotion of ABC drug transporters Breast cancer resistance protein (BCRP), MDR1, and MRP1 and through maintenance of quiescence. Additionally, we explore the PI3K/AKT cell survival pathway that may support refractory cancer by promoting CSCs and activating both HIF1α and HIF2α. Accordingly, HIF1α and HIF2α inhibition, potentially via PI3K/AKT inhibitors, could reduce chemotherapy resistance and prevent cancer relapse. © 2016 John Wiley & Sons Australia, Ltd.

Mild hypoxia-induced cardiomyocyte hypertrophy via up-regulation of HIF-1α-mediated TRPC signalling

PubMed Central

Chu, Wenfeng; Wan, Lin; Zhao, Dan; Qu, Xuefeng; Cai, Fulai; Huo, Rong; Wang, Ning; Zhu, Jiuxin; Zhang, Chun; Zheng, Fangfang; Cai, Ruijun; Dong, Deli; Lu, Yanjie; Yang, Baofeng

2012-01-01

Hypoxia-inducible factor-1 alpha (HIF-1α) is a central transcriptional regulator of hypoxic response. The present study was designed to investigate the role of HIF-1α in mild hypoxia-induced cardiomyocytes hypertrophy and its underlying mechanism. Mild hypoxia (MH, 10% O2) caused hypertrophy in cultured neonatal rat cardiac myocytes, which was accompanied with increase of HIF-1α mRNA and accumulation of HIF-1α protein in nuclei. Transient receptor potential canonical (TRPC) channels including TRPC3 and TRPC6, except for TRPC1, were increased, and Ca2+-calcineurin signals were also enhanced in a time-dependent manner under MH condition. MH-induced cardiomyocytes hypertrophy, TRPC up-regulation and enhanced Ca2+-calcineurin signals were inhibited by an HIF-1α specific blocker, SC205346 (30 μM), whereas promoted by HIF-1α overexpression. Electrophysiological voltage-clamp demonstrated that DAG analogue, OAG (30 μM), induced TRPC current by as much as 170% in neonatal rat cardiomyocytes overexpressing HIF-1α compared to negative control. These results implicate that HIF-1α plays a key role in development of cardiac hypertrophy in responses to hypoxic stress. Its mechanism is associated with up-regulating TRPC3, TRPC6 expression, activating TRPC current and subsequently leading to enhanced Ca2+-calcineurin signals. PMID:22129453

HIF-2α mediates hypoxia-induced LIF expression in human colorectal cancer cells

PubMed Central

Zhao, Yuhan; Zhang, Cen; Wang, Jiabei; Yue, Xuetian; Yang, Qifeng; Hu, Wenwei

2015-01-01

Leukemia inhibitory factor (LIF), a multi-functional cytokine, has a complex role in cancer. While LIF induces the differentiation of several myeloid leukemia cells and inhibits their growth, it also promotes tumor progression, metastasis and chemoresistance in many solid tumors. LIF is frequently overexpressed in a variety of human tumors and its overexpression is often associated with poor prognosis of patients. Currently, the mechanism for LIF overexpression in tumor cells is not well-understood. Here, we report that hypoxia, a hallmark of solid tumors, induced LIF mRNA expression in human colorectal cancer cells. Analysis of LIF promoter revealed several hypoxia-responsive elements (HREs) that can specifically interact with and be transactivated by HIF-2α but not HIF-1α. Consistently, ectopic expression of HIF-2α but not HIF-1α transcriptionally induced LIF expression levels in cells. Knockdown of endogenous HIF-2α but not HIF-1α by siRNA largely abolished the induction of LIF by hypoxia in cells. Furthermore, there is a strong association of HIF-2α overexpression with LIF overexpression in human colorectal cancer specimens. In summary, results from this study demonstrate that hypoxia induces LIF expression in human cancer cells mainly through HIF-2α, which could be an important underlying mechanism for LIF overexpression in human cancers. PMID:25726527

Inhibition of hypoxia inducible factors combined with all-trans retinoic acid treatment enhances glial transdifferentiation of neuroblastoma cells.

PubMed

Cimmino, Flora; Pezone, Lucia; Avitabile, Marianna; Acierno, Giovanni; Andolfo, Immacolata; Capasso, Mario; Iolascon, Achille

2015-06-09

Neuroblastoma (NBL) is a heterogeneous tumor characterized by a wide range of clinical manifestations. A high tumor cell differentiation grade correlates to a favorable stage and positive outcome. Expression of the hypoxia inducible factors HIF1-α (HIF1A gene) and HIF2-α (EPAS1 gene) and/or hypoxia-regulated pathways has been shown to promote the undifferentiated phenotype of NBL cells. Our hypothesis is that HIF1A and EPAS1 expression represent one of the mechanisms responsible for the lack of responsiveness of NBL to differentiation therapy. Clinically, high levels of HIF1A and EPAS1 expression were associated with inferior survival in two NBL microarray datasets, and patient subgroups with lower expression of HIF1A and EPAS1 showed significant enrichment of pathways related to neuronal differentiation. In NBL cell lines, the combination of all-trans retinoic acid (ATRA) with HIF1A or EPAS1 silencing led to an acquired glial-cell phenotype and enhanced expression of glial-cell differentiation markers. Furthermore, HIF1A or EPAS1 silencing might promote cell senescence independent of ATRA treatment. Taken together, our data suggest that HIF inhibition coupled with ATRA treatment promotes differentiation into a more benign phenotype and cell senescence in vitro. These findings open the way for additional lines of attack in the treatment of NBL minimal residue disease.

Inhibition of hypoxia inducible factors combined with all-trans retinoic acid treatment enhances glial transdifferentiation of neuroblastoma cells

PubMed Central

Cimmino, Flora; Pezone, Lucia; Avitabile, Marianna; Acierno, Giovanni; Andolfo, Immacolata; Capasso, Mario; Iolascon, Achille

2015-01-01

Neuroblastoma (NBL) is a heterogeneous tumor characterized by a wide range of clinical manifestations. A high tumor cell differentiation grade correlates to a favorable stage and positive outcome. Expression of the hypoxia inducible factors HIF1-α (HIF1A gene) and HIF2-α (EPAS1 gene) and/or hypoxia-regulated pathways has been shown to promote the undifferentiated phenotype of NBL cells. Our hypothesis is that HIF1A and EPAS1 expression represent one of the mechanisms responsible for the lack of responsiveness of NBL to differentiation therapy. Clinically, high levels of HIF1A and EPAS1 expression were associated with inferior survival in two NBL microarray datasets, and patient subgroups with lower expression of HIF1A and EPAS1 showed significant enrichment of pathways related to neuronal differentiation. In NBL cell lines, the combination of all-trans retinoic acid (ATRA) with HIF1A or EPAS1 silencing led to an acquired glial-cell phenotype and enhanced expression of glial-cell differentiation markers. Furthermore, HIF1A or EPAS1 silencing might promote cell senescence independent of ATRA treatment. Taken together, our data suggest that HIF inhibition coupled with ATRA treatment promotes differentiation into a more benign phenotype and cell senescence in vitro. These findings open the way for additional lines of attack in the treatment of NBL minimal residue disease. PMID:26057707

Oral delivery of prolyl hydroxylase inhibitor: AKB-4924 promotes localized mucosal healing in a mouse model of colitis.

PubMed

Marks, Ellen; Goggins, Bridie J; Cardona, Jocelle; Cole, Siobhan; Minahan, Kyra; Mateer, Sean; Walker, Marjorie M; Shalwitz, Robert; Keely, Simon

2015-02-01

Pharmacological induction of hypoxia-inducible factor (HIF), a global transcriptional regulator of the hypoxic response, by prolyl hydroxylase inhibitors (PHDi) is protective in murine models of colitis, and epithelial cells are critical for the observed therapeutic efficacy. Because systemic HIF activation may lead to potentially negative off-target effects, we hypothesized that targeting epithelial HIF through oral delivery of PHDi would be sufficient to protect against colitis in a mouse model. Using a chemically induced trinitrobenzene sulfonic acid murine model of colitis, we compared the efficacy of oral and intraperitoneal (i.p.) delivery of the PHDi; AKB-4924 in preventing colitis, as measured by endoscopy, histology, barrier integrity, and immune profiling. Furthermore, we measured potential off-target effects, examining HIF and HIF target genes in the heart and kidney, as well as erythropoietin and hematocrit levels. Oral administration of AKB-4924 exhibited mucosal protection comparable i.p. dosing. Oral delivery of PHDi led to reduced colonic epithelial HIF stabilization compared with i.p. delivery, but this was still sufficient to induce transcription of downstream HIF targets. Furthermore, oral delivery of PHDi led to reduced stabilization of HIF and activation of HIF targets in extraintestinal organs. Oral delivery of PHDi therapies to this intestinal mucosa protects against colitis in animal models and represents a potential therapeutic strategy for inflammatory bowel disease, which also precludes unwanted extraintestinal effects.

Maternal intestinal HIF-2α is necessary for sensing iron demands of lactation in mice

PubMed Central

Ramakrishnan, Sadeesh K.; Anderson, Erik R.; Martin, Angelical; Centofanti, Brook; Shah, Yatrik M.

2015-01-01

The mechanisms that are essential for the maintenance of nutrient status in breast milk are unclear. Our data demonstrate that the intestine via hypoxia-inducible factor (HIF)-2α is an essential regulatory mechanism for maintaining the quality of breast milk. During lactation, intestinal HIF-2α is highly increased, leading to an adaptive induction of apical and basolateral iron transport genes. Disruption of intestinal HIF-2α (but not HIF-1α) or the downstream target gene divalent metal transporter (DMT)-1 in lactating mothers did not alter systemic iron homeostasis in the mothers, but led to anemia, decreased growth, and truncal alopecia in pups which was restored following weaning. Moreover, pups born from mothers with a disruption of intestinal HIF-2α led to long-term cognitive defects. Cross-fostering experiments and micronutrient profiling of breast milk demonstrated that the defects observed were due to decreased maternal iron delivery via milk. Increasing intestinal iron absorption by activation of HIF-2α or parenteral administration of iron-dextran in HIF-2α knockout mothers ameliorated anemia and restored neonatal development and adult cognitive functions. The present work details the importance of breast milk iron in neonatal development and uncovers an unexpected molecular mechanism for the regulation of nutritional status of breast milk through intestinal HIF-2α. PMID:26124130

HIF-1α regulates Cx40-dependent vasodilatation following hemorrhagic shock in rats

PubMed Central

Duan, Chenyang; Chen, Ken; Yang, Guangming; Li, Tao; Liu, Liangming

2017-01-01

HIF-1α plays an essential role in hemorrhagic shock-induced vasoconstriction. However, the underlying mechanisms remain poorly understood. Here, we studied both the role of HIF-1α in regulating vasodilatation, and the involvement of Cx40 in this process. We found that endothelium-dependent vasodilatation exhibited an overall decline after hemorrhagic shock: at the beginning of shock vasodilatation reactivity significantly decreased, followed by a slight increase from 0.5 h to 2 h after shock. After 2 h vasodilatation dropped again. Throughout this process, protein levels of HIF-1α gradually increased. In the late period of shock, vasodilatation reactivity was enhanced by oligomycin, an HIF-1α inhibitor, suggesting that HIF-1α may promote vasoconstriction. Moreover, in the late period of shock Cx40 levels gradually increased and exhibited a negative correlation with endothelium-dependent vasoconstriction reactivity. Furthermore, Cx40 AODN significantly improved vasoconstriction reactivity and could be regulated by either an HIF-1α inhibitor or an agonist. Together, these data suggest that HIF-1α may inhibit endothelium-dependent vasodilatation reactivity following hemorrhagic shock by up-regulating Cx40, especially in the late period of shock. PMID:28386353

Sulforaphane inhibits hypoxia-induced HIF-1α and VEGF expression and migration of human colon cancer cells.

PubMed

Kim, Dong Hwan; Sung, Bokyung; Kang, Yong Jung; Hwang, Seong Yeon; Kim, Min Jeong; Yoon, Jeong-Hyun; Im, Eunok; Kim, Nam Deuk

2015-12-01

The effects of sulforaphane (a natural product commonly found in broccoli) was investigated on hypoxia inducible factor-1α (HIF-1α) expression in HCT116 human colon cancer cells and AGS human gastric cancer cells. We found that hypoxia-induced HIF-1α protein expression in HCT116 and AGS cells, while treatment with sulforaphane markedly and concentration-dependently inhibited HIF-1α expression in both cell lines. Treatment with sulforaphane inhibited hypoxia-induced vascular endothelial growth factor (VEGF) expression in HCT116 cells. Treatment with sulforaphane modulated the effect of hypoxia on HIF-1α stability. However, degradation of HIF-1α by sulforaphane was not mediated through the 26S proteasome pathway. We also found that the inhibition of HIF-1α by sulforaphane was not mediated through AKT and extracellular signal-regulated kinase phosphorylation under hypoxic conditions. Finally, hypoxia-induced HCT116 cell migration was inhibited by sulforaphane. These data suggest that sulforaphane may inhibit human colon cancer progression and cancer cell angiogenesis by inhibiting HIF-1α and VEGF expression. Taken together, these results indicate that sulforaphane is a new and potent chemopreventive drug candidate for treating patients with human colon cancer.

Hydrologic Instrumentation Facility of the U.S. Geological Survey, annual report for fiscal year 1993

USGS Publications Warehouse

Latkovich, V.J.; Tracey, Debra C.

1994-01-01

The Hydrologic lnstrumentation Facility (HIF) of the U.S. Geological Survey (USGS) has nationwide responsibility for all aspects of hydrologic field instrumentation in support of Survey data-collection programs. Each year the HIF publishes a report to inform Water Resources Division (WRD) personnel of progress made by the HIF in fulfilling its mission to improve instrumentation services to the Division. The report for fiscal year 1993 (FY93) describes the activities of the HIF, including major accomplish- ments for the year; personnel actions; active projects (reported by section--Technical Services Section, Administrative Services Section, Field Coordination, Applications and Development Section, Test and Evaluation Section, Field Service and Supply Section); and planned activities for the coming year. Also presented in the appendixes are detailed listings of the memberships of the Instrumentation Committee and the Instrumentation Technical Advisory Subcommittee; district, sub- district, and field office visits by HIF personnel; professional and technical meetings attended by HIF personnel; vendor visits; and reports prepared by HIF personnel.

The interplay between HIF-1 and calcium signalling in cancer.

PubMed

Azimi, Iman

2018-04-01

The interplay between hypoxia-inducible factor-1 (HIF-1) and calcium in cancer has begun to be unravelled with recent findings demonstrating the relationships between the two in different cancer types. This is an area of significance considering the crucial roles of both HIF-1 and calcium signalling in cancer progression and metastasis. This review summarises the experimental evidence of the crosstalk between HIF-1 and specific calcium channels, pumps and regulators in the context of cancer. HIF-1 as a master regulator of hypoxic transcriptional responses, mediates transcription of several calcium modulators. On the other hand, specific calcium channels and pumps regulate HIF-1 activity through controlling its transcription, translation, stabilisation, or nuclear translocation. Identifying the interplay between HIF-1 and components of the calcium signal will give new insights into mechanisms underlying cellular responses to physiological and pathophysiological cues, and may provide novel and more efficient therapeutic strategies for the control of cancer progression. Copyright © 2018 Elsevier Ltd. All rights reserved.

ERα upregulates Phd3 to ameliorate HIF-1 induced fibrosis and inflammation in adipose tissue

PubMed Central

Kim, Min; Neinast, Michael D.; Frank, Aaron P.; Sun, Kai; Park, Jiyoung; Zehr, Jordan A.; Vishvanath, Lavanya; Morselli, Eugenia; Amelotte, Mason; Palmer, Biff F.; Gupta, Rana K.; Scherer, Philipp E.; Clegg, Deborah J.

2014-01-01

Hypoxia Inducible Factor 1 (HIF-1) promotes fibrosis and inflammation in adipose tissues, while estrogens and Estrogen Receptor α (ERα) have the opposite effect. Here we identify an Estrogen Response Element (ERE) in the promoter of Phd3, which is a negative regulatory enzyme of HIF-1, and we demonstrate HIF-1α is ubiquitinated following 17-β estradiol (E2)/ERα mediated Phd3 transcription. Manipulating ERα in vivo increases Phd3 transcription and reduces HIF-1 activity, while addition of PHD3 ameliorates adipose tissue fibrosis and inflammation. Our findings outline a novel regulatory relationship between E2/ERα, PHD3 and HIF-1 in adipose tissues, providing a mechanistic explanation for the protective effect of E2/ERα in adipose tissue. PMID:25161887

An efficient way of studying protein-protein interactions involving HIF-α, c-Myc, and Sp1.

PubMed

To, Kenneth K-W; Huang, L Eric

2013-01-01

Protein-protein interaction is an essential biochemical event that mediates various cellular processes including gene expression, intracellular signaling, and intercellular interaction. Understanding such interaction is key to the elucidation of mechanisms of cellular processes in biology and diseases. The hypoxia-inducible transcription factor HIF-1α possesses a non-transcriptional activity that competes with c-Myc for Sp1 binding, whereas its isoform HIF-2α lacks Sp1-binding activity due to phosphorylation. Here, we describe the use of in vitro translation to effectively investigate the dynamics of protein-protein interactions among HIF-1α, c-Myc, and Sp1 and to demonstrate protein phosphorylation as a molecular determinant that functionally distinguishes HIF-2α from HIF-1α.

Turbulence structure of the near-surface boundary layer in complex terrain

NASA Astrophysics Data System (ADS)

Sfyri, Eleni; Rotach, Mathias Walter; Stiperski, Ivana; Bosveld, Fred; Lehner, Manuela; Obleitner, Friedrich

2017-04-01

Monin-Obukhov Similarity Theory (MOST) is evaluated in two cases: truly complex terrain (CT) and horizontally inhomogeneous and flat (HIF) terrain. CT data are derived from 5 measurement sites, which differ in terms of slope, orientation and surface roughness at the Inn Valley of Austria (i-Box) and HIF data come from one measurement site at the Cabauw experimental site (Netherlands). The applicability of the surface-layer, 'ideal' similarity relations is examined for both data-sets and the non-dimensional variances of temperature and humidity as a function of stability (z/L, where L is the Obukhov length) are compared for each type of terrain. Large deviations from the reference curves in case of temperature are observed in both CT and HIF, leading to the conclusion that these deviations are not due to the complex terrain but due to inappropriate near-neutral description of the reference curves. It is found here that the non-dimensional temperature variance exhibits a -1 slope in the near-neutral region, for both CT and HIF datasets. In addition, the constant-fluxes hypothesis of the MOST is evaluated at one i-Box site. It is found that only about 1% of the data show constant momentum, sensible and latent heat fluxes with height. Therefore, local scaling instead of surface layer scaling is being used in this study.

GDC-0941 inhibits metastatic characteristics of thyroid carcinomas by targeting both the phosphoinositide-3 kinase (PI3K) and hypoxia-inducible factor-1α (HIF-1α) pathways.

PubMed

Burrows, Natalie; Babur, Muhammad; Resch, Julia; Ridsdale, Sophie; Mejin, Melissa; Rowling, Emily J; Brabant, Georg; Williams, Kaye J

2011-12-01

Phosphoinositide 3-kinase (PI3K) regulates the transcription factor hypoxia-inducible factor-1 (HIF-1) in thyroid carcinoma cells. Both pathways are associated with aggressive phenotype in thyroid carcinomas. Our objective was to assess the effects of the clinical PI3K inhibitor GDC-0941 and genetic inhibition of PI3K and HIF on metastatic behavior of thyroid carcinoma cells in vitro and in vivo. Vascular endothelial growth factor ELISA, HIF activity assays, proliferation studies, and scratch-wound migration and cell spreading assays were performed under various O(2) tensions [normoxia, hypoxia (1 and 0.1% O(2)), and anoxia] with or without GDC-0941 in a panel of four thyroid carcinoma cell lines (BcPAP, WRO, FTC133, and 8505c). Genetic inhibition was achieved by overexpressing phosphatase and tensin homolog (PTEN) into PTEN-null cells and by using a dominant-negative variant of HIF-1α (dnHIF). In vivo, human enhanced green fluorescence protein-expressing follicular thyroid carcinomas (FTC) were treated with GDC-0941 (orally). Spontaneous lung metastasis was confirmed by viewing enhanced green fluorescence protein-positive colonies cultured from lung tissue. GDC-0941 inhibited hypoxia/anoxia-induced HIF-1α and HIF-2α expression and HIF activity in thyroid carcinoma cells. Basal (three of four cell lines) and/or hypoxia-induced (four of four) secreted vascular endothelial growth factor was inhibited by GDC-0941, whereas selective HIF targeting predominantly affected hypoxia/anoxia-mediated secretion (P < 0.05-0.0001). Antiproliferative effects of GDC-0941 were more pronounced in PTEN mutant compared with PTEN-restored cells (P < 0.05). Hypoxia increased migration in papillary cells and cell spreading/migration in FTC cells (P < 0.01). GDC-0941 reduced spreading and migration in all O(2) conditions, whereas dnHIF had an impact only on hypoxia-induced migration (P < 0.001). In vivo, GDC-0941 reduced expression of HIF-1α, phospho-AKT, GLUT-1, and lactate dehydrogenase A in FTC xenografts. DnHIF expression and GDC-0941 reduced FTC tumor growth and metastatic lung colonization (P < 0.05). PI3K plays a prominent role in the metastatic behavior of thyroid carcinoma cells irrespective of O(2) tension and appears upstream of HIF activation. GDC-0941 significantly inhibited the metastatic phenotype, supporting the clinical development of PI3K inhibition in thyroid carcinomas.

MTA1 and MTA3 Regulate HIF1a Expression in Hypoxia-Treated Human Trophoblast Cell Line HTR8/Svneo

PubMed Central

Wang, Kai; Chen, Ying; Ferguson, Susan D.; Leach, Richard E.

2015-01-01

Hypoxia plays an important role in placental trophoblast differentiation and function during early pregnancy. Hypoxia-inducible factor 1 alpha (HIF1a) is known to regulate cellular adaption to hypoxic conditions. However, our current understanding of the role of HIF1a in trophoblast physiology is far from complete. Metastasis Associated Protein 1 and 3 (MTA1 and MTA3) are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, a chromatin remodeling complex, and are highly expressed in term placental trophoblasts. However, the role of MTA1 and MTA3 in the hypoxic placental environment of early pregnancy is unknown. In the present study, we examined the association among MTA1, MTA3 and HIF1a expression under hypoxic conditions in trophoblasts both in vivo and in vitro. We first investigated the localization of MTA1 and MTA3 with HIF1a expression in the placental trophoblast of 1st trimester placenta via immunohistochemistry. Our data reveals that under physiologically hypoxic environment, MTA1 and MTA3 along with HIF1a are highly expressed by villous trophoblasts. Next, we investigated the effect of hypoxia on these genes in vitro using the first trimester-derived HTR8/SVneo cell line and observed up-regulation of MTA1 and MTA3 as well as HIF1a protein following hypoxia treatment. To investigate the direct effect of MTA1 and MTA3 upon HIF1a, we over-expressed MTA1 and MTA3 genes in HTR8/SVneo cells respectively and examined protein levels of HIF1a via Western blot as well as HIF1a target gene expression using a luciferase assay driven by a hypoxia-response element promoter (HRE-luciferase). We found that over-expressions of MTA1 and MTA3 up-regulate both HIF1a protein level and HRE-luciferase activity under hypoxic condition. In summary, both MTA1 and MTA3 are induced by hypoxia and up-regulate HIF1a expression and HIF1a target gene expression in trophoblasts. These data suggest that MTA1 and MTA3 play critical roles in trophoblast function and differentiation during early pregnancy. PMID:25705708

HIF-1α induces VE-cadherin expression and modulates vasculogenic mimicry in esophageal carcinoma cells

PubMed Central

Tang, Na-Na; Zhu, Hong; Zhang, Hong-Jie; Zhang, Wei-Feng; Jin, Hai-Lin; Wang, Lu; Wang, Pin; He, Gui-Jun; Hao, Bo; Shi, Rui-Hua

2014-01-01

AIM: To investigate whether hypoxia inducible factor (HIF)-1α modulates vasculogenic mimicry (VM) by upregulating VE-cadherin expression in esophageal squamous cell carcinoma (ESCC). METHODS: Esophageal squamous cancer cell lines Eca109 and TE13 were transfected with plasmids harboring small interfering RNAs targeting HIF-1α or VE-cadherin. The proliferation and invasion of esophageal carcinoma cells were detected by MTT and Transwell migration assays. The formation of tubular networks of cells was analyzed by 3D culture in vitro. BALB/c nude mice were used to observe xenograft tumor formation. The relationship between the expression of HIF-1α and VE-cadherin, ephrinA2 (EphA2) and laminin5γ2 (LN5γ2) was measured by Western blot and real-time polymerase chain reaction. RESULTS: Knockdown of HIF-1α inhibited cell proliferation (32.3% ± 6.1% for Eca109 cells and 38.6% ± 6.8% for TE13 cells, P < 0.05). Both Eca109 and TE13 cells formed typical tubular networks. The number of tubular networks markedly decreased when HIF-1α or VE-cadherin was knocked down. Expression of VE-cadherin, EphA2 and LN5γ2 was dramatically inhibited, but the expression of matrix metalloproteinase 2 had no obvious change in HIF-1α-silenced cells. Knockdown of VE-cadherin significantly decreased expression of both EphA2 and LN5γ2 (P < 0.05), while HIF-1α expression was unchanged. The time for xenograft tumor formation was 6 ± 1.2 d for Eca109 cells and Eca109 cells transfected with HIF-1α Neo control short hairpin RNA (shRNA) vector, and 8.4 ± 2.1 d for Eca109 cells transfected with an shRNA against HIF-1α. Knockdown of HIF-1α inhibited vasculogenic mimicry (VM) and tumorigenicity in vivo. CONCLUSION: HIF-1α may modulate VM in ESCC by regulating VE-cadherin expression, which affects VM formation through EphA2 and LN5γ2. PMID:25548487

Advanced Glycated End-Products Affect HIF-Transcriptional Activity in Renal Cells

PubMed Central

Bondeva, Tzvetanka; Heinzig, Juliane; Ruhe, Carola

2013-01-01

Advanced glycated end-products (AGEs) are ligands of the receptor for AGEs and increase in diabetic disease. MAPK organizer 1 (Morg1) via its binding partner prolyl-hydroxylase domain (PHD)-3 presumably plays a role in the regulation of hypoxia-inducible factor (HIF)-1α and HIF-2α transcriptional activation. The purpose of this study was to analyze the influence of AGEs on Morg1 expression and its correlation to PHD3 activity and HIF-transcriptional activity in various renal cell types. The addition of glycated BSA (AGE-BSA) significantly up-regulated Morg1 mRNA levels in murine mesangial cells and down-regulated it in murine proximal tubular cells and differentiated podocytes. These effects were reversible when the cells were preincubated with a receptor for α-AGE antibody. AGE-BSA treatment induced a relocalization of the Morg1 cellular distribution compared with nonglycated control-BSA. Analysis of PHD3 activity demonstrated an elevated PHD3 enzymatic activity in murine mesangial cells but an inhibition in murine proximal tubular cells and podocytes after the addition of AGE-BSA. HIF-transcriptional activity was also affected by AGE-BSA treatment. Reporter gene assays and EMSAs showed that AGEs regulate HIF- transcriptional activity under nonhypoxic conditions in a cell type-specific manner. In proximal tubular cells, AGE-BSA stimulation elevated mainly HIF-1α transcriptional activity and to a lesser extent HIF-2α. We also detected an increased expression of the HIF-1α and the HIF-2α proteins in kidneys from Morg1 heterozygous (HZ) placebo mice compared with the Morg1 wild-type (WT) placebo-treated mice, and the HIF-1α protein expression in the Morg1 HZ streptozotocin-treated mice was significantly higher than the WT streptozotocin-treated mice. Analysis of isolated mesangial cells from Morg1 HZ (±) and WT mice showed an inhibited PHD3 activity and an increased HIF-transcriptional activity in cells with only one Morg1 allele. These findings are important for a better understanding of the molecular mechanisms of diabetic nephropathy. PMID:24030251

Persisting mild hypothermia suppresses hypoxia-inducible factor-1alpha protein synthesis and hypoxia-inducible factor-1-mediated gene expression.

PubMed

Tanaka, Tomoharu; Wakamatsu, Takuhiko; Daijo, Hiroki; Oda, Seiko; Kai, Shinichi; Adachi, Takehiko; Kizaka-Kondoh, Shinae; Fukuda, Kazuhiko; Hirota, Kiichi

2010-03-01

The transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in regulating gene expression in response to hypoxia-ischemia. Ischemia causes the tissue not only to be hypoxic but also to be hypothermic because of the hypoperfusion under certain circumstances. On the other hand, the induced hypothermia is one of the most common therapeutic modalities to extend tolerance to hypoxia. Although hypoxia elicits a variety of cellular and systemic responses at different organizational levels in the body, little is known about how hypoxia-induced responses are affected by low temperature. We examined the influence of mild hypothermic conditions (28-32 degrees C) on HIF-1 in both in vitro and in vivo settings. In vitro experiments adopting cultured cells elucidated that hypoxia-induced HIF-1 activation was resistant to 4-h exposure to the low temperature. In contrast, exposure to the low temperature as long as 24 h suppressed HIF-1 activation and the subsequent upregulation of HIF-1 target genes such as VEGF or GLUT-1. HIF-1alpha protein stability in the cell was not affected by hypothermic treatment. Furthermore, intracellular ATP content was reduced under 1% O(2) conditions but was not largely affected by hypothermic treatment. The evidence indicates that reduction of oxygen consumption is not largely involved in suppression of HIF-1. In addition, we demonstrated that HIF-1 DNA-binding activity and HIF-1-dependent gene expressions induced under 10% O(2) atmosphere in mouse brain were not influenced by treatment under 3-h hypothermic temperature but were inhibited under 5-h treatment. On the other hand, we indicated that warming ischemic legs of mice for 24 h preserved HIF-1 activity. In this report we describe for the first time that persisting low temperature significantly reduced HIF-1alpha neosynthesis under hypoxic conditions, leading to a decrease in gene expression for adaptation to hypoxia in both in vitro and in vivo settings.

The fruit juice of Morinda citrifolia (noni) downregulates HIF-1α protein expression through inhibition of PKB, ERK-1/2, JNK-1 and S6 in manganese-stimulated A549 human lung cancer cells.

PubMed

Jang, Byeong-Churl

2012-03-01

High exposure of manganese is suggested to be a risk factor for many lung diseases. Evidence suggests anticancerous and antiangiogenic effects by products derived from Morinda citrifolia (noni) fruit. In this study, we investigated the effect of noni fruit juice (NFJ) on the expression of HIF-1α, a tumor angiogenic transcription factor in manganese-chloride (manganese)-stimulated A549 human lung carcinoma cells. Treatment with manganese largely induced expression of HIF-1α protein but did not affect HIF-1α mRNA expression in A549 cells, suggesting the metal-mediated co- and/or post-translational HIF-1α upregulation. Manganese treatment also led to increased phosphorylation of extracellular-regulated protein kinase-1/2 (ERK-1/2), c-Jun N-terminal kinase-1 (JNK-1), protein kinase B (PKB), S6 and eukaryotic translation initiation factor-2α (eIF-2α) in A549 cells. Of note, the exposure of NFJ inhibited the manganese-induced HIF-1α protein upregulation in a concentration-dependent manner. Importantly, as assessed by results of pharmacological inhibition and siRNA transfection studies, the effect of NFJ on HIF-1α protein downregulation seemed to be largely associated with the ability of NFJ to interfere with the metal's signaling to activate PKB, ERK-1/2, JNK-1 and S6 in A549 cells. It was further shown that NFJ could repress the induction of HIF-1α protein by desferoxamine or interleukin-1β (IL-1β), another HIF-1α inducer in A549 cells. Thus, the present study provides the first evidence that NFJ has the ability to strongly downregulate manganese-induced HIF-1α protein expression in A549 human lung cancer cells, which may suggest the NFJ-mediated beneficial effects on lung pathologies in which manganese and HIF-1α overexpression play pathogenic roles.

The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

PubMed

Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

2002-09-01

Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

Defect of Adaptation to Hypoxia in Patients With COPD Due to Reduction of Histone Deacetylase 7

PubMed Central

To, Masako; Yamamura, Satoshi; Akashi, Kenichi; Charron, Catherine E.; Barnes, Peter J.

2012-01-01

Background: Hypoxia inducible factor (HIF)-1 plays an important role in cellular adaptation to hypoxia by activating oxygen-regulated genes such as vascular endothelial growth factor (VEGF) and erythropoietin. Sputum VEGF levels are reported to be decreased in COPD, despite hypoxia. Here we show that patients with COPD fail to induce HIF-1α and VEGF under hypoxic condition because of a reduction in histone deacetylase (HDAC) 7. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with moderate to severe COPD (n = 21), smokers without COPD (n = 12), and nonsmokers (n = 15). PBMCs were exposed to hypoxia (1% oxygen, 5% CO2, and 94% N2) for 24 h, and HIF-1α and HDAC7 protein expression in nuclear extracts were determined by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE)/Western blotting. Results: HIF-1α was significantly induced by hypoxia in each group when compared with the normoxic condition (12-fold induction in nonsmokers, 24-fold induction in smokers without COPD, fourfold induction in COPD), but induction of HIF-1α under hypoxia was significantly lower in patients with COPD than in nonsmokers and smokers without COPD (P < .05 and P < .01, respectively). VEGF messenger RNA detected by quantitative real-time polymerase chain reaction was correlated with HIF-1α protein in nuclei (r = 0.79, P < .05), and HDAC7 protein expression was correlated with HIF-1α protein in nuclei (r = 0.46, P < .05). HDAC7 knockdown inhibited hypoxia-induced HIF-1α activity in U937 cells, and HIF-1α nuclear translocation and HIF-1α binding to the VEGF promoter in A549 cells. Conclusions: HDAC7 reduction in COPD causes a defect of HIF-1α induction response to hypoxia with impaired VEGF gene expression. This poor cellular adaptation might play a role in the pathogenesis of COPD. PMID:22172637

Role of Compartmentalization on HiF-1α Degradation Dynamics during Changing Oxygen Conditions: A Computational Approach

PubMed Central

Bedessem, Baptiste; Stéphanou, Angélique

2014-01-01

HiF-1α is the central protein driving the cellular response to hypoxia. Its accumulation in cancer cells is linked to the appearance of chemoresistant and aggressive tumor phenotypes. As a consequence, understanding the regulation of HiF-1α dynamics is a major issue to design new anti-cancer therapies. In this paper, we propose a model of the hypoxia pathway, involving HiF-1α and its inhibitor pVHL. Based on data from the literature, we made the hypothesis that the regulation of HiF-1α involves two compartments (nucleus and cytoplasm) and a constitutive shuttle of the pVHL protein between them. We first show that this model captures correctly the main features of HiF-1α dynamics, including the bi-exponential degradation profile in normoxia, the kinetics of induction in hypoxia, and the switch-like accumulation. Second, we simulated the effects of a hypoxia/reoxygenation event, and show that it generates a strong instability of HiF-1α. The protein concentration rapidly increases 3 hours after the reoxygenation, and exhibits an oscillating pattern. This effect vanishes if we do not consider compartmentalization of HiF-1α. This result can explain various counter-intuitive observations about the specific molecular and cellular response to the reoxygenation process. Third, we simulated the HiF-1α dynamics in the tumor case. We considered different types of mutations associated with tumorigenesis, and we compared their consequences on HiF-1α dynamics. Then, we tested different therapeutics strategies. We show that a therapeutic decrease of HiF-1α nuclear level is not always correlated with an attenuation of reoxygenation-induced instabilities. Thus, it appears that the design of anti-HiF-1α therapies have to take into account these two aspects to maximize their efficiency. PMID:25338163

Downregulation of HIF-1a sensitizes U251 glioma cells to the temozolomide (TMZ) treatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Jun-Hai; Ma, Zhi-Xiong; Huang, Guo-Hao

Purpose: The aim of this study was to investigate the effect of downregulation of HIF-1α gene on human U251 glioma cells and examine the consequent changes of TMZ induced effects and explore the molecular mechanisms. Methods: U251 cell line stably expressing HIF-1α shRNA was acquired via lentiviral vector transfection. The mRNA and protein expression alterations of genes involved in our study were determined respectively by qRT-PCR and Western blot. Cell proliferation was measured by MTT assay and colony formation assay, cell invasion/migration capacity was determined by transwell invasion assay/wound healing assay, and cell apoptosis was detected by flow cytometry. Results:more » We successfully established a U251 cell line with highly efficient HIF-1α knockdown. HIF-1a downregulation sensitized U251 cells to TMZ treatment and enhanced the proliferation-inhibiting, invasion/migration-suppressing, apoptosis-inducing and differentiation-promoting effects exerted by TMZ. The related molecular mechanisms demonstrated that expression of O{sup 6}-methylguanine DNA methyltransferase gene (MGMT) and genes of Notch1 pathway were significantly upregulated by TMZ treatment. However, this upregulation was abrogated by HIF-1α knockdown. We further confirmed important regulatory roles of HIF-1α in the expression of MGMT and activation of Notch1 pathways. Conclusion: HIF-1α downregulation sensitizes U251 glioma cells to the temozolomide treatment via inhibiting MGMT expression and Notch1 pathway activation. - Highlights: • TMZ caused more significant proliferation inhibition and apoptosis in U251 cells after downregulating HIF-1α. • Under TMZ treatment, HIF-1 downregulated U251 cells exhibited weaker mobility and more differentiated state. • TMZ caused MGMT over-expression and Notch1 pathway activation, which could be abrogated by HIF-1α downregulation.« less

Activated PAR-2 regulates pancreatic cancer progression through ILK/HIF-α-induced TGF-α expression and MEK/VEGF-A-mediated angiogenesis.

PubMed

Chang, Li-Hsun; Pan, Shiow-Lin; Lai, Chin-Yu; Tsai, An-Chi; Teng, Che-Ming

2013-08-01

Tissue factor initiates the process of thrombosis and activates cell signaling through protease-activated receptor-2 (PAR-2). The aim of this study was to investigate the pathological role of PAR-2 signaling in pancreatic cancer. We first demonstrated that activated PAR-2 up-regulated the protein expression of both hypoxia-inducible factor-1α (HIF-1α) and HIF-2α, resulting in enhanced transcription of transforming growth factor-α (TGF-α). Down-regulation of HIFs-α by siRNA or YC-1, an HIF inhibitor, resulted in depleted levels of TGF-α protein. Furthermore, PAR-2, through integrin-linked kinase (ILK) signaling, including the p-AKT, promoted HIF protein expression. Diminishing ILK by siRNA decreased the levels of PAR-2-induced p-AKT, HIFs-α, and TGF-α; our results suggest that ILK is involved in the PAR-2-mediated TGF-α via an HIF-α-dependent pathway. Furthermore, the culture medium from PAR-2-treated pancreatic cancer cells enhanced human umbilical vein endothelial cell proliferation and tube formation, which was blocked by the MEK inhibitor, PD98059. We also found that activated PAR-2 enhanced tumor angiogenesis through the release of vascular endothelial growth factor-A (VEGF-A) from cancer cells, independent of the ILK/HIFs-α pathways. Consistent with microarray analysis, activated PAR-2 induced TGF-A and VEGF-A gene expression. In conclusion, the activation of PAR-2 signaling induced human pancreatic cancer progression through the induction of TGF-α expression by ILK/HIFs-α, as well as through MEK/VEGF-A-mediated angiogenesis, and it plays a role in the interaction between cancer progression and cancer-related thrombosis. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Hypoxia inducible factor 1 links fast-patterned muscle activity and fast muscle phenotype in rats.

PubMed

Lunde, Ida G; Anton, Siobhan L; Bruusgaard, Jo C; Rana, Zaheer A; Ellefsen, Stian; Gundersen, Kristian

2011-03-15

Exercise influences muscle phenotype by the specific pattern of action potentials delivered to the muscle, triggering intracellular signalling pathways. PO2 can be reduced by an order of magnitude in working muscle. In humans, carriers of a hyperactive polymorphism of the transcription factor hypoxia inducible factor 1α (HIF-1α) have 50% more fast fibres, and this polymorphism is prevalent among strength athletes. We have investigated the putative role of HIF-1α in mediating activity changes in muscle.When rat muscles were stimulated with short high frequency bursts of action potentials known to induce a fast muscle phenotype, HIF-1α increased by about 80%. In contrast, a pattern consisting of long low frequency trains known to make fast muscles slow reduced the HIF-1α level of the fast extensor digitorum longus (EDL) muscle by 44%. Nuclear protein extracts from normal EDL contained 2.3-fold more HIF-1α and 4-fold more HIF-1β than the slow soleus muscle, while von-Hippel-Lindau protein was 4.8-fold higher in slow muscles. mRNA displayed a reciprocal pattern; thus FIH-1 mRNA was almost 2-fold higher in fast muscle, while the HIF-1α level was half, and consequently protein/mRNA ratio for HIF-1α was more than 4-fold higher in the fast muscle, suggesting that HIF-1α is strongly suppressed post-transcriptionally in slow muscles.When HIF-1α was overexpressed for 14 days after somatic gene transfer in adult rats, a slow-to-fast transformation was observed, encompassing an increase in fibre cross sectional area, oxidative enzyme activity and myosin heavy chain. The latter was shown to be regulated at the mRNA level in C2C12 myotubes.

New HIF2α inhibitors: potential implications as therapeutics for advanced pheochromocytomas and paragangliomas.

PubMed

Toledo, Rodrigo Almeida

2017-09-01

Two recent independent studies published in Nature show robust responses of clear cell renal cell carcinoma (ccRCC) cell lines, preclinical ccRCC xenograft models and, remarkably, a patient with progressive ccRCC despite receiving multiple lines of treatment, to the long-awaited, recently developed inhibitors of hypoxia-inducible factor 2-alpha (HIF2α). This commentary published in Endocrine-Related Cancer is based on the recognition of similar molecular drivers in ccRCC and the endocrine neoplasias pheochromocytomas and paragangliomas (PPGLs), ultimately leading to stabilization of HIFs. HIF-stabilizing mutations have been detected in the von Hippel-Lindau (VHL) gene, as well as in other genes, such as succinate dehydrogenase (SDHx), fumarate hydratase (FH) and transcription elongation factor B subunit 1 (TCEB1), as well as the gene that encodes HIF2α itself: EPAS1 HIF2α Importantly, the recent discovery of EPAS1 mutations in PPGLs and the results of comprehensive in vitro and in vivo studies revealing their oncogenic roles characterized a hitherto unknown direct mechanism of HIF2α activation in human cancer. The now available therapeutic opportunity to successfully inhibit HIF2α pharmacologically with PT2385 and PT2399 will certainly spearhead a series of investigations in several types of cancers, including patients with SDHB -related metastatic PPGL for whom limited therapeutic options are currently available. Future studies will determine the efficacy of these promising drugs against the hotspot EPAS1 mutations affecting HIF2α amino acids 529-532 (in PPGLs) and amino acids 533-540 (in erythrocytosis type 4), as well as against HIF2α protein activated by VHL , SDHx and FH mutations in PPGL-derived chromatin cells. © 2017 Society for Endocrinology.

SU-G-TeP3-10: Radiation Induces Prompt Live-Cell Metabolic Fluxes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campos, D; Peeters, W; Bussink, J

2016-06-15

Purpose: To compare metabolic dynamics and HIF-1α expression following radiation between a cancerous cell line (UM-SCC-22B) and a normal, immortalized cell line, NOK (Normal Oral Keratinocyte). HIF-1 is a key factor in metabolism and radiosensitivity. A better understanding of how radiation affects the interplay of metabolism and HIF-1 might give a better understanding of the mechanisms responsible for radiosensitivity. Methods: Changes in cellular metabolism in response to radiation are tracked by fluorescence lifetime of NADH. Expression of HIF-1α was measured by immunofluorescence for both cell lines with and without irradiation. Radiation response is also monitored with additional treatment of amore » HIF-1α inhibitor (chrysin) as well as a radical scavenger (glutathione). Changes in oxygen consumption and respiratory capacity are also monitored using the Seahorse XF analyzer. Results: An increase in HIF-1α was found to be in response to radiation for the cancer cell line, but not the normal cell line. Radiation was found to shift metabolism toward glycolytic pathways in cancer cells as measured by oxygen consumption and respiratory capacity. Radiation response was found to be muted by addition of glutathione to cell media. HIF-1α inhibition similarly muted radiation response in cancer. Conclusion: The HIF-1 protein complex is a key regulator cellular metabolism through the regulation of glycolysis and glucose transport enzymes. Moreover, HIF-1 has shown radio-protective effects in tumor vascular endothelia, and has been implicated in metastatic aggression. Monitoring interplay between metabolism and the HIF-1 protein complex can give a more fundamental understanding of radiotherapy response.« less

Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMP-activated protein kinase in DU145 human prostate cancer cells.

PubMed

Jung, Seung-Nam; Yang, Woo Kyeom; Kim, Joungmok; Kim, Hak Su; Kim, Eun Ju; Yun, Hee; Park, Hyunsung; Kim, Sung Soo; Choe, Wonchae; Kang, Insug; Ha, Joohun

2008-04-01

Hypoxia-inducible factor (HIF-1) plays a central role in the cellular adaptive response to hypoxic conditions, which are closely related to pathophysiological conditions, such as cancer. Although reactive oxygen species (ROS) have been implicated in the regulation of hypoxic and non-hypoxic induction of HIF-1 under various conditions, the role of ROS is quite controversial, and the mechanism underlying the HIF-1 regulation by ROS is not completely understood yet. Here, we investigated the biochemical mechanism for the ROS-induced HIF-1 by revealing a novel role of adenosine monophosphate-activated protein kinase (AMPK) and the upstream signal components. AMPK plays an essential role as energy-sensor under adenosine triphosphate-deprived conditions. Here we report that ROS induced by a direct application of H(2)O(2) and menadione to DU145 human prostate carcinoma resulted in accumulation of HIF-1alpha protein by attenuation of its degradation and activation of its transcriptional activity in an AMPK-dependent manner. By way of contrast, AMPK was required only for the transcriptional activity of HIF-1 under hypoxic condition, revealing a differential role of AMPK in these two stimuli. Furthermore, our data show that inhibition of AMPK enhances HIF-1alpha ubiquitination under ROS condition. Finally, we show that the regulation of HIF-1 by AMPK in response to ROS is under the control of c-Jun N-terminal kinase and Janus kinase 2 pathways. Collectively, our findings identify AMPK as a key determinant of HIF-1 functions in response to ROS and its possible role in the sophisticated HIF-1 regulatory mechanisms.

Mosaicism in HIF2A-related polycythemia-paraganglioma syndrome.

PubMed

Buffet, Alexandre; Smati, Sarra; Mansuy, Ludovic; Ménara, Mélanie; Lebras, Maëlle; Heymann, Marie-Françoise; Simian, Christophe; Favier, Judith; Murat, Arnaud; Cariou, Bertrand; Gimenez-Roqueplo, Anne-Paule

2014-02-01

HIF2A germline mutations were known to cause congenital polycythemia. Recently, HIF2A somatic mutations were found in several patients with polycythemia and paraganglioma, pheochromocytoma, or somatostatinoma, suggesting the occurrence of a de novo postzygotic HIF2A mutation that has not been demonstrated clearly. Patient 1 is a woman suffering from polycythemia diagnosed at the age of 16 years. She was operated on for a pheochromocytoma at 45 years and for two abdominal paragangliomas at 59 years. She was also diagnosed with somatostatinoma. Patient 2 is a young boy who suffered from polycythemia since infancy. He underwent surgery for a nonfunctional adrenal paraganglioma at the age of 9 years. We sequenced by Sanger and next-generation sequencing the HIF2A gene in DNA extracted from tumors, leukocytes, and buccal cells. In patient 1, we identified a somatic HIF2A mutation (c.1586T>C; p.Leu529Pro) in DNA extracted from both paragangliomas. The mutation was detected as a somatic mosaic in DNA extracted from somatostatinoma and was absent from germline DNA. In patient 2, we found an HIF2A heterozygous mutation (c.1625T>C; p.Leu542Pro) in the paraganglioma, but the mutation was also present as a mosaic in leukocyte DNA and in DNA extracted from buccal cells (3.3 and 8.96% of sequencing reads, respectively). Both mutations disrupt the hydroxylation domain of the HIF2α protein. Our study shows that HIF2A-related tumors are caused by postzygotic mutations occurring in early developmental stages. Potential germline mosaicism should be considered during the familial genetic counseling when an individual has been diagnosed with HIF2A-related polycythemia-paraganglioma syndrome.

Differential pattern of HIF-1α expression in HNSCC cancer stem cells after carbon ion or photon irradiation: one molecular explanation of the oxygen effect.

PubMed

Wozny, Anne-Sophie; Lauret, Alexandra; Battiston-Montagne, Priscillia; Guy, Jean-Baptiste; Beuve, Michael; Cunha, Micaela; Saintigny, Yannick; Blond, Emilie; Magne, Nicolas; Lalle, Philippe; Ardail, Dominique; Alphonse, Gersende; Rodriguez-Lafrasse, Claire

2017-05-09

Head and neck squamous cell carcinoma (HNSCC) are resistant to standard treatments, partly due to cancer stem cells (CSCs) localised in hypoxic niches. Compared to X-rays, carbon ion irradiation relies on better ballistic properties, higher relative biological effectiveness and the absence of oxygen effect. Hypoxia-inducible factor-1α (HIF-1α) is involved in the resistance to photons, whereas its role in response to carbon ions remains unclear. Two HNSCC cell lines and their CSC sub-population were studied in response to photons or carbon ion irradiation, in normoxia or hypoxia, after inhibition or not of HIF-1α. Under hypoxia, compared to non-CSCs, HIF-1α is expressed earlier in CSCs. A combined effect photons/hypoxia, less observed with carbon ions, results in a synergic and earlier HIF-1α expression in both subpopulations. The diffuse ROS production by photons is concomitant with HIF-1α expression and essential to its activation. There is no oxygen effect in response to carbon ions and the ROS localised in the track might be insufficient to stabilise HIF-1α. Finally, in hypoxia, cells were sensitised to both types of radiations after HIF-1α inhibition. Hypoxia-inducible factor-1α plays a main role in the response of CSCs and non-CSCs to carbon ion and photon irradiations, which makes the HIF-1α targeting an attractive therapeutic challenge.

Kruppel-like factor 2 inhibits hypoxia-inducible factor 1alpha expression and function in the endothelium.

PubMed

Kawanami, Daiji; Mahabeleshwar, Ganapati H; Lin, Zhiyong; Atkins, G Brandon; Hamik, Anne; Haldar, Saptarsi M; Maemura, Koji; Lamanna, Joseph C; Jain, Mukesh K

2009-07-31

Hypoxia-inducible factor 1 (HIF-1) is a central regulator of the hypoxic response in many cell types. In endothelial cells, HIF-1 induces the expression of key proangiogenic factors to promote angiogenesis. Recent studies have identified Kruppel-like factor 2 (KLF2) as a potent inhibitor of angiogenesis. However, the role of KLF2 in regulating HIF-1 expression and function has not been evaluated. KLF2 expression was induced acutely by hypoxia in endothelial cells. Adenoviral overexpression of KLF2 inhibited hypoxia-induced expression of HIF-1alpha and its target genes such as interleukin 8, angiopoietin-2, and vascular endothelial growth factor in endothelial cells. Conversely, knockdown of KLF2 increased expression of HIF-1alpha and its targets. Furthermore, KLF2 inhibited hypoxia-induced endothelial tube formation, whereas endothelial cells from mice with haploinsufficiency of KLF2 showed increased tube formation in response to hypoxia. Consistent with this ex vivo observation, KLF2 heterozygous mice showed increased microvessel density in the brain. Mechanistically, KLF2 promoted HIF-1alpha degradation in a von Hippel-Lindau protein-independent but proteasome-dependent manner. Finally, KLF2 disrupted the interaction between HIF-1alpha and its chaperone Hsp90, suggesting that KLF2 promotes degradation of HIF-1alpha by affecting its folding and maturation. These observations identify KLF2 as a novel inhibitor of HIF-1alpha expression and function. Therefore, KLF2 may be a target for modulating the angiogenic response in disease states.

Knockdown of HIF-1α and IL-8 induced apoptosis of hepatocellular carcinoma triggers apoptosis of vascular endothelial cells.

PubMed

Choi, Sung Hoon; Park, Jun Yong; Kang, Wonseok; Kim, Seung Up; Kim, Do Young; Ahn, Sang Hoon; Ro, Simon Wonsang; Han, Kwang-Hyub

2016-01-01

A local hypoxic microenvironment is one of the most important characteristics of solid tumors. Hypoxia inducible factor-1α (HIF-1α) and Interleukin-8 (IL-8) activate tumor survival from hypoxic-induced apoptosis in each pathway. This study aimed to evaluate whether knockdown of HIF-1α and IL-8 induced apoptosis of the hepatocellular carcinoma (HCC) and endothelial cell lines. HCC cell lines were infected with adenovirus-expressing shRNA for HIF-1α and IL-8 and maintained under hypoxic conditions (1% O2, 24 h). The expression levels of HIF-1α and both apoptotic and growth factors were examined by real-time quantitative PCR and western blot. We also investigated apoptosis by TUNEL assay (FACS and Immunofluorescence) and measured the concentration of cytochrome C. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while downregulating anti-apoptotic factors simultaneously. Knockdown of HIF-1α and IL-8 increased the concentration of cytochrome C and enhanced DNA fragmentation in HCC cell lines. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in human umbilical vein endothelial cells under hypoxia, and the expression of variable apoptotic ligand increased from HCC cell lines, time-dependently. These data suggest that adenovirus-mediated knockdown of HIF-1α and IL-8 induced apoptosis in HCC cells and triggered apoptosis of vascular endothelial cells.

Sensors, transmitters, and targets in mitochondrial oxygen shortage-a hypoxia-inducible factor relay story.

PubMed

Dehne, Nathalie; Brüne, Bernhard

2014-01-10

Cells sense and respond to a shortage of oxygen by activating the hypoxia-inducible transcription factors HIF-1 and HIF-2 and evoking adaptive responses. Mitochondria are at the center of a hypoxia sensing and responding relay system. Under normoxia, reactive oxygen species (ROS) and nitric oxide (NO) are HIF activators. As their individual flux rates determine their diffusion-controlled interaction, predictions how these radicals affect HIF appear context-dependent. Considering that the oxygen requirement for NO formation limits its role in activating HIF to conditions of ambient oxygen tension. Given the central role of mitochondrial complex IV as a NO target, especially under hypoxia, allows inhibition of mitochondrial respiration by NO to spare oxygen thus, raising the threshold for HIF activation. HIF targets seem to configure a feedback-signaling circuit aimed at gradually adjusting mitochondrial function. In hypoxic cancer cells, mitochondria redirect Krebs cycle intermediates to preserve their biosynthetic ability. Persistent HIF activation lowers the entry of electron-delivering compounds into mitochondria to reduce Krebs cycle fueling and β-oxidation, attenuates the expression of electron transport chain components, limits mitochondria biosynthesis, and provokes their removal by autophagy. Mitochondria can be placed central in a hypoxia sensing-hypoxia responding circuit. We need to determine to which extent and how mitochondria contribute to sense hypoxia, explore whether modulating their oxygen-consuming capacity redirects hypoxic responses in in vivo relevant disease conditions, and elucidate how the multiple HIF targets in mitochondria shape conditions of acute versus chronic hypoxia.

Transient Ureteral Obstruction Prevents against Kidney Ischemia/Reperfusion Injury via Hypoxia-Inducible Factor (HIF)-2α Activation

PubMed Central

Chen, Xiao-Song; Zhang, Ming; Xu, Long-Mei; Zhang, Jian-Jun; Xia, Qiang

2012-01-01

Although the protective effect of transient ureteral obstruction (UO) prior to ischemia on subsequent renal ischemia/reperfusion (I/R) injury has been documented, the underlying molecular mechanism remains to be understood. We showed in the current study that 24 h of UO led to renal tubular hypoxia in the ipsilateral kidney in mice, with the accumulation of hypoxia-inducible factor (HIF)-2α, which lasted for a week after the release of UO. To address the functions of HIF-2α in UO-mediated protection of renal IRI, we utilized the Mx-Cre/loxP recombination system to knock out target genes. Inactivation of HIF-2α, but not HIF-1α blunted the renal protective effects of UO, as demonstrated by much higher serum creatinine level and severer histological damage. UO failed to prevent postischemic neutrophil infiltration and apoptosis induction in HIF-2α knockout mice, which also diminished the postobstructive up-regulation of the protective molecule, heat shock protein (HSP)-27. The renal protective effects of UO were associated with the improvement of the postischemic recovery of intra-renal microvascular blood flow, which was also dependent on the activation of HIF-2α. Our results demonstrated that UO protected the kidney via activation of HIF-2α, which reduced tubular damages via preservation of adequate renal microvascular perfusion after ischemia. Thus, preconditional HIF-2α activation might serve as a novel therapeutic strategy for the treatment of ischemic acute renal failure. PMID:22295069

Intestine-specific Disruption of Hypoxia-inducible Factor (HIF)-2α Improves Anemia in Sickle Cell Disease.

PubMed

Das, Nupur; Xie, Liwei; Ramakrishnan, Sadeesh K; Campbell, Andrew; Rivella, Stefano; Shah, Yatrik M

2015-09-25

Sickle cell disease (SCD) is caused by genetic defects in the β-globin chain. SCD is a frequently inherited blood disorder, and sickle cell anemia is a common type of hemoglobinopathy. During anemia, the hypoxic response via the transcription factor hypoxia-inducible factor (HIF)-2α is highly activated in the intestine and is essential in iron absorption. Intestinal disruption of HIF-2α protects against tissue iron accumulation in iron overload anemias. However, the role of intestinal HIF-2α in regulating anemia in SCD is currently not known. Here we show that in mouse models of SCD, disruption of intestinal HIF-2α significantly decreased tissue iron accumulation. This was attributed to a decrease in intestinal iron absorptive genes, which were highly induced in a mouse model of SCD. Interestingly, disruption of intestinal HIF-2α led to a robust improvement in anemia with an increase in RBC, hemoglobin, and hematocrit. This was attributed to improvement in RBC survival, hemolysis, and insufficient erythropoiesis, which is evident from a significant decrease in serum bilirubin, reticulocyte counts, and serum erythropoietin following intestinal HIF-2α disruption. These data suggest that targeting intestinal HIF-2α has a significant therapeutic potential in SCD pathophysiology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

Greenald, David; Wilson, Garrick K.; Peron, Margherita; Markham, Eleanor; Sinnakaruppan, Mathavan; Matthews, Laura C.; McKeating, Jane A.; Argenton, Francesco; van Eeden, Fredericus J. M.

2017-01-01

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. PMID:28851829

Hypoxia inducible factor-1α-induced interleukin-33 expression in intestinal epithelia contributes to mucosal homeostasis in inflammatory bowel disease.

PubMed

Sun, M; He, C; Wu, W; Zhou, G; Liu, F; Cong, Y; Liu, Z

2017-03-01

Intestinal epithelial cells (IECs), an important barrier to gut microbiota, are subject to low oxygen tension, particularly during intestinal inflammation. Hypoxia inducible factor-1α (HIF-1α) is expressed highly in the inflamed mucosa of inflammatory bowel disease (IBD) and functions as a key regulator in maintenance of intestinal homeostasis. However, how IEC-derived HIF-1α regulates intestinal immune responses in IBD is still not understood completely. We report here that the expression of HIF-1α and IL-33 was increased significantly in the inflamed mucosa of IBD patients as well as mice with colitis induced by dextran sulphate sodium (DSS). The levels of interleukin (IL)-33 were correlated positively with that of HIF-1α. A HIF-1α-interacting element was identified in the promoter region of IL-33, indicating that HIF-1α activity regulates IL-33 expression. Furthermore, tumour necrosis factor (TNF) facilitated the HIF-1α-dependent IL-33 expression in IEC. Our data thus demonstrate that HIF-1α-dependent IL-33 in IEC functions as a regulatory cytokine in inflamed mucosa of IBD, thereby regulating the intestinal inflammation and maintaining mucosal homeostasis. © 2016 British Society for Immunology.

Dexamethasone impairs hypoxia-inducible factor-1 function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagner, A.E.; Huck, G.; Stiehl, D.P.

2008-07-25

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of {alpha}- and {beta}-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1{alpha} levels in the cytosol of HepG2 cells, while nuclear HIF-1{alpha} levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in amore » reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.« less

Expression of hypoxia-inducible factor-1α during ovarian follicular growth and development in Sprague-Dawley rats.

PubMed

Zhang, Z H; Chen, L Y; Wang, F; Wu, Y Q; Su, J Q; Huang, X H; Wang, Z C; Cheng, Y

2015-06-01

Hypoxia-inducible factor-1α (HIF-1α) has been identified as a transcription factor that is involved in diverse physiological and pathological processes in the ovary. In this study, we examined whether HIF-1α is expressed in a cell- and stage-specific manner during follicular growth and development in the mammalian ovaries. Using immunohistochemistry and Western blot analysis, HIF-1α expression was observed in granulosa cells specifically and was significantly increased during the follicular growth and development of postnatal rats. Furthermore, pregnant mare serum gonadotropin also induced HIF-1α expression in granulosa cells and ovaries during the follicular development of immature rats primed with gonadotropin. Moreover, we also examined proliferation cell nuclear antigen, a cell proliferation marker, during follicular growth and development and found that its expression pattern was similar to that of HIF-1α protein. Granulosa cell culture experiments revealed that proliferation cell nuclear antigen expression may be regulated by HIF-1α. These results indicated that HIF-1α plays an important role in the follicular growth and development of these 2 rat models. The HIF-1α-mediated signaling pathway may be an important mechanism regulating follicular growth and development in mammalian ovaries in vivo.

PITX1, a specificity determinant in the HIF-1α-mediated transcriptional response to hypoxia

PubMed Central

Mudie, Sharon; Bandarra, Daniel; Batie, Michael; Biddlestone, John; Moniz, Sonia; Ortmann, Brian; Shmakova, Alena; Rocha, Sonia

2014-01-01

Hypoxia is an important developmental cue for multicellular organisms but it is also a contributing factor for several human pathologies, such as stroke, cardiovascular diseases and cancer. In cells, hypoxia activates a major transcriptional program coordinated by the Hypoxia Inducible Factor (HIF) family. HIF can activate more than one hundred targets but not all of them are activated at the same time, and there is considerable cell type variability. In this report we identified the paired-like homeodomain pituitary transcription factor (PITX1), as a transcription factor that helps promote specificity in HIF-1α dependent target gene activation. Mechanistically, PITX1 associates with HIF-1β and it is important for the induction of certain HIF-1 dependent genes but not all. In particular, PITX1 controls the HIF-1α-dependent expression of the histone demethylases; JMJD2B, JMJD2A, JMJD2C and JMJD1B. Functionally, PITX1 is required for the survival and proliferation responses in hypoxia, as PITX1 depleted cells have higher levels of apoptotic markers and reduced proliferation. Overall, our study identified PITX1 as a key specificity factor in HIF-1α dependent responses, suggesting PITX1 as a protein to target in hypoxic cancers. PMID:25558831

Genetic responses to seasonal variation in altitudinal stress: whole-genome resequencing of great tit in eastern Himalayas

PubMed Central

Qu, Yanhua; Tian, Shilin; Han, Naijian; Zhao, Hongwei; Gao, Bin; Fu, Jun; Cheng, Yalin; Song, Gang; Ericson, Per G. P.; Zhang, Yong E.; Wang, Dawei; Quan, Qing; Jiang, Zhi; Li, Ruiqiang; Lei, Fumin

2015-01-01

Species that undertake altitudinal migrations are exposed to a considerable seasonal variation in oxygen levels and temperature. How they cope with this was studied in a population of great tit (Parus major) that breeds at high elevations and winters at lower elevations in the eastern Himalayas. Comparison of population genomics of high altitudinal great tits and those living in lowlands revealed an accelerated genetic selection for carbohydrate energy metabolism (amino sugar, nucleotide sugar metabolism and insulin signaling pathways) and hypoxia response (PI3K-akt, mTOR and MAPK signaling pathways) in the high altitudinal population. The PI3K-akt, mTOR and MAPK pathways modulate the hypoxia-inducible factors, HIF-1α and VEGF protein expression thus indirectly regulate hypoxia induced angiogenesis, erythropoiesis and vasodilatation. The strategies observed in high altitudinal great tits differ from those described in a closely related species on the Tibetan Plateau, the sedentary ground tit (Parus humilis). This species has enhanced selection in lipid-specific metabolic pathways and hypoxia-inducible factor pathway (HIF-1). Comparative population genomics also revealed selection for larger body size in high altitudinal great tits. PMID:26404527

Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts.

PubMed

Rapisarda, Annamaria; Zalek, Jessica; Hollingshead, Melinda; Braunschweig, Till; Uranchimeg, Badarch; Bonomi, Carrie A; Borgel, Suzanne D; Carter, John P; Hewitt, Stephen M; Shoemaker, Robert H; Melillo, Giovanni

2004-10-01

We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

Hostile takeover: Manipulation of HIF-1 signaling in pathogen-associated cancers (Review).

PubMed

Zhu, Caixia; Zhu, Qing; Wang, Chong; Zhang, Liming; Wei, Fang; Cai, Qiliang

2016-10-01

Hypoxia-inducible factor (HIF)-1 is a central regulator in the adaptation process of cell response to hypoxia (low oxygen). Emerging evidence has demonstrated that HIF-1 plays an important role in the development and progression of many types of human diseases, including pathogen-associated cancers. In the present review, we summarize the recent understandings of how human pathogenic agents including viruses, bacteria and parasites deregulate cellular HIF-1 signaling pathway in their associated cancer cells, and highlight the common molecular mechanisms of HIF-1 signaling activated by these pathogenic infection, which could act as potential diagnostic markers and new therapeutic strategies against human infectious cancers.

Hypoxia in Tumor Angiogenesis and Metastasis: Evaluation of VEGF and MMP Over-expression and Down-Regulation of HIF-1alpha with RNAi in Hypoxic Tumor Cells

NASA Astrophysics Data System (ADS)

Shah, Shruti

Background: As tumor mass grows beyond a few millimeters in diameter, the angiogenic "switch" is turned on leading to recruitment of blood vessels from surrounding artery and veins. However, the tumor mass is poorly perfused and there are pockets of hypoxia or lower oxygen concentrations relative to normal tissue. Hypoxia-inducing factor-1a (HIF-1a), a transcription factor, is activated when the oxygen concentration is low. Upon activation of HIF-1a, a number of other genes also turn on that allows the tumor to become more aggressive and resistant to therapy. Purpose: The main objectives of this study were to evaluate the effect of hypoxia-induced HIF-1a followed by over-expression of angiogenic and metastatic markers in tumor cells and down-regulation of HIF-1a using nanoparticle-delivered RNA interference therapy. Methods: Human ovarian (SKOV3) and breast (MDA-MB-231) adenocarcinoma cells were incubated under normoxic and hypoxic conditions. Following hypoxia treatment of the cells, HIF-1α, vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), and MMP-9 expression was analyzed qualitatively and quantitatively. For intracellular delivery of HIF-1a gene silencing small interfering RNA (siRNA), type B gelatin nanoparticles were fabricated using the solvent displacement method and the surface was modified with poly(ethylene glycol) (PEG, Mol. wt. 2kDa). Cellular uptake and distribution of the nanoparticles was observed with Cy3-siRNA loaded, FITC-conjugated gelatin nanoparticles. Cytotoxicity of the nanoparticle formulations was evaluated in both the cell lines. siRNA was transfected in the gelatin nanoparticles under hypoxic conditions. Total cellular protein and RNA were extracted for analysis of HIF1a, VEGF, MMP-2 and MMP-9 expression. Results: MDA-MB-231 and SKOV3 cells show increased expression of HIF1a under hypoxic conditions compared to baseline levels at normoxic conditions. ELISA and western blots of VEGF, MMP-2 and MMP-9 appear to increase with the increase in the HIF1α levels. Gelatin and PEG-modified gelatin nanoparticles were successfully prepared having a particle size in the range of 200 to 300 nm in diameter. Cell uptake studies showed that both types of nanoparticles could be efficiently internalized in tumor cells with a maximum intracellular concentrations reaching after 6 hours of incubation. Cytotoxicity analysis using MTS (formazan) assay showed that there was no significant change in cell viability upon treatment with any of the nanoparticle formulations relative to untreated control. Comparative analysis of gelatin nanoparticles and lipofectamine transfection of HIF1α-siRNA, shows a higher HIF1α knockdown for gelatin nanoparticles. Conclusion: Use of HIF1α siRNA for the treatment of cancer cells that overly express HIF1α has shown great therapeutic potential as shown by the results. The expression of VEGF, MMP-2 and MMP-9 decreases with the decrease of HIF1α expression. This may indicate the reversal of the aggressive phenotype of the tumors with HIF1α knockdown. HIF1α-siRNA, hence shows great potential for the therapy of aggressive tumors, however in vivo studies need to be carried out to validate these findings.

The vacuolar-ATPase complex and assembly factors, TMEM199 and CCDC115, control HIF1α prolyl hydroxylation by regulating cellular iron levels.

PubMed

Miles, Anna L; Burr, Stephen P; Grice, Guinevere L; Nathan, James A

2017-03-15

Hypoxia Inducible transcription Factors (HIFs) are principally regulated by the 2-oxoglutarate and Iron(II) prolyl hydroxylase (PHD) enzymes, which hydroxylate the HIFα subunit, facilitating its proteasome-mediated degradation. Observations that HIFα hydroxylation can be impaired even when oxygen is sufficient emphasise the importance of understanding the complex nature of PHD regulation. Here, we use an unbiased genome-wide genetic screen in near-haploid human cells to uncover cellular processes that regulate HIF1α. We identify that genetic disruption of the Vacuolar H+ ATPase (V-ATPase), the key proton pump for endo-lysosomal acidification, and two previously uncharacterised V-ATPase assembly factors, TMEM199 and CCDC115, stabilise HIF1α in aerobic conditions. Rather than preventing the lysosomal degradation of HIF1α, disrupting the V-ATPase results in intracellular iron depletion, thereby impairing PHD activity and leading to HIF activation. Iron supplementation directly restores PHD catalytic activity following V-ATPase inhibition, revealing important links between the V-ATPase, iron metabolism and HIFs.

Limitation of HIF-1α with pentoxifillyne on renal tubular ischemia result of hiperoxaluria and ESWL.

PubMed

Erturhan, Sakip; Bayrak, Omer; Seckiner, Ilker; Celik, Mehmet; Karakok, Metin

2014-03-01

To evaluate hypoxia-inducible factor 1 subunit α (HIF-1α) expression during the performance of extracorporeal shock wave lithotripsy (ESWL) and to investigate the effects of pentoxyphylline on HIF-1α expression. One hundred New Zealand Albino rabbit were used in the study divided in 5 groups. There were 20 rabbits in each group. The groups were divided in two parts: early (7 days) and late period (14 days) according to follow up duration. Immunohistochemical analyses were performed using nuclear staining to show HIF-1α expression in rabbit renal tissue sample. HIF-1α expression was higher in rabbits undergoing ESWL (group 4). In the hyperoxaluria group taking pentoxyphylline before ESWL (group 5), HIF-1α expression was lower in both early and late period subgroups (p < 0.05) CONCLUSION: In this study we evaluated HIF-1α expression and showed that ESWL may cause renal cell injury. Our results suggest that pentoxyphylline, as a circulatory regulator agent, may prevent renal cell injury induced by ESWL.

Global Metabolic Profiling Identifies a Pivotal Role of Proline and Hydroxyproline Metabolism in Supporting Hypoxic Response in Hepatocellular Carcinoma.

PubMed

Tang, Ling; Zeng, Jun; Geng, Pengyu; Fang, Chengnan; Wang, Yang; Sun, Mingju; Wang, Changsong; Wang, Jiao; Yin, Peiyuan; Hu, Chunxiu; Guo, Lei; Yu, Jane; Gao, Peng; Li, Enyou; Zhuang, Zhengping; Xu, Guowang; Liu, Yang

2018-01-15

Purpose: Metabolic reprogramming is frequently identified in hepatocellular carcinoma (HCC), which is the most common type of liver malignancy. The reprogrammed cellular metabolisms promote tumor cell survival, proliferation, angiogenesis, and metastasis. However, the mechanisms of this process remain unclear in HCC. Experimental Design: The global nontargeted metabolic study in 69 paired hepatic carcinomas and adjacent tissue specimens was performed using capillary electrophoresis-time of flight mass spectrometry-based approach. Key findings were validated by targeted metabolomic approach. Biological studies were also performed to investigate the role of proline biosynthesis in HCC pathogenesis. Results: Proline metabolism was markedly changed in HCC tumor tissue, characterized with accelerated consumption of proline and accumulation of hydroxyproline, which significantly correlated with α-fetoprotein levels and poor prognosis in HCC. In addition, we found that hydroxyproline promoted hypoxia- and HIF-dependent phenotype in HCC. Moreover, we demonstrated that hypoxia activated proline biosynthesis via upregulation of ALDH18A1 , subsequently leading to accumulation of hydroxyproline via attenuated PRODH2 activity. More importantly, we showed that glutamine, proline, and hydroxyproline metabolic axis supported HCC cell survival through modulating HIF1α stability in response to hypoxia. Finally, inhibition of proline biosynthesis significantly enhanced cytotoxicity of sorafenib in vitro and in vivo Conclusions: Our results demonstrate that hypoxic microenvironment activates proline metabolism, resulting in accumulation of hydroxyproline that promotes HCC tumor progression and sorafenib resistance through modulating HIF1α. These findings provide the proof of concept for targeting proline metabolism as a potential therapeutic strategy for HCC. Clin Cancer Res; 24(2); 474-85. ©2017 AACR . ©2017 American Association for Cancer Research.

Bioactivity-Guided Identification and Cell Signaling Technology to Delineate the Lactate Dehydrogenase A Inhibition Effects of Spatholobus suberectus on Breast Cancer

PubMed Central

Wang, Zhiyu; Wang, Dongmei; Han, Shouwei; Wang, Neng; Mo, Feizhi; Loo, Tjing Yung; Shen, Jiangang; Huang, Hui; Chen, Jianping

2013-01-01

Aerobic glycolysis is an important feature of cancer cells. In recent years, lactate dehydrogenase A (LDH-A) is emerging as a novel therapeutic target for cancer treatment. Seeking LDH-A inhibitors from natural resources has been paid much attention for drug discovery. Spatholobus suberectus (SS) is a common herbal medicine used in China for treating blood-stasis related diseases such as cancer. This study aims to explore the potential medicinal application of SS for LDH-A inhibition on breast cancer and to determine its bioactive compounds. We found that SS manifested apoptosis-inducing, cell cycle arresting and anti-LDH-A activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cell. Oral herbal extracts (1 g/kg/d) administration attenuated tumor growth and LDH-A expression in both breast cancer xenografts. Bioactivity-guided fractionation finally identified epigallocatechin as a key compound in SS inhibiting LDH-A activity. Further studies revealed that LDH-A plays a critical role in mediating the apoptosis-induction effects of epigallocatechin. The inhibited LDH-A activities by epigallocatechin is attributed to disassociation of Hsp90 from HIF-1α and subsequent accelerated HIF-1α proteasome degradation. In vivo study also demonstrated that epigallocatechin could significantly inhibit breast cancer growth, HIF-1α/LDH-A expression and trigger apoptosis without bringing toxic effects. The preclinical study thus suggests that the potential medicinal application of SS for inhibiting cancer LDH-A activity and the possibility to consider epigallocatechin as a lead compound to develop LDH-A inhibitors. Future studies of SS for chemoprevention or chemosensitization against breast cancer are thus warranted. PMID:23457597

HIF Transcription Factors, Inflammation, and Immunity

PubMed Central

Palazon, Asis; Goldrath, Ananda; Nizet, Victor

2015-01-01

The hypoxic response in cells and tissues is mediated by the family of hypoxia-inducible factor (HIF) transcription factors that play an integral role in the metabolic changes that drive cellular adaptation to low oxygen availability. HIF expression and stabilization in immune cells can be triggered by hypoxia, but also by other factors associated with pathological stress: e.g., inflammation, infectious microorganisms, and cancer. HIF induces a number of aspects of host immune function, from boosting phagocyte microbicidal capacity to driving T cell differentiation and cytotoxic activity. Cellular metabolism is emerging as a key regulator of immunity, and it constitutes another layer of fine-tuned immune control by HIF that can dictate myeloid cell and lymphocyte development, fate, and function. Here we discuss how oxygen sensing in the immune microenvironment shapes immunological response and examine how HIF and the hypoxia pathway control innate and adaptive immunity. PMID:25367569

HIF transcription factors, inflammation, and immunity.

PubMed

Palazon, Asis; Goldrath, Ananda W; Nizet, Victor; Johnson, Randall S

2014-10-16

The hypoxic response in cells and tissues is mediated by the family of hypoxia-inducible factor (HIF) transcription factors; these play an integral role in the metabolic changes that drive cellular adaptation to low oxygen availability. HIF expression and stabilization in immune cells can be triggered by hypoxia, but also by other factors associated with pathological stress: e.g., inflammation, infectious microorganisms, and cancer. HIF induces a number of aspects of host immune function, from boosting phagocyte microbicidal capacity to driving T cell differentiation and cytotoxic activity. Cellular metabolism is emerging as a key regulator of immunity, and it constitutes another layer of fine-tuned immune control by HIF that can dictate myeloid cell and lymphocyte development, fate, and function. Here we discuss how oxygen sensing in the immune microenvironment shapes immunological response and examine how HIF and the hypoxia pathway control innate and adaptive immunity.

Engineered myocardium model to study the roles of HIF-1α and HIF1A-AS1 in paracrine-only signaling under pathological level oxidative stress.

PubMed

Acun, Aylin; Zorlutuna, Pinar

2017-08-01

Studying heart tissue is critical for understanding and developing treatments for cardiovascular diseases. In this work, we fabricated precisely controlled and biomimetic engineered model tissues to study how cell-cell and cell-matrix interactions influence myocardial cell survival upon exposure to pathological level oxidative stress. Specifically, the interactions of endothelial cells (ECs) and cardiomyocytes (CMs), and the role of hypoxia inducible factor-1α (HIF-1α), with its novel alternative regulator, HIF-1α antisense RNA1 (HIF1A-AS1), in these interactions were investigated. We encapsulated CMs in photo-crosslinkable, biomimetic hydrogels with or without ECs, then exposed to oxidative stress followed by normoxia. With precisely controlled microenvironment provided by the model tissues, cell-cell interactions were restricted to be solely through the secreted factors. CM survival after oxidative stress was significantly improved, in the presence of ECs, when cells were in the model tissues that were functionalized with cell attachment motifs. Importantly, the cardioprotective effect of ECs was reduced when HIF-1α expression was knocked down suggesting that HIF-1α is involved in cardioprotection from oxidative damage, provided through secreted factors conferred by the ECs. Using model tissues, we showed that cell survival increased with increased cell-cell communication and enhanced cell-matrix interactions. In addition, whole genome transcriptome analysis showed, for the first time to our knowledge, a possible role for HIF1A-AS1 in oxidative regulation of HIF-1α. We showed that although HIF1A-AS1 knockdown helps CM survival, its effect is overridden by CM-EC bidirectional interactions as we showed that the conditioned media taken from the CM-EC co-cultures improved CM survival, regardless of HIF1A-AS1 expression. Cardiovascular diseases, most of which are associated with oxidative stress, is the most common cause of death worldwide. Thus, understanding the molecular events as well as the role of intercellular communication under oxidative stress is upmost importance in its prevention. In this study we used 3D engineered tissue models to investigate the role of HIF-1α and its regulation in EC-mediated cardioprotection. We showed that EC-mediated protection is only possible when there is a bidirectional crosstalk between ECs and CMs even without physical cell-cell contact. In addition, this protective effect is at least partially related to cell-ECM interactions and HIF-1α, which is regulated by HIF1A-AS1 under oxidative stress. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Somatic Pairing of Chromosome 19 in Renal Oncocytoma Is Associated with Deregulated ELGN2-Mediated Oxygen-Sensing Response

PubMed Central

Petillo, David; Westphal, Michael; Koelzer, Katherine; Metcalf, Julie L.; Zhang, Zhongfa; Matsuda, Daisuke; Dykema, Karl J.; Houseman, Heather L.; Kort, Eric J.; Furge, Laura L.; Kahnoski, Richard J.; Richard, Stéphane; Vieillefond, Annick; Swiatek, Pamela J.; Teh, Bin Tean; Ohh, Michael; Furge, Kyle A.

2008-01-01

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase ELGN2, a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of ELGN2 in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma. PMID:18773095

Deregulation of HIF1-alpha and hypoxia-regulated pathways in hepatocellular carcinoma and corresponding non-malignant liver tissue--influence of a modulated host stroma on the prognosis of HCC.

PubMed

Simon, Frank; Bockhorn, Maximilian; Praha, Christian; Baba, Hideo A; Broelsch, Christoph E; Frilling, Andrea; Weber, Frank

2010-04-01

The aim of this study was to elucidate the role of HIF1A expression in hepatocellular carcinoma (HCC) and the corresponding non-malignant liver tissue and to correlate it with the clinical outcome of HCC patients after curative liver resection. HIF1A expression was determined by quantitative RT-PCR in HCC and corresponding non-malignant liver tissue of 53 patients surgically treated for HCC. High-density gene expression analysis and pathway analysis was performed on a selected subset of patients with high and low HIF1A expression in the non-malignant liver tissue. HIF1A over-expression in the apparently non-malignant liver tissue was a predictor of tumor recurrence and survival. The estimated 1-year and 5-year disease-free survival was significantly better in patients with low HIF1A expression in the non-malignant liver tissue when compared to those patients with high HIF1 expression (88.9% vs. 67.9% and 61.0% vs. 22.6%, respectively, p = 0.008). Based on molecular pathway analysis utilizing high-density gene-expression profiling, HIF1A related molecular networks were identified that contained genes involved in cell migration, cell homing, and cell-cell interaction. Our study identified a potential novel mechanism contributing to prognosis of HCC. The deregulation of HIF1A and its related pathways in the apparently non-malignant liver tissue provides for a modulated environment that potentially enhances or allows for HCC recurrence after curative resection.

Gene Expression Profiling of the Hypoxia Signaling Pathway in Hypoxia-Inducible Factor 1α Null Mouse Embryonic Fibroblasts

PubMed Central

Vengellur, Ajith; Woods, Barbara G.; Ryan, Heather E.; Johnson, Randall S.; Lapres, John J.

2003-01-01

Hypoxia is defined as a deficiency of oxygen reaching the tissues of the body, and it plays a critical role in development and pathological conditions, such as cancer. Once tumors outgrow their blood supply, their central portion becomes hypoxic and the tumor stimulates angiogenesis through the activation of the hypoxia-inducible factors (HIFs). HIFs are transcription factors that are regulated in an oxygen-dependent manner by a group of prolyl hydroxylases (known as PHDs or HPHs). Our understanding of hypoxia signaling is limited by our incomplete knowledge of HIF target genes. cDNA microarrays and a cell line lacking a principal HIF protein, HIF1α, were used to identify a more complete set of hypoxia-regulated genes. The microarrays identified a group of 286 clones that were significantly influenced by hypoxia and 54 of these were coordinately regulated by cobalt chloride. The expression profile of HIF1α −/− cells also identified a group of downregulated genes encoding enzymes involved in protecting cells from oxidative stress, offering an explanation for the increased sensitivity of HIF1α −/− cells to agents that promote this type of response. The microarray studies confirmed the hypoxia-induced expression of the HIF regulating prolyl hydroxylase, PHD2. An analysis of the members of the PHD family revealed that they are differentially regulated by cobalt chloride and hypoxia. These results suggest that HIF1α is the predominant isoform in fibroblasts and that it regulates a wide battery of genes critical for normal cellular function and survival under various stresses. PMID:14686790

Hypoxia-Inducible Factor-1α (HIF-1α) Expression on Endothelial Cells in Juvenile Nasopharyngeal Angiofibroma: A Review of 70 cases and Tissue Microarray Analysis.

PubMed

Song, Xiaole; Yang, Chenhe; Zhang, Huankang; Wang, Jingjing; Sun, Xicai; Hu, Li; Liu, Zhuofu; Wang, Dehui

2018-06-01

To examine the expression of hypoxia-inducible factor-1α (HIF-1α) and its related molecules (cellular repressor of E1A-stimulated genes [CREG], osteopontin [OPN], proto-oncogene tyrosine-protein kinase Src [c-Src], and vascular endothelial growth factor [VEGF]) in juvenile nasopharyngeal angiofibroma (JNA) and explore the correlation between clinical prognosis and HIF-1α expression. The study performed a retrospective review of the clinical records of patients with JNA treated between 2003 and 2007. Specimens were analyzed by immunohistochemistry for HIF-1α, CREG, OPN, c-Src, and VEGF expression, and microvessel density (MVD) was assessed by tissue microarray. The correlation between expression levels and clinicopathological features including age, tumor stage, intraoperative blood loss, and recurrence was analyzed. HIF-1α, CREG, OPN, c-Src, and VEGF were upregulated in endothelial cells (ECs) of patients with JNA, and strong correlations in the expression of these molecules were observed. HIF-1α expression was higher in young patients ( P = .032) and in recurrent cases ( P = .01). Survival analysis showed that low HIF-1α levels in ECs predicted longer time to recurrence (log rank test P = .006). Receiver operating characteristic curve analysis showed that HIF-1α was a prognostic factor for recurrence (area under the curve = 0.690, P = .019). No correlation was found between the expression of molecules and Radkowski stage or intraoperative blood loss. In cases of JNA treated surgically, HIF-1α expression in ECs is a useful prognostic factor for tumor recurrence.

Sevoflurane pretreatment enhance HIF-2α expression in mice after renal ischemia/reperfusion injury

PubMed Central

Zheng, Beijie; Zhan, Qionghui; Chen, Jue; Xu, Huan; He, Zhenzhou

2015-01-01

Ischemia/reperfusion (I/R) injury often occurs, which is one of the major causes of acute kidney injury, thus increasing in-hospital mortality. HIF-2α has a protective role against ischemia of the kidney. Renal ischemia/reperfusion under sevoflurane anesthesia resulted in drastic improvements in renal function. We hypothesized that underlying mechanism responsible for renal protection from sevoflurane pretreatment involves the upregulation of HIF-2α. Sevoflurane pretreatment were performed on WT and HIF-2α knockout mice before renal ischemia/reperfusion. Levels of blood urea nitrogen (BUN) and serum creatinine (Cr) were determined with a standard clinical automatic analyzer. The left kidneys were taken for morphological examination. Expression of HIF-2α in kidney tissue was examined by western blotting. In WT mice, group I/R injury had significantly higher BUN and Cr levels than group control, whereas group I/R + Sev had significantly lower BUN and Cr levels than group I/R injury. Renal HIF-2α expression levels were significantly higher in WT mice of group I/R + Sev than group control and group I/R. In HIF-2α-/- mice, group I/R + Sev showed much higher BUN and Cr levels and severer histological damage than group I/R and group control. Renal HIF-2α expression levels were significantly higher in WT mice of group I/R + Sev than group control and group I/R. Our findings suggested that HIF-2α might contribute to the beneficial effect of sevoflurane in renal ischemia/reperfusion injury. PMID:26722509

Disturbed hypoxic responses as a pathogenic mechanism of diabetic foot ulcers.

PubMed

Catrina, Sergiu-Bogdan; Zheng, Xiaowei

2016-01-01

Diabetic foot ulceration (DFU) is a chronic complication of diabetes that is characterized by impaired wound healing in the lower extremities. DFU remains a major clinical challenge because of poor understanding of its pathogenic mechanisms. Impaired wound healing in diabetes is characterized by decreased angiogenesis, reduced bone marrow-derived endothelial progenitor cell (EPC) recruitment, and decreased fibroblast and keratinocyte proliferation and migration. Recently, increasing evidence has suggested that increased hypoxic conditions and impaired cellular responses to hypoxia are essential pathogenic factors of delayed wound healing in DFU. Hypoxia-inducible factor-1 (HIF-1, a heterodimer of HIF-1α and HIF-1β) is a master regulator of oxygen homeostasis that mediates the adaptive cellular responses to hypoxia by regulating the expression of genes involved in angiogenesis, metabolic changes, proliferation, migration, and cell survival. However, HIF-1 signalling is inhibited in diabetes as a result of hyperglycaemia-induced HIF-1α destabilization and functional repression. Increasing HIF-1α expression and activity using various approaches promotes angiogenesis, EPC recruitment, and granulation, thereby improving wound healing in experimental diabetes. The mechanisms underlying HIF-1α regulation in diabetes and the therapeutic strategies targeting HIF-1 signalling for the treatment of diabetic wounds are discussed in this review. Further investigations of the pathways involved in HIF-1α regulation in diabetes are required to advance our understanding of the mechanisms underlying impaired wound healing in diabetes and to provide a foundation for developing novel therapeutic approaches to treat DFU. Copyright © 2016 John Wiley & Sons, Ltd.

β-N-oxalyl-L-α, β- diaminopropionic acid induces HRE expression by inhibiting HIF-prolyl hydroxylase-2 in normoxic conditions.

PubMed

Eslavath, Ravi Kumar; Sharma, Deepshikha; Bin Omar, Nabil A M; Chikati, Rajasekhar; Teli, Mahesh Kumar; Rajanikant, G K; Singh, Surya S

2016-11-15

Hypoxia inducible factor (HIF)-1α, a subunit of HIF transcription factor, regulates cellular response to hypoxia. In normoxic conditions, it is hydroxylated by prolyl hydroxylase (PHD)-2 and targeted for proteosomal degradation. Drugs which inhibit PHD-2 have implications in conditions arising from insufficient blood supply. β-ODAP (β-N- oxalyl-L-α, β- diaminopropionic acid), a non-protein excitatory amino acid present in Lathyrus sativus, is an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor agonist known to activate conventional protein kinase C and stabilize HIF-1α under normoxic conditions. However, the mechanism of HIF-1α stabilization by this compound is unknown. In silico approach was used to understand the mechanism of stabilization of HIF-1α which revealed β-ODAP interacts with key amino acid residues and Fe 2+ at the catalytic site of PHD-2. These results were further corroborated with luciferase HRE (hypoxia response element) reporter system in HeLa cells. Different chemical modulators of PHD-2 activity and HIF-1α levels were included in the study for comparison. Results obtained indicate that β-ODAP inhibits PHD-2 and facilitates HIF dependent HRE expression and hence, might be helpful in conditions arising from hypoxia. Copyright © 2016 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pedersen, Malin; Loefstedt, Tobias; Sun Jianmin

Signaling by the receptor for stem cell factor (SCF), c-Kit, is of major importance for hematopoiesis, melanogenesis and reproduction, and the biological responses are commonly proliferation and cell survival. Thus, constitutive activation due to c-Kit mutations is involved in the pathogenesis of several forms of cancer, e.g. leukemias, gastrointestinal stromal tumors and testicular tumors. Tumor survival requires oxygen supply through induced neovascularization, a process largely mediated by the vascular endothelial growth factor (VEGF), a prominent target of the transcription factors hypoxia-inducible factor-1 (HIF-1) and HIF-2. Using Affymetrix microarrays we have identified genes that are upregulated following SCF stimulation. Interestingly, manymore » of the genes induced were found to be related to a hypoxic response. These findings were corroborated by our observation that SCF stimulation of the hematopoietic cell lines M-07e induces HIF-1{alpha} and HIF-2{alpha} protein accumulation at normoxia. In addition, SCF-induced HIF-1{alpha} was transcriptionally active, and transcribed HIF-1 target genes such as VEGF, BNIP3, GLUT1 and DEC1, an effect that could be reversed by siRNA against HIF-1{alpha}. We also show that SCF-induced accumulation of HIF-1{alpha} is dependent on both the PI-3-kinase and Ras/MEK/Erk pathways. Our data suggest a novel mechanism of SCF/c-Kit signaling in angiogenesis and tumor progression.« less

A Small-Molecule Antagonist of HIF2α Is Efficacious in Preclinical Models of Renal Cell Carcinoma.

PubMed

Wallace, Eli M; Rizzi, James P; Han, Guangzhou; Wehn, Paul M; Cao, Zhaodan; Du, Xinlin; Cheng, Tzuling; Czerwinski, Robert M; Dixon, Darryl D; Goggin, Barry S; Grina, Jonas A; Halfmann, Megan M; Maddie, Melissa A; Olive, Sarah R; Schlachter, Stephen T; Tan, Huiling; Wang, Bin; Wang, Keshi; Xie, Shanhai; Xu, Rui; Yang, Hanbiao; Josey, John A

2016-09-15

More than 90% of clear cell renal cell carcinomas (ccRCC) exhibit inactivation of the von Hippel-Lindau (pVHL) tumor suppressor, establishing it as the major underlying cause of this malignancy. pVHL inactivation results in stabilization of the hypoxia-inducible transcription factors, HIF1α and HIF2α, leading to expression of a genetic program essential for the initiation and progression of ccRCC. Herein, we describe the potent, selective, and orally active small-molecule inhibitor PT2385 as a specific antagonist of HIF2α that allosterically blocks its dimerization with the HIF1α/2α transcriptional dimerization partner ARNT/HIF1β. PT2385 inhibited the expression of HIF2α-dependent genes, including VEGF-A, PAI-1, and cyclin D1 in ccRCC cell lines and tumor xenografts. Treatment of tumor-bearing mice with PT2385 caused dramatic tumor regressions, validating HIF2α as a pivotal oncogenic driver in ccRCC. Notably, unlike other anticancer agents that inhibit VEGF receptor signaling, PT2385 exhibited no adverse effect on cardiovascular performance. Thus, PT2385 represents a novel class of therapeutics for the treatment of RCC with potent preclincal efficacy as well as improved tolerability relative to current agents that target the VEGF pathway. Cancer Res; 76(18); 5491-500. ©2016 AACR. ©2016 American Association for Cancer Research.

Albendazole inhibits HIF-1α-dependent glycolysis and VEGF expression in non-small cell lung cancer cells.

PubMed

Zhou, Fang; Du, Jin; Wang, Jianjun

2017-04-01

Albendazole (ABZ) has an anti-tumor ability and inhibits HIF-1α activity. HIF-1α is associated with glycolysis and vascular endothelial cell growth factor (VEGF) expression, which plays an important role in cancer progression. These clues indicate that ABZ exerts an anti-cancer effect by regulating glycolysis and VEGF expression. The aim of this study is to clarify the effects of ABZ on non-small cell lung cancer (NSCLC) cells and explore the underlying molecular mechanisms. The expression levels of HIF-1α and VEGF were detected using western blot analysis, and the effect of ABZ on glycolysis was evaluated by measuring the relative activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) and detecting the production of lactate in A549 and H1299 cells. The results showed that ABZ decreased the expression levels of HIF-1α and VEGF and suppressed glycolysis in under hypoxia, but not normoxic condition. Inhibiting HIF-1α also suppressed glycolysis and VEGF expression. Additionally, ABZ inhibited the volume and weight, decreased the relative activities of HK, PK, and LDH, and reduced the levels of HIF-1α and VEGF of A549 xenografts in mouse models. In conclusion, ABZ inhibited growth of NSCLC cells by suppressing HIF-1α-dependent glycolysis and VEGF expression.

Hypoxia-inducible factor-1α plays roles in Epstein-Barr virus’s natural life cycle and tumorigenesis by inducing lytic infection through direct binding to the immediate-early BZLF1 gene promoter

PubMed Central

Kraus, Richard J.; Cordes, Blue-leaf A.; Nawandar, Dhananjay M.; Ma, Shidong; McChesney, Kyle G.; Lin, Zhen; Makielski, Kathleen R.; Lee, Denis L.; Lambert, Paul F.; Johannsen, Eric C.; Kenney, Shannon C.

2017-01-01

When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs). We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV). Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE) located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK) and gingival epithelial (hGET) cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV’s natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for development of a lytic-induction therapy for treating some EBV-associated malignancies. PMID:28617871

Helicobacter pylori Induced Phosphatidylinositol-3-OH Kinase/mTOR Activation Increases Hypoxia Inducible Factor-1α to Promote Loss of Cyclin D1 and G0/G1 Cell Cycle Arrest in Human Gastric Cells.

PubMed

Canales, Jimena; Valenzuela, Manuel; Bravo, Jimena; Cerda-Opazo, Paulina; Jorquera, Carla; Toledo, Héctor; Bravo, Denisse; Quest, Andrew F G

2017-01-01

Helicobacter pylori ( H. pylori ) is a human gastric pathogen that has been linked to the development of several gastric pathologies, such as gastritis, peptic ulcer, and gastric cancer. In the gastric epithelium, the bacterium modifies many signaling pathways, resulting in contradictory responses that favor both proliferation and apoptosis. Consistent with such observations, H. pylori activates routes associated with cell cycle progression and cell cycle arrest. H. pylori infection also induces the hypoxia-induced factor HIF-1α, a transcription factor known to promote expression of genes that permit metabolic adaptation to the hypoxic environment in tumors and angiogenesis. Recently, however, also roles for HIF-1α in the repair of damaged DNA and inhibition of gene expression were described. Here, we investigated signaling pathways induced by H. pylori in gastric cells that favor HIF-1α expression and the consequences thereof in infected cells. Our results revealed that H. pylori promoted PI3K/mTOR-dependent HIF-1α induction, HIF-1α translocation to the nucleus, and activity as a transcription factor as evidenced using a reporter assay. Surprisingly, however, transcription of known HIF-1α effector genes evaluated by qPCR analysis, revealed either no change (LDHA and GAPDH), statistically insignificant increases SLC2A1 (GLUT-1) or greatly enhance transcription (VEGFA), but in an HIF-1α-independent manner, as quantified by PCR analysis in cells with shRNA-mediated silencing of HIF-1α. Instead, HIF-1α knockdown facilitated G1/S progression and increased Cyclin D1 protein half-life, via a post-translational pathway. Taken together, these findings link H. pylori -induced PI3K-mTOR activation to HIF-1α induced G0/G1 cell cycle arrest by a Cyclin D1-dependent mechanism. Thus, HIF-1α is identified here as a mediator between survival and cell cycle arrest signaling activated by H. pylori infection.

High Nuclear Hypoxia-Inducible Factor 1 Alpha Expression Is a Predictor of Distant Recurrence in Patients With Resected Pancreatic Adenocarcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colbert, Lauren E.; Winship Cancer Institute, Emory University, Atlanta, Georgia; Memorial Sloan-Kettering Cancer Center, New York, New York

Purpose: To evaluate nuclear hypoxia-inducible factor 1α (HIF-1α) expression as a prognostic factor for distant recurrence (DR) and local recurrence (LR) after pancreatic adenocarcinoma resection. Methods and Materials: Tissue specimens were collected from 98 patients with pancreatic adenocarcinoma who underwent resection without neoadjuvant therapy between January 2000 and December 2011. Local recurrence was defined as radiographic or pathologic evidence of progressive disease in the pancreas, pancreatic bed, or associated nodal regions. Distant recurrence was defined as radiographically or pathologically confirmed recurrent disease in other sites. Immunohistochemical staining was performed and scored by an independent pathologist blinded to patient outcomes. Highmore » HIF-1α overall expression score was defined as high percentage and intensity staining and thus score >1.33. Univariate analysis was performed for HIF-1α score with LR alone and with DR. Multivariate logistic regression was used to determine predictors of LR and DR. Results: Median follow-up time for all patients was 16.3 months. Eight patients (8%) demonstrated isolated LR, 26 patients (26.5%) had isolated DR, and 13 patients had both LR and DR. Fifty-three patients (54%) had high HIF-1α expression, and 45 patients (46%) had low HIF-1α expression. High HIF-1α expression was significantly associated with DR (P=.03), and low HIF-1α expression was significantly associated with isolated LR (P=.03). On multivariate logistic regression analysis, high HIF-1α was the only significant predictor of DR (odds ratio 2.46 [95% confidence interval 1.06-5.72]; P=.03). In patients with a known recurrence, an HIF-1α score ≥2.5 demonstrated a specificity of 100% for DR. Conclusions: High HIF-1α expression is a significant predictor of distant failure versus isolated local failure in patients undergoing resection of pancreatic adenocarcinoma. Expression of HIF-1α may have utility in determining candidates for adjuvant local radiation therapy and systemic chemotherapy.« less

Clinically relevant HIF-1α-dependent metabolic reprogramming in oropharyngeal squamous cell carcinomas includes coordinated activation of CAIX and the miR-210/ISCU signaling axis, but not MCT1 and MCT4 upregulation

PubMed Central

Sáenz-de-Santa-María, Inés; Bernardo-Castiñeira, Cristóbal; Secades, Pablo; Bernaldo-de-Quirós, Sandra; Rodrigo, Juan Pablo; Astudillo, Aurora; Chiara, María-Dolores

2017-01-01

Metabolic reprogramming is a very heterogeneous phenomenon in cancer. It mostly consists on increased glycolysis, lactic acid formation and extracellular acidification. These events have been associated to increased activity of the hypoxia inducible factor, HIF-1α. This study aimed at defining the metabolic program activated by HIF-1α in oropharyngeal squamous cell carcinomas (SCC) and assessing its clinical impact. Global gene/miRNA expression was analyzed in SCC-derived cells exposed to hypoxia. Expression of HIF-1α, the carbonic anhydrase CAIX, and the lactate/H+ transporters MCT1 and MCT4 were analyzed by immunohistochemistry in 246 SCCs. Cell-based analysis revealed that HIF-1α-driven metabolic program includes over-expression of glycolytic enzymes and the microRNA miR-210 coupled to down-regulation of its target, the iron-sulfur cluster assembly protein, ISCU. pH-regulator program entailed over-expression of CAIX, but not MCT1 or MCT4. Accordingly, significant overlapping exists between over-expression of HIF-1α and CAIX, but not HIF-1α and MCT1 or MCT4, in tumor cells. Increased miR-210 and concomitant decreased ISCU RNA levels were found in ~40% of tumors and this was significantly associated with HIF-1α and CAIX, but not MCT1 or MCT4, over-expression. HIF-1α and/or CAIX over-expression was associated with high recurrence rate and low overall survival of surgically treated patients. By contrast, clinically significant correlations were not found in tumors with MCT1 or MCT4 over-expression. This is the first study that provides in vivo evidences of coordinated activation of HIF-1α, CAIX, miR-210 and ISCU in carcinoma and association with poor prognosis, a finding with important implications for the development of metabolic-targeting therapies against hypoxia. PMID:28099149

Clinically relevant HIF-1α-dependent metabolic reprogramming in oropharyngeal squamous cell carcinomas includes coordinated activation of CAIX and the miR-210/ISCU signaling axis, but not MCT1 and MCT4 upregulation.

PubMed

Sáenz-de-Santa-María, Inés; Bernardo-Castiñeira, Cristóbal; Secades, Pablo; Bernaldo-de-Quirós, Sandra; Rodrigo, Juan Pablo; Astudillo, Aurora; Chiara, María-Dolores

2017-02-21

Metabolic reprogramming is a very heterogeneous phenomenon in cancer. It mostly consists on increased glycolysis, lactic acid formation and extracellular acidification. These events have been associated to increased activity of the hypoxia inducible factor, HIF-1α. This study aimed at defining the metabolic program activated by HIF-1α in oropharyngeal squamous cell carcinomas (SCC) and assessing its clinical impact. Global gene/miRNA expression was analyzed in SCC-derived cells exposed to hypoxia. Expression of HIF-1α, the carbonic anhydrase CAIX, and the lactate/H+ transporters MCT1 and MCT4 were analyzed by immunohistochemistry in 246 SCCs. Cell-based analysis revealed that HIF-1α-driven metabolic program includes over-expression of glycolytic enzymes and the microRNA miR-210 coupled to down-regulation of its target, the iron-sulfur cluster assembly protein, ISCU. pH-regulator program entailed over-expression of CAIX, but not MCT1 or MCT4. Accordingly, significant overlapping exists between over-expression of HIF-1α and CAIX, but not HIF-1α and MCT1 or MCT4, in tumor cells. Increased miR-210 and concomitant decreased ISCU RNA levels were found in ~40% of tumors and this was significantly associated with HIF-1α and CAIX, but not MCT1 or MCT4, over-expression. HIF-1α and/or CAIX over-expression was associated with high recurrence rate and low overall survival of surgically treated patients. By contrast, clinically significant correlations were not found in tumors with MCT1 or MCT4 over-expression. This is the first study that provides in vivo evidences of coordinated activation of HIF-1α, CAIX, miR-210 and ISCU in carcinoma and association with poor prognosis, a finding with important implications for the development of metabolic-targeting therapies against hypoxia.

Hypoxia-inducible factor 1-alpha release after intracoronary versus intramyocardial stem cell therapy in myocardial infarction.

PubMed

Gyöngyösi, Mariann; Hemetsberger, Rayyan; Posa, Aniko; Charwat, Silvia; Pavo, Noemi; Petnehazy, Ors; Petrasi, Zsolt; Pavo, Imre J; Hemetsberger, Hani; Benedek, Imre; Benedek, Teodora; Benedek, Istvan; Kovacs, Istvan; Kaun, Christoph; Maurer, Gerald

2010-04-01

We have investigated the effect of stem cell delivery on the release of hypoxia-inducible factor 1 alpha (HIF-1alpha) in peripheral circulation and myocardium in experimental myocardial ischemia. Closed-chest, reperfused myocardial infarction (MI) was created in domestic pigs. Porcine mesenchymal stem cells (MSCs) were cultured and delivered (9.8 +/- 1.2 x 10(6)) either percutaneously NOGA-guided transendocardially (Group IM) or intracoronary (Group IC) 22 +/- 4 days post-MI. Pigs without MSC delivery served as sham control (Group S). Plasma HIF-1alpha was measured at baseline, immediately post- and at follow-up (FUP; 2 h or 24 h) post-MSC delivery by ELISA kit. Myocardial HIF-1alpha expression of infarcted, normal myocardium, or border zone was determined by Western blot. Plasma level of HIF-1alpha increased immediately post-MI (from 278 +/- 127 to 631 +/- 375 pg/ml, p < 0.05). Cardiac delivery of MSCs elevated the plasma levels of HIF-1alpha significantly (p < 0.05) in groups IC and IM immediately post-MSC delivery, and returned to baseline level at FUP, without difference between the groups IC and IM. The myocardial tissue HIF-1alpha expression in the infarcted area was higher in Group IM than in Group IC or S (1,963 +/- 586 vs. 1,307 +/- 392 vs. 271 +/- 110 activity per square millimeter, respectively, p < 0.05), while the border zone contained similarly lower level of HIF-1alpha, but still significantly higher as compared with Group S. Trend towards increase in myocardial expression of HIF-1alpha was measured in Group IM at 24 h, in contrast to Group IC. In conclusion, both stem cell delivery modes increase the systemic and myocardial level of HIF-1alpha. Intramyocardial delivery of MSC seems to trigger the release of angiogenic HIF-1alpha more effectively than does intracoronary delivery.

The association of the placental Hypoxia-inducible factor1-α polymorphisms and HIF1-α mRNA expression with preeclampsia.

PubMed

Harati-Sadegh, Mahdiyeh; Kohan, Leila; Teimoori, Batool; Mehrabani, Mehrnaz; Salimi, Saeedeh

2018-07-01

Evidence has confirmed that placental/fetal hypoxia plays a key role in both endothelial cell dysfunction and PE pathogenesis. The aim of the present study was to investigate whether maternal/placental hypoxia-inducible factor1-α (HIF1-α) C1772T (rs11549465) and/or G1790A (rs11549467) polymorphisms and HIF1-α mRNA expression are associated with PE development. The blood samples of 203 PE and 202 control women and the placenta of 86 PE and 84 control women were collected after delivery. The HIF1-α polymorphisms were genotyped using PCR- RFLP method. The mRNA expression levels were measured by Quantitative Real -Time PCR. The present study found no association between maternal HIF1-α rs11549465 and rs11549467 and placental rs11549467 polymorphisms and PE. However, the placental rs11549465 polymorphism was associated with PE in the dominant model. The CT/GG combined genotypes and TG haplotype of placental rs11549465 and rs11549467 polymorphisms were associated with higher risk of PE. The HIF1-α mRNA expression was 3-fold higher in the PE women. The rs11549465 TT genotype was associated with higher HIF1-α mRNA expression in PE women and in total population and rs11549467 GA genotype was associated with higher mRNA expression in total population. The relative mRNA expression of HIF1-α gene was higher in presence of CC/GA, TT/GG and TT/GA combined genotypes. This study found an association between placental but not maternal HIF1-α rs11549465 polymorphism and PE in the dominant model. The HIF1-α mRNA expression was higher in the placenta of PE women and was associated with rs11549465 and rs11549467 polymorphisms. Copyright © 2018. Published by Elsevier Ltd.

CD44 Interacts with HIF-2α to Modulate the Hypoxic Phenotype of Perinecrotic and Perivascular Glioma Cells.

PubMed

Johansson, Elinn; Grassi, Elisa S; Pantazopoulou, Vasiliki; Tong, Bei; Lindgren, David; Berg, Tracy J; Pietras, Elin J; Axelson, Håkan; Pietras, Alexander

2017-08-15

Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic phenotype of stem-like glioma cells is achieved by stabilization of HIF-2α through interaction with CD44, independently of oxygen. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth.

PubMed

Pacary, Emilie; Tixier, Emmanuelle; Coulet, Florence; Roussel, Simon; Petit, Edwige; Bernaudin, Myriam

2007-07-01

This study demonstrates that the Rho-kinase (ROCK) inhibitor, Y-27632, potentiates not only the effect of cobalt chloride (CoCl(2)) but also that of deferoxamine, another HIF-1 inducer, on mesenchymal stem cell (MSC) neuronal differentiation. HIF-1 is essential for CoCl(2)+/-Y-27632-induced MSC neuronal differentiation, since agents inhibiting HIF-1 abolish the changes of morphology and cell cycle arrest-related gene or protein expressions (p21, cyclin D1) and the increase of neuronal marker expressions (Tuj1, NSE). Y-27632 potentiates the CoCl(2)-induced decrease of cyclin D1 and nestin expressions, the increase of HIF-1 activation and EPO expression, and decreases pVHL expression. Interestingly, CoCl(2) decreases RhoA expression, an effect potentiated by Y-27632, revealing crosstalk between HIF-1 and RhoA/ROCK pathways. Moreover, we demonstrate a synergistic effect of CoCl(2) and Y-27632 on neurosphere differentiation into neurons and PC12 neurite outgrowth underlining that a co-treatment targeting both HIF-1 and ROCK pathways might be relevant to differentiate stem cells into neurons.

Hypoxia-inducible factor 1α is a critical downstream mediator for hypoxia-induced mitogenic factor (FIZZ1/RELMα)-induced pulmonary hypertension

PubMed Central

Johns, Roger A.; Takimoto, Eiki; Meuchel, Lucas W.; Elsaigh, Esra; Zhang, Ailan; Heller, Nicola M.; Semenza, Gregg L.; Yamaji-Kegan, Kazuyo

2017-01-01

Objective Pulmonary hypertension (PH) is characterized by progressive elevation of pulmonary vascular resistance, right ventricular failure, and ultimately death. We have shown that in rodents, hypoxia-induced mitogenic factor (HIMF; also known as FIZZ1 or RELMα) causes PH by initiating lung vascular inflammation. We hypothesized that hypoxia-inducible factor-1 (HIF-1) is a critical downstream signal mediator of HIMF during PH development. Approach and Results In this study, we compared the degree of HIMF-induced pulmonary vascular remodeling and PH development in wild-type (HIF-1α+/+) and HIF-1α heterozygous null (HIF-1α+/−) mice. HIMF-induced PH was significantly diminished in HIF-1α+/− mice and was accompanied by a dysregulated VEGF-A–VEGF receptor 2 pathway. HIF-1α was critical for bone marrow-derived cell migration and vascular tube formation in response to HIMF. Furthermore, HIMF and its human homolog, resistin-like molecule-β (RELMβ), significantly increased IL-6 in macrophages and lung resident cells through a mechanism dependent on HIF-1α and, at least to some extent, on nuclear factor κB. Conclusions Our results suggest that HIF-1α is a critical downstream transcription factor for HIMF-induced pulmonary vascular remodeling and PH development. Importantly, both HIMF and human RELMβ significantly increased IL-6 in lung resident cells and increased perivascular accumulation of IL-6–expressing macrophages in the lungs of mice. These data suggest that HIMF can induce HIF-1, VEGF-A, and interleukin-6, which are critical mediators of both hypoxic inflammation and PH pathophysiology. PMID:26586659

Activation of Dll4/Notch Signaling and Hypoxia-Inducible Factor-1 Alpha Facilitates Lymphangiogenesis in Lacrimal Glands in Dry Eye.

PubMed

Min, Ji Hwan; Lee, Chul Hee; Ji, Yong Woo; Yeo, Areum; Noh, Hyemi; Song, Insil; Kim, Eung Kweon; Lee, Hyung Keun

2016-01-01

By using hypoxia-inducible factor-1 alpha conditional knockout (HIF-1α CKO) mice and a dry eye (DE) mouse model, we aimed to determine the role played by delta-like ligand 4 (Dll4)/Notch signaling and HIF-1α in the lymphangiogenesis of lacrimal glands (LGs). C57BL/6 mice were housed in a controlled-environment chamber for DE induction. During DE induction, the expression level of Dll4/Notch signaling and lymphangiogenesis in LGs was measured by quantitative RT-PCR, immunoblot, and immunofluorescence staining. Next, lymphangiogenesis was measured after Dll4/Notch signal inhibition by anti-Dll4 antibody or γ-secretase inhibitor. Using HIF-1α CKO mice, the expression of Dll4/Notch signaling and lymphangiogenesis in LGs of DE-induced HIF-1α CKO mice were assessed. Additionally, the infiltration of CD45+ cells in LGs was assessed by immunohistochemical (IHC) staining and flow cytometry for each condition. DE significantly upregulated Dll4/Notch and lymphangiogenesis in LGs. Inhibition of Dll4/Notch significantly suppressed lymphangiogenesis in LGs. Compared to wild-type (WT) mice, DE induced HIF-1α CKO mice showed markedly low levels of Dll4/Notch and lymphangiogenesis. Inhibition of lymphangiogenesis by Dll4/Notch suppression resulted in increased CD45+ cell infiltration in LGs. Likewise, CD45+ cells infiltrated more in the LGs of HIF-1α CKO DE mice than in non-DE HIF-1α CKO mice. Dll4/Notch signaling and HIF-1α are closely related to lymphangiogenesis in DE-induced LGs. Lymphangiogenesis stimulated by Dll4/Notch and HIF-1α may play a role in protecting LGs from DE-induced inflammation by aiding the clearance of immune cells from LGs.

Minoxidil Induction of VEGF Is Mediated by Inhibition of HIF-Prolyl Hydroxylase

PubMed Central

Yum, Soohwan; Jeong, Seongkeun; Kim, Dohoon; Lee, Sunyoung; Kim, Wooseong; Yoo, Jin-Wook; Kwon, Oh Sang; Kim, Dae-Duk; Min, Do Sik; Jung, Yunjin

2017-01-01

The topical application of minoxidil may achieve millimolar concentrations in the skin. We investigated whether millimolar minoxidil could induce vascular endothelial growth factor (VEGF), a possible effector for minoxidil-mediated hair growth, and how it occurred at the molecular level. Cell-based experiments were performed to investigate a molecular mechanism underlying the millimolar minoxidil induction of VEGF. The inhibitory effect of minoxidil on hypoxia-inducible factor (HIF) prolyl hydroxylase-2 (PHD-2) was tested by an in vitro von Hippel–Lindau protein (VHL) binding assay. To examine the angiogenic potential of millimolar minoxidil, a chorioallantoic membrane (CAM) assay was used. In human keratinocytes and dermal papilla cells, millimolar minoxidil increased the secretion of VEGF, which was not attenuated by a specific adenosine receptor antagonist that inhibits the micromolar minoxidil induction of VEGF. Millimolar minoxidil induced hypoxia-inducible factor-1α (HIF-1α), and the induction of VEGF was dependent on HIF-1. Moreover, minoxidil applied to the dorsal area of mice increased HIF-1α and VEGF in the skin. In an in vitro VHL binding assay, minoxidil directly inhibited PHD-2, thus preventing the hydroxylation of cellular HIF-1α and VHL-dependent proteasome degradation and resulting in the stabilization of HIF-1α protein. Minoxidil inhibition of PHD-2 was reversed by ascorbate, a cofactor of PHD-2, and the minoxidil induction of cellular HIF-1α was abrogated by the cofactor. Millimolar minoxidil promoted angiogenesis in the CAM assay, an in vivo angiogenic test, and this was nullified by the specific inhibition of VEGF. Our data demonstrate that PHD may be the molecular target for millimolar minoxidil-mediated VEGF induction via HIF-1. PMID:29295567

Reciprocal regulation by hypoxia-inducible factor-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in osteoarthritis.

PubMed

Oh, H; Kwak, J-S; Yang, S; Gong, M-K; Kim, J-H; Rhee, J; Kim, S K; Kim, H-E; Ryu, J-H; Chun, J-S

2015-12-01

Hypoxia-inducible factor-2α (HIF-2α) transcriptionally upregulates Nampt in articular chondrocytes. NAMPT, which exhibits nicotinamide phosphoribosyltransferase activity, in turn causes osteoarthritis (OA) in mice by stimulating the expression of matrix-degrading enzymes. Here, we sought to elucidate whether HIF-2α activates the NAMPT-NAD(+)-SIRT axis in chondrocytes and thereby contributes to the pathogenesis of OA. Assays of NAD levels, SIRT activity, reporter gene activity, mRNA, and protein levels were conducted in primary cultured mouse articular chondrocytes. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1) or NAMPT (Ad-Nampt). The functions of SIRT in OA were examined by IA co-injection of SIRT inhibitors or adenovirus expressing individual SIRT isoforms or shRNA targeting specific SIRT isoforms. HIF-2α activated the NAMPT-NAD(+)-SIRT axis in chondrocytes by upregulating NAMPT, which stimulated NAD(+) synthesis and thereby activated SIRT family members. The activated NAMPT-SIRT pathway, in turn, promoted HIF-2α protein stability by negatively regulating its hydroxylation and 26S proteasome-mediated degradation, resulting in increased HIF-2α transcriptional activity. Among SIRT family members (SIRT1-7), SIRT2 and SIRT4 were positively associated with HIF-2α stability and transcriptional activity in chondrocytes. This reciprocal regulation was required for the expression of catabolic matrix metalloproteinases (MMP3, MMP12, and MMP13) and OA cartilage destruction caused by IA injection of Ad-Epas1 Ad-Nampt. The reciprocal regulation of HIF-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in OA cartilage destruction caused by HIF-2α or NAMPT. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Reactive oxygen species-generating mitochondrial DNA mutation up-regulates hypoxia-inducible factor-1alpha gene transcription via phosphatidylinositol 3-kinase-Akt/protein kinase C/histone deacetylase pathway.

PubMed

Koshikawa, Nobuko; Hayashi, Jun-Ichi; Nakagawara, Akira; Takenaga, Keizo

2009-11-27

Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1alpha mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1alpha transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1alpha mRNA overexpression. Stimulation of HIF-1alpha transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1alpha transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1alpha mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1alpha transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1alpha transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1alpha protein accumulation in hypoxic tumor cells.

Development of unidentified dna-specific hif 1α gene of lizard (hemidactylus platyurus) which plays a role in tissue regeneration process

NASA Astrophysics Data System (ADS)

Novianti, T.; Sadikin, M.; Widia, S.; Juniantito, V.; Arida, E. A.

2018-03-01

Development of unidentified specific gene is essential to analyze the availability these genes in biological process. Identification unidentified specific DNA of HIF 1α genes is important to analyze their contribution in tissue regeneration process in lizard tail (Hemidactylus platyurus). Bioinformatics and PCR techniques are relatively an easier method to identify an unidentified gene. The most widely used method is BLAST (Basic Local Alignment Sequence Tools) method for alignment the sequences from the other organism. BLAST technique is online software from website https://blast.ncbi.nlm.nih.gov/Blast.cgi that capable to generate the similar sequences from closest kinship to distant kindship. Gecko japonicus is a species that it has closest kinship with H. platyurus. Comparing HIF 1 α gene sequence of G. japonicus with the other species used multiple alignment methods from Mega7 software. Conserved base areas were identified using Clustal IX method. Primary DNA of HIF 1 α gene was design by Primer3 software. HIF 1α gene of lizard (H. platyurus) was successfully amplified using a real-time PCR machine by primary DNA that we had designed from Gecko japonicus. Identification unidentified gene of HIF 1a lizard has been done successfully with multiple alignment method. The study was conducted by analyzing during the growth of tail on day 1, 3, 5, 7, 10, 13 and 17 of lizard tail after autotomy. Process amplification of HIF 1α gene was described by CT value in real time PCR machine. HIF 1α expression of gene is quantified by Livak formula. Chi-square statistic test is 0.000 which means that there is a different expression of HIF 1 α gene in every growth day treatment.

Hypoxia induced E-cadherin involving regulators of Hippo pathway due to HIF-1α stabilization/nuclear translocation in bone metastasis from breast carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maroni, Paola; Matteucci, Emanuela; Drago, Lorenzo

The present study deals with the molecular mechanisms involved in the regulation of E-cadherin expression under hypoxia, because the adjustment of the amount of E-cadherin due to physical stimuli of the microenvironment might influence the colonization of metastasis to skeleton. We analyzed the effect of 1% oxygen tension, that is similar to that encountered in the bone marrow by metastatic cells spreading from breast carcinoma. The purpose was to evaluate the hypoxia-orchestrated control of E-cadherin transactivation via hypoxia inducible factor-1 (HIF-1) and peroxisome proliferator activated receptor-γ (PPARγ), and the involvement of Hippo pathway members, as regulators of transcription factors. Tomore » give a translational significance to the study, we took into consideration human pair-matched ductal breast carcinoma and bone metastasis: E-cadherin and Wwox were expressed in bone metastasis but not in breast carcinoma, while HIF-1α and TAZ seemed localized principally in nuclei of metastasis and were found in all cell compartments of breast carcinoma. A close examination of the regulatory mechanisms underlying E-cadherin expression in bone metastasis was done in 1833 clone derived from MDA-MB231 cells. Hypoxia induced E-cadherin only in 1833 clone, but not in parental cells, through HIF-1 and PPARγ activities, while Wwox decreased. Since Wwox was highly expressed in bone metastasis, the effect of ectopic Wwox was evaluated, and we showed E-cadherin transactivation and enhanced invasiveness in WWOX transfected 1833 cells. Also, hypoxia was additive with ectopic Wwox remarkably enhancing HIF-1α nuclear shuttle and accumulation due to the lengthening of the half-life of HIF-1α protein; under this experimental condition HIF-1α appeared as a slower migrated band compared with control, in agreement with the phosphorylation state. The in vitro data strongly supported the almost exclusive presence of HIF-1α in nuclei of human-bone metastasis. Thus, we identified Wwox as a novel molecule in the HIF-1α-HDM2 regulatory loop, necessary for the dynamic regulation of the HIF-1α amount, and we suggested that the reduction of endogenous Wwox free pool under hypoxia might also be due to the interaction with HDM2, sequestering the E3 ubiquitin ligase. We highlighted the importance of nuclear HIF-1α in the biology of metastasis for the mesenchymal-epithelial transition: this phenotype was regulated by Wwox plus hypoxia through E-cadherin target gene, playing a pivotal role in bone metastasis colonization. - Highlights: • E-cadherin accumulates in hypoxic bone metastasis opposite to primary carcinoma. • HIF-1 and PPARγ cooperate in inducing E-cadherin under hypoxia in metastatic cells. • Wwox regulates HIF-1α phosphorylation and nuclear translocation. • Hypoxia plus Wwox prevent HIF-1α degradation via HDM2 forming a regulatory loop.« less

Feedback regulation via AMPK and HIF-1 mediates ROS-dependent longevity in Caenorhabditis elegans

PubMed Central

Hwang, Ara B.; Ryu, Eun-A; Artan, Murat; Chang, Hsin-Wen; Kabir, Mohammad Humayun; Nam, Hyun-Jun; Lee, Dongyeop; Yang, Jae-Seong; Kim, Sanguk; Mair, William B.; Lee, Cheolju; Lee, Siu Sylvia; Lee, Seung-Jae

2014-01-01

Mild inhibition of mitochondrial respiration extends the lifespan of many species. In Caenorhabditis elegans, reactive oxygen species (ROS) promote longevity by activating hypoxia-inducible factor 1 (HIF-1) in response to reduced mitochondrial respiration. However, the physiological role and mechanism of ROS-induced longevity are poorly understood. Here, we show that a modest increase in ROS increases the immunity and lifespan of C. elegans through feedback regulation by HIF-1 and AMP-activated protein kinase (AMPK). We found that activation of AMPK as well as HIF-1 mediates the longevity response to ROS. We further showed that AMPK reduces internal levels of ROS, whereas HIF-1 amplifies the levels of internal ROS under conditions that increase ROS. Moreover, mitochondrial ROS increase resistance to various pathogenic bacteria, suggesting a possible association between immunity and long lifespan. Thus, AMPK and HIF-1 may control immunity and longevity tightly by acting as feedback regulators of ROS. PMID:25288734

Hypoxia and Inflammation in Cancer, Focus on HIF and NF-κB

PubMed Central

D’Ignazio, Laura; Batie, Michael; Rocha, Sonia

2017-01-01

Cancer is often characterised by the presence of hypoxia and inflammation. Paramount to the mechanisms controlling cellular responses under such stress stimuli, are the transcription factor families of Hypoxia Inducible Factor (HIF) and Nuclear Factor of κ-light-chain-enhancer of activated B cells (NF-κB). Although, a detailed understating of how these transcription factors respond to their cognate stimulus is well established, it is now appreciated that HIF and NF-κB undergo extensive crosstalk, in particular in pathological situations such as cancer. Here, we focus on the current knowledge on how HIF is activated by inflammation and how NF-κB is modulated by hypoxia. We summarise the evidence for the possible mechanism behind this activation and how HIF and NF-κB function impacts cancer, focusing on colorectal, breast and lung cancer. We discuss possible new points of therapeutic intervention aiming to harness the current understanding of the HIF-NF-κB crosstalk. PMID:28536364

Comparative systems pharmacology of HIF stabilization in the prevention of retinopathy of prematurity

PubMed Central

Hoppe, George; Yoon, Suzy; Gopalan, Banu; Savage, Alexandria R.; Brown, Rebecca; Case, Kelsey; Vasanji, Amit; Chan, E. Ricky; Silver, Randi B.; Sears, Jonathan E.

2016-01-01

Retinopathy of prematurity (ROP) causes 100,000 new cases of childhood blindness each year. ROP is initiated by oxygen supplementation necessary to prevent neonatal death. We used organ systems pharmacology to define the transcriptomes of mice that were cured of oxygen-induced retinopathy (OIR, ROP model) by hypoxia-inducible factor (HIF) stabilization via HIF prolyl hydroxylase inhibition using the isoquinolone Roxadustat or the 2-oxoglutarate analog dimethyloxalylglycine (DMOG). Although both molecules conferred a protective phenotype, gene expression analysis by RNA sequencing found that Roxadustat can prevent OIR by two pathways: direct retinal HIF stabilization and induction of aerobic glycolysis or indirect hepatic HIF-1 stabilization and increased serum angiokines. As predicted by pathway analysis, Roxadustat rescued the hepatic HIF-1 knockout mouse from retinal oxygen toxicity, whereas DMOG could not. The simplicity of systemic treatment that targets both the liver and the eye provides a rationale for protecting the severely premature infant from oxygen toxicity. PMID:27091985

The last common ancestor of animals lacked the HIF pathway and respired in low-oxygen environments

PubMed Central

Vargas, Sergio; Larsen, Morten; Elemans, Coen PH; Canfield, Donald E

2018-01-01

Animals have a carefully orchestrated relationship with oxygen. When exposed to low environmental oxygen concentrations, and during periods of increased energy expenditure, animals maintain cellular oxygen homeostasis by enhancing internal oxygen delivery, and by enabling the anaerobic production of ATP. These low-oxygen responses are thought to be controlled universally across animals by the hypoxia-inducible factor (HIF). We find, however, that sponge and ctenophore genomes lack key components of the HIF pathway. Since sponges and ctenophores are likely sister to all remaining animal phyla, the last common ancestor of extant animals likely lacked the HIF pathway as well. Laboratory experiments show that the marine sponge Tethya wilhelma maintains normal transcription under oxygen levels down to 0.25% of modern atmospheric saturation, the lowest levels we investigated, consistent with the predicted absence of HIF or any other HIF-like pathway. Thus, the last common ancestor of all living animals could have metabolized aerobically under very low environmental oxygen concentrations. PMID:29402379

Molecular-targeted antitumor agents. 19. Furospongolide from a marine Lendenfeldia sp. sponge inhibits hypoxia-inducible factor-1 activation in breast tumor cells.

PubMed

Liu, Yang; Liu, Rui; Mao, Shui-Chun; Morgan, J Brian; Jekabsons, Mika B; Zhou, Yu-Dong; Nagle, Dale G

2008-11-01

A natural product chemistry-based approach was employed to discover small-molecule inhibitors of the important tumor-selective molecular target hypoxia-inducible factor-1 (HIF-1). Bioassay-guided isolation of an active lipid extract of a Saipan collection of the marine sponge Lendenfeldia sp. afforded the terpene-derived furanolipid furospongolide as the primary inhibitor of hypoxia-induced HIF-1 activation (IC(50) 2.9 μM, T47D breast tumor cells). The active component of the extract also contained one new cytotoxic scalarane sesterterpene and two previously reported scalaranes. Furospongolide blocked the induction of the downstream HIF-1 target secreted vascular endothelial growth factor (VEGF) and was shown to suppress HIF-1 activation by inhibiting the hypoxic induction of HIF-1α protein. Mechanistic studies indicate that furospongolide inhibits HIF-1 activity primarily by suppressing tumor cell respiration via the blockade of NADH-ubiquinone oxidoreductase (complex I)-mediated mitochondrial electron transfer.

Hypoxia and hypoxia inducible factor-1α are required for normal endometrial repair during menstruation.

PubMed

Maybin, Jacqueline A; Murray, Alison A; Saunders, Philippa T K; Hirani, Nikhil; Carmeliet, Peter; Critchley, Hilary O D

2018-01-23

Heavy menstrual bleeding (HMB) is common and debilitating, and often requires surgery due to hormonal side effects from medical therapies. Here we show that transient, physiological hypoxia occurs in the menstrual endometrium to stabilise hypoxia inducible factor 1 (HIF-1) and drive repair of the denuded surface. We report that women with HMB have decreased endometrial HIF-1α during menstruation and prolonged menstrual bleeding. In a mouse model of simulated menses, physiological endometrial hypoxia occurs during bleeding. Maintenance of mice under hyperoxia during menses decreases HIF-1α induction and delays endometrial repair. The same effects are observed upon genetic or pharmacological reduction of endometrial HIF-1α. Conversely, artificial induction of hypoxia by pharmacological stabilisation of HIF-1α rescues the delayed endometrial repair in hypoxia-deficient mice. These data reveal a role for HIF-1 in the endometrium and suggest its pharmacological stabilisation during menses offers an effective, non-hormonal treatment for women with HMB.

TLX controls angiogenesis through interaction with the von Hippel-Lindau protein.

PubMed

Zeng, Zhao-Jun; Johansson, Erik; Hayashi, Amiko; Chavali, Pavithra L; Akrap, Nina; Yoshida, Takeshi; Kohno, Kimitoshi; Izumi, Hiroto; Funa, Keiko

2012-06-15

TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-α) for binding to pVHL, which contributes to the stabilization of HIF-2α in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2α, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2α and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2α will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2α is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2α. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis.

TLX controls angiogenesis through interaction with the von Hippel-Lindau protein

PubMed Central

Zeng, Zhao-jun; Johansson, Erik; Hayashi, Amiko; Chavali, Pavithra L.; Akrap, Nina; Yoshida, Takeshi; Kohno, Kimitoshi; Izumi, Hiroto; Funa, Keiko

2012-01-01

Summary TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-α) for binding to pVHL, which contributes to the stabilization of HIF-2α in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2α, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2α and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2α will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2α is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2α. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis. PMID:23213445

Overexpression of HIF-1α in mesenchymal stem cells contributes to repairing hypoxic-ischemic brain damage in rats.

PubMed

Lin, Deju; Zhou, Liping; Wang, Biao; Liu, Lizhen; Cong, Li; Hu, Chuanqin; Ge, Tingting; Yu, Qin

2017-01-01

Preclinical researches on mesenchymal stem cells (MSCs) transplantation, which is used to treat hypoxic-ischemic (HI) brain damage, have received inspiring achievements. However, the insufficient migration of active cells to damaged tissues has limited their potential therapeutic effects. There are some evidences that hypoxia inducible factor-1 alpha (HIF-1α) promotes the viability and migration of the cells. Here, we aim to investigate whether overexpression of HIF-1α in MSCs could improve the viability and migration capacity of cells, and its therapeutic efficiency on HI brain damage. In the study, MSCs with HIF-1α overexpression was achieved by recombinant lentiviral vector and transplanted to the rats subsequent to HI. Our data indicated that overexpression of HIF-1α promoted the viability and migration of MSCs, HIF-1α overexpressed MSCs also had a stronger therapeutic efficiency on HI brain damaged treatment by mitigating the injury on behavioral and histological changes evoked by HI insults, accompanied with more MSCs migrating to cerebral damaged area. This study demonstrated that HIF-1α overexpression could increase the MSCs' therapeutic efficiency in HI and the promotion of the cells' directional migration to cerebral HI area by overexpression may be responsible for it, which showed that transplantation of MSCs with HIF-1α overexpression is an attractive therapeutic option to treat HI-induced brain injury in the future. Copyright © 2016 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Inducing a visceral organ to protect a peripheral capillary bed: stabilizing hepatic HIF-1α prevents oxygen-induced retinopathy.

PubMed

Hoppe, George; Lee, Tamara J; Yoon, Suzy; Yu, Minzhong; Peachey, Neal S; Rayborn, Mary; Zutel, M Julieta; Trichonas, George; Au, John; Sears, Jonathan E

2014-06-01

Activation of hypoxia-inducible factor (HIF) can prevent oxygen-induced retinopathy in rodents. Here we demonstrate that dimethyloxaloylglycine (DMOG)-induced retinovascular protection is dependent on hepatic HIF-1 because mice deficient in liver-specific HIF-1α experience hyperoxia-induced damage even with DMOG treatment, whereas DMOG-treated wild-type mice have 50% less avascular retina (P < 0.0001). Hepatic HIF stabilization protects retinal function because DMOG normalizes the b-wave on electroretinography in wild-type mice. The localization of DMOG action to the liver is further supported by evidence that i) mRNA and protein erythropoietin levels within liver and serum increased in DMOG-treated wild-type animals but are reduced by 60% in liver-specific HIF-1α knockout mice treated with DMOG, ii) triple-positive (Sca1/cKit/VEGFR2), bone-marrow-derived endothelial precursor cells increased twofold in DMOG-treated wild-type mice (P < 0.001) but are unchanged in hepatic HIF-1α knockout mice in response to DMOG, and iii) hepatic luminescence in the luciferase oxygen-dependent degradation domain mouse was induced by subcutaneous and intraperitoneal DMOG. These findings uncover a novel endocrine mechanism for retinovascular protection. Activating HIF in visceral organs such as the liver may be a simple strategy to protect capillary beds in the retina and in other peripheral tissues. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Regulation of HIF-1α signaling and chemoresistance in acute lymphocytic leukemia under hypoxic conditions of the bone marrow microenvironment

PubMed Central

Frolova, Olga; Samudio, Ismael; Benito, Juliana Maria; Jacamo, Rodrigo; Kornblau, Steven M.; Markovic, Ana; Schober, Wendy; Lu, Hongbo; Qiu, Yi Hua; Buglio, Daniela; McQueen, Teresa; Pierce, Sherry; Shpall, Elizabeth; Konoplev, Sergej; Thomas, Deborah; Kantarjian, Hagop; Lock, Richard; Andreeff, Michael; Konopleva, Marina

2012-01-01

Overcoming resistance to chemotherapy is the main therapeutic challenge in the treatment of acute lymphocytic leukemia (ALL). Interactions between leukemia cells and the microenvironment promote leukemia cell survival and confer resistance to chemotherapy. Hypoxia is an integral component of bone marrow (BM) microenvironment. Hypoxia-inducible factor-1α (HIF-1), a key regulator of the cellular response to hypoxia, regulates cell growth and metabolic adaptation to hypoxia. HIF-1α expression, analyzed by Reverse Phase Protein Arrays in 92 specimens from newly diagnosed patients with pre-B-ALL, had a negative prognostic impact on survival (p = 0.0025). Inhibition of HIF-1α expression by locked mRNA antagonist (LNA) promoted chemosensitivity under hypoxic conditions, while pharmacological or genetic stabilization of HIF-1α under normoxia inhibited cell growth and reduced apoptosis induction by chemotherapeutic agents. Co-culture of pre-B ALL or REH cells with BM-derived mesenchymal stem cells (MSC) under hypoxia resulted in further induction of HIF-1α protein and acquisition of the glycolytic phenotype, in part via stroma-induced AKT/mTOR signaling. mTOR blockade with everolimus reduced HIF-1α expression, diminished glucose uptake and glycolytic rate and partially restored the chemosensitivity of ALL cells under hypoxia/stroma co-cultures. Hence, mTOR inhibition or blockade of HIF-1α-mediated signaling may play an important role in chemosensitization of ALL cells under hypoxic conditions of the BM microenvironment. PMID:22785211

c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription

PubMed Central

Alfranca, Arántzazu; Gutiérrez, M. Dolores; Vara, Alicia; Aragonés, Julián; Vidal, Felipe; Landázuri, Manuel O.

2002-01-01

Under low-oxygen conditions, cells develop an adaptive program that leads to the induction of several genes, which are transcriptionally regulated by hypoxia-inducible factor 1 (HIF-1). On the other hand, there are other factors which modulate the HIF-1-mediated induction of some genes by binding to cis-acting motifs present in their promoters. Here, we show that c-Jun functionally cooperates with HIF-1 transcriptional activity in different cell types. Interestingly, a dominant-negative mutant of c-Jun which lacks its transactivation domain partially inhibits HIF-1-mediated transcription. This cooperative effect is not due to an increase in the nuclear amount of the HIF-1α subunit, nor does it require direct binding of c-Jun to DNA. c-Jun and HIF-1α are able to associate in vivo but not in vitro, suggesting that this interaction involves the participation of additional proteins and/or a posttranslational modification of these factors. In this context, hypoxia induces phosphorylation of c-Jun at Ser63 in endothelial cells. This process is involved in its cooperative effect, since specific blockade of the JNK pathway and mutation of c-Jun at Ser63 and Ser73 impair its functional cooperation with HIF-1. The functional interplay between c-Jun and HIF-1 provides a novel insight into the regulation of some genes, such as the one for VEGF, which is a key regulator of tumor angiogenesis. PMID:11739718

Hypoxia-mediated sorafenib resistance can be overcome by EF24 through Von Hippel-Lindau tumor suppressor-dependent HIF-1α inhibition in hepatocellular carcinoma.

PubMed

Liang, Yingjian; Zheng, Tongsen; Song, Ruipeng; Wang, Jiabei; Yin, Dalong; Wang, Luoluo; Liu, Haitao; Tian, Lantian; Fang, Xiang; Meng, Xianzhi; Jiang, Hongchi; Liu, Jiaren; Liu, Lianxin

2013-05-01

The increasing incidence of hepatocellular carcinoma (HCC) is of great concern not only in the United States but throughout the world. Although sorafenib, a multikinase inhibitor with antiangiogenic and antiproliferative effects, currently sets the new standard for advanced HCC, tumor response rates are usually quite low. An understanding of the underlying mechanisms for sorafenib resistance is critical if outcomes are to be improved. In this study we tested the hypothesis that hypoxia caused by the antiangiogenic effects of sustained sorafenib therapy could induce sorafenib resistance as a cytoprotective adaptive response, thereby limiting sorafenib efficiency. We found that HCCs, clinically resistant to sorafenib, exhibit increased intratumor hypoxia compared with HCCs before treatment or HCCs sensitive to sorafenib. Hypoxia protected HCC cells against sorafenib and hypoxia-inducible factor 1 (HIF-1α) was required for the process. HCC cells acquired increased P-gp expression, enhanced glycolytic metabolism, and increased nuclear factor kappa B (NF-κB) activity under hypoxia. EF24, a molecule having structural similarity to curcumin, could synergistically enhance the antitumor effects of sorafenib and overcome sorafenib resistance through inhibiting HIF-1α by sequestering it in cytoplasm and promoting degradation by way of up-regulating Von Hippel-Lindau tumor suppressor (VHL). Furthermore, we found that sustained sorafenib therapy led to increased intratumor hypoxia, which was associated with sorafenib sensitivity in HCC subcutaneous mice tumor models. The combination of EF24 and sorafenib showed synergistically effects against metastasis both in vivo and in vitro. Synergistic tumor growth inhibition effects were also observed in subcutaneous and orthotopic hepatic tumors. Hypoxia induced by sustained sorafenib treatment confers sorafenib resistance to HCC through HIF-1α and NF-κB activation. EF24 overcomes sorafenib resistance through VHL-dependent HIF-1α degradation and NF-κB inactivation. EF24 in combination with sorafenib represents a promising strategy for HCC. Copyright © 2013 American Association for the Study of Liver Diseases.

Ascorbic acid, but not dehydroascorbic acid increases intracellular vitamin C content to decrease Hypoxia Inducible Factor -1 alpha activity and reduce malignant potential in human melanoma.

PubMed

Fischer, Adam P; Miles, Sarah L

2017-02-01

Accumulation of hypoxia inducible factor-1 alpha (HIF-1α) in malignant tissue is known to contribute to oncogenic progression and is inversely associated with patient survival. Ascorbic acid (AA) depletion in malignant tissue may contribute to aberrant normoxic activity of HIF-1α. While AA supplementation has been shown to attenuate HIF-1α function in malignant melanoma, the use of dehydroascorbic acid (DHA) as a therapeutic means to increase intracellular AA and modulate HIF-1α function is yet to be evaluated. Here we compared the ability of AA and DHA to increase intracellular vitamin C content and decrease the malignant potential of human melanoma by reducing the activity of HIF-1α. HIF-1α protein accumulation was evaluated by western blot and transcriptional activity was evaluated by reporter gene assay using a HIF-1 HRE-luciferase plasmid. Protein expressions and subcellular localizations of vitamin C transporters were evaluated by western blot and confocal imaging. Intracellular vitamin C content following AA, ascorbate 2-phosphate (A2P), or DHA supplementation was determined using a vitamin C assay. Malignant potential was accessed using a 3D spheroid Matrigel invasion assay. Data was analyzed by One or Two-way ANOVA with Tukey's multiple comparisons test as appropriate with p<0.05 considered significant. Melanoma cells expressed both sodium dependent vitamin C (SVCT) and glucose (GLUT) transporters for AA and DHA transport respectively, however advanced melanomas responded favorably to AA, but not DHA. Physiological glucose conditions significantly impaired intracellular vitamin C accumulation following DHA treatment. Consequently, A2P and AA, but not DHA treated cells demonstrated lower HIF-1α protein expression and activity, and reduced malignant potential. The ability of AA to regulate HIF-1α was dependent on SVCT2 function and SVCT2 was not significantly inhibited at pH representative of the tumor microenvironment. The use of ascorbic acid as an adjuvant cancer therapy remains under investigated. While AA and A2P were capable of modulating HIF-1α protein accumulation/activity, DHA supplementation resulted in minimal intracellular vitamin C activity with decreased ability to inhibit HIF-1α activity and malignant potential in advanced melanoma. Restoring AA dependent regulation of HIF-1α in malignant cells may prove beneficial in reducing chemotherapy resistance and improving treatment outcomes. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs)

PubMed Central

2013-01-01

Introduction Carcinoma-associated fibroblasts (CAFs) play a pivotal role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors possess a unique microenvironment characterized by local hypoxia, which induces gene expression changes and biological features leading to poor outcomes. Hypoxia Inducible Factor 1 (HIF-1) is the main transcription factor that mediates the cell response to hypoxia through different mechanisms that include the regulation of genes strongly associated with cancer aggressiveness. Among the HIF-1 target genes, the G-protein estrogen receptor (GPER) exerts a stimulatory role in diverse types of cancer cells and in CAFs. Methods We evaluated the regulation and function of the key angiogenic mediator vascular endothelial growth factor (VEGF) in CAFs exposed to hypoxia. Gene expression studies, Western blotting analysis and immunofluorescence experiments were performed in CAFs and breast cancer cells in the presence of cobalt chloride (CoCl2) or cultured under low oxygen tension (2% O2), in order to analyze the involvement of the HIF-1α/GPER signaling in the biological responses to hypoxia. We also explored the role of the HIF-1α/GPER transduction pathway in functional assays like tube formation in human umbilical vein endothelial cells (HUVECs) and cell migration in CAFs. Results We first determined that hypoxia induces the expression of HIF-1α and GPER in CAFs, then we ascertained that the HIF-1α/GPER signaling is involved in the regulation of VEGF expression in breast cancer cells and in CAFs exposed to hypoxia. We also assessed by ChIP assay that HIF-1α and GPER are both recruited to the VEGF promoter sequence and required for VEGF promoter stimulation upon hypoxic condition. As a biological counterpart of these findings, conditioned medium from hypoxic CAFs promoted tube formation in HUVECs in a HIF-1α/GPER dependent manner. The functional cooperation between HIF-1α and GPER in CAFs was also evidenced in the hypoxia-induced cell migration, which involved a further target of the HIF-1α/GPER signaling like connective tissue growth factor (CTGF). Conclusions The present results provide novel insight into the role elicited by the HIF-1α/GPER transduction pathway in CAFs towards the hypoxia-dependent tumor angiogenesis. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression. PMID:23947803

HIF-1α promoted vasculogenic mimicry formation in hepatocellular carcinoma through LOXL2 up-regulation in hypoxic tumor microenvironment.

PubMed

Wang, Meili; Zhao, Xiulan; Zhu, Dongwang; Liu, Tieju; Liang, Xiaohui; Liu, Fang; Zhang, Yanhui; Dong, Xueyi; Sun, Baocun

2017-04-27

The incidence and mortality rates of hepatocellular carcinoma (HCC) have steadily increased in recent years. A hypoxic microenvironment is one of the most important characteristics of solid tumors which has been shown to promote tumor metastasis, epithelial-mesenchymal transition and angiogenesis. Epithelial-mesenchymal transition and vasculogenic mimicry have been regarded as crucial contributing factors to cancer progression. HIF-1α functions as a master transcriptional regulator in the adaptive response to hypoxia. Lysyl oxidases like 2 (LOXL2) is a member of the lysyl oxidase family, which main function is to catalyze the covalent cross-linkages of collagen and elastin in the extracellular matrix. Recent work has demonstrated that HIF-1α promotes the expression of LOXL2, which is believed to amplify tumor aggressiveness. LOXL2 has shown to promote metastasis and is correlated with poor prognosis in hepatocellular carcinoma. The purpose of our study is to explore the role of HIF-1α in progression and metastasis of hepatocellular carcinoma by promoting the expression of LOXL2 as well as the potential regulatory mechanism. HIF-1α, LOXL2 expression and CD31/periodic acid-Schiff double staining in HCC patient samples were examined by immunohistochemical staining. shRNA plasmids against HIF-1α was used to determine whether LOXL2 been increased by HIF-1α. We monitored a series of rescue assays to demonstrate our hypothesis that LOXL2 is required and sufficient for HIF-1α induced EMT and VM formation, which mediates cellular transformation and takes effect in cellular invasion. Then we performed GeneChip® Human Transcriptome Array (HTA) 2.0 in HepG2 cells, HepG2 cells overexpressed LOXL2 and HepG2 cells treated with CoCl 2 . In clinical HCC tissues, it confirmed a positive relationship between HIF-1α and LOXL2 protein. Importantly, HIF-1α and LOXL2 high expression and the presence of vasculogenic mimicry were correlated to poor prognosis. HIF-1α was found to induce EMT, HCC cell migration, invasion and VM formation by regulating LOXL2. The results of microarray assays were analyzed. HIF-1α plays an important role in the development of HCC by promoting HCC metastasis, EMT and VM through up-regulating LOXL2. This study highlights the potential therapeutic value of targeting LOXL2 for suppression of HCC metastasis and progression.

The presence and expression of the HIF-1α in the respiratory intestine of the bronze Corydoras Corydoras aeneus (Callichthyidae Teleostei).

PubMed

Satora, Leszek; Mytych, Jennifer; Bilska-Kos, Anna

2018-05-23

Bronze corydoras (Corydoras aeneus) is a small diurnal activity fish from South America. Under hypoxia conditions, it uses the posterior part of the intestine as an accessory respiratory organ. The present PCR studies demonstrated higher expression of HIF-1α (hypoxia-inducible factor) gene in the respiratory than that in digestive part of bronze corydoras intestine. Further, immunolocalization studies using antibodies specific to HIF-1α and transmission electron microscopy (TEM) revealed the presence of HIF-1α epitopes in the intestine of Corydoras aeneus. In the respiratory intestine, the numerous clusters of gold particles visualizing HIF-1α antibody were observed within fibroblasts, whereas in the digestive tract of this species, single gold grains in the epithelial cells were noted. On the other hand, the presence of HIF-1α and the cytoplasmic domain of the epidermal growth factor receptor (EGFR) in the respiratory intestine of bronze corydoras assumes their interactions in the system where these factors appeared for the first time. The non-obligatory air-breathing fishes using their digestive tract as an accessory respiratory organ during hypoxia conditions are very interesting for the studies of the processes that control HIF-1α expression and squamous cell proliferation.

Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1α–mediated tumor apoptotic switch

PubMed Central

Magnon, Claire; Opolon, Paule; Ricard, Marcel; Connault, Elisabeth; Ardouin, Patrice; Galaup, Ariane; Métivier, Didier; Bidart, Jean-Michel; Germain, Stéphane; Perricaudet, Michel; Schlumberger, Martin

2007-01-01

Tumor radioresponsiveness depends on endothelial cell death, which leads in turn to tumor hypoxia. Radiation-induced hypoxia was recently shown to trigger tumor radioresistance by activating angiogenesis through hypoxia-inducible factor 1–regulated (HIF-1–regulated) cytokines. We show here that combining targeted radioiodide therapy with angiogenic inhibitors, such as canstatin, enhances direct tumor cell apoptosis, thereby overcoming radio-induced HIF-1–dependent tumor survival pathways in vitro and in vivo. We found that following dual therapy, HIF-1α increases the activity of the canstatin-induced αvβ5 signaling tumor apoptotic pathway and concomitantly abrogates mitotic checkpoint and tetraploidy triggered by radiation. Apoptosis in conjunction with mitotic catastrophe leads to lethal tumor damage. We discovered that HIF-1 displays a radiosensitizing activity that is highly dependent on treatment modalities by regulating key apoptotic molecular pathways. Our findings therefore support a crucial role for angiogenesis inhibitors in shifting the fate of radiation-induced HIF-1α activity from hypoxia-induced tumor radioresistance to hypoxia-induced tumor apoptosis. This study provides a basis for developing new biology-based clinically relevant strategies to improve the efficacy of radiation oncology, using HIF-1 as an ally for cancer therapy. PMID:17557121

Bacterial vaginosis-associated microflora isolated from the female genital tract activates HIV-1 expression.

PubMed

Al-Harthi, L; Roebuck, K A; Olinger, G G; Landay, A; Sha, B E; Hashemi, F B; Spear, G T

1999-07-01

Alteration of cervicovaginal microbial flora can lead to vaginosis, which is associated with an increased risk of HIV-1 transmission. We recently characterized a soluble HIV-inducing factor (HIF) from the cervicovaginal lavage (CVL) samples of women. The goals of this study were to determine the effect of cervicovaginal microflora on HIV-1 expression and to elucidate the relationship between HIF activity and microflora. Physiologically relevant microorganisms, Mycoplasma, diphtheroid-like bacteria, Gardnerella vaginalis, Streptococcus agalactiae, and Streptococcus constellatus, cultured from the CVL of a representative woman with a clinical condition of bacterial vaginosis and possessing HIF activity, induced HIV-1 expression. The magnitude of virus induction varied widely with the greatest stimulation induced by diphtheroid-like bacteria and Mycoplasma. The transcriptional induction by Mycoplasma was mediated by activation of the KB enhancer, an activation mechanism shared with HIF. Also as with HIF, Mycoplasma induced AP-1 dependent transcription. Polymerase chain reaction (PCR)-based speciation showed that the isolate was M. hominis. Our data indicate that bacterial vaginosis-associated microflora can enhance HIV-1 transcription and replication and identify M. hominis as a potential source for HIF activity. The virus-enhancing activities associated with the microflora and HIF may increase genital tract viral load, potentially contributing to HIV transmission.

Tetrahydrocurcumin induces mesenchymal-epithelial transition and suppresses angiogenesis by targeting HIF-1α and autophagy in human osteosarcoma

PubMed Central

Zhang, Yan; Liu, Ying; Zou, Jilong; Yan, Lixin; Du, Wei; Zhang, Yafeng; Sun, Hanliang; Lu, Peng; Geng, Shuo; Gu, Rui; Zhang, Hongyue; Bi, Zhenggang

2017-01-01

Human osteosarcoma is considered a malignant tumor with poor prognosis that readily metastasizes. Tetrahydrocurcumin (THC) has been reported to have anti-tumor activity in numerous tumors. In addition, hypoxia-inducible factor-1α (HIF-1α) has been demonstrated to be associated with tumor metastasis by regulating epithelial-mesenchymal transition (EMT). However, the role of THC in osteosarcoma remains uncertain. Therefore, this study aimed to elucidate the potential mechanisms. We found that THC significantly reduced the growth of osteosarcoma cells and suppressed migration and invasion, as tested in a nude mouse lung metastasis model. Additionally, the mesenchymal-epithelial transition (MET) process was facilitated by THC. Mechanistically, our study showed that HIF-1α had a pivotal role in the anti-metastatic effect of THC. Importantly, HIF-1α expression was downregulated by THC by inhibiting Akt/mTOR and p38 MAPK pathways. Moreover, THC exhibited a remarkable inhibitory effect on HIF-1α expression and angiogenesis under hypoxic conditions. Furthermore, THC activated autophagy and induced MET and suppressed angiogenesis in a HIF-1α-related manner. Taken together, our findings suggest that THC suppresses metastasis and invasion and this may be associated with HIF-1α and autophagy, which would potentially provide therapeutic strategies for human osteosarcoma. PMID:29207631

Hypoxia inducible factor-1 (HIF-1)–flavin containing monooxygenase-2 (FMO-2) signaling acts in silver nanoparticles and silver ion toxicity in the nematode, Caenorhabditis elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eom, Hyun-Jeong; Ahn, Jeong-Min; Kim, Younghun

2013-07-15

In the present study, nanotoxicity mechanism associated with silver nanoparticles (AgNPs) exposure was investigated on the nematode, Caenorhabditis elegans focusing on the hypoxia response pathway. In order to test whether AgNPs-induced hypoxia inducible factor-1 (HIF-1) activation was due to hypoxia or to oxidative stress, depletion of dissolved oxygen (DO) in the test media and a rescue effect using an antioxidant were investigated, respectively. The results suggested that oxidative stress was involved in activation of the HIF-1 pathway. We then investigated the toxicological implications of HIF-1 activation by examining the HIF-1 mediated transcriptional response. Of the genes tested, increased expression ofmore » the flavin containing monooxygenase-2 (FMO-2) gene was found to be the most significant as induced by AgNPs exposure. We found that AgNPs exposure induced FMO-2 activation in a HIF-1 and p38 MAPK PMK-1 dependent manner, and oxidative stress was involved in it. We conducted all experiments to include comparison of AgNPs and AgNO{sub 3} in order to evaluate whether any observed toxicity was due to dissolution or particle specific. The AgNPs and AgNO{sub 3} did not produce any qualitative differences in terms of exerting toxicity in the pathways observed in this study, however, considering equal amount of silver mass, in every endpoint tested the AgNPs were found to be more toxic than AgNO{sub 3}. These results suggest that Ag nanotoxicity is dependent not only on dissolution of Ag ion but also on particle specific effects and HIF-1–FMO-2 pathway seems to be involved in it. - Highlights: • HIF-1 signaling was investigated in C. elegans exposed to AgNPs and AgNO{sub 3}. • HIF-1 and PMK-1 were needed for AgNPs- and AgNO{sub 3}-induced fmo-2 gene expression. • PMK-1–HIF-1–FMO-2 pathway was dependent on oxidative stress. • AgNPs and AgNO{sub 3} did not produce any qualitative differences in HIF-1 signaling. • AgNPs were more toxic than an equal amount of silver mass contained in AgNO{sub 3}.« less

Identification of the Ah-Receptor Structural Determinants for Ligand Preferences

PubMed Central

Xing, Yongna

2012-01-01

The aryl hydrocarbon receptor (AHR) is a transcription factor that responds to diverse ligands and plays a critical role in toxicology, immune function, and cardiovascular physiology. The structural basis of the AHR for ligand promiscuity and preferences is critical for understanding AHR function. Based on the structure of a closely related protein HIF2α, we modeled the AHR ligand binding domain (LBD) bound to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo(a)pyrene (BaP) and identified residues that control ligand preferences by shape and H-bond potential. Mutations to these residues, particularly Q377 and G298, resulted in robust and opposite changes in the potency of TCDD and BaP and up to a 20-fold change in the ratio of TCDD/BaP efficacy. The model also revealed a flexible “belt” structure; molecular dynamic (MD) simulation suggested that the “belt” and several other structural elements in the AHR-LBD are more flexible than HIF2α and likely contribute to ligand promiscuity. Molecular docking of TCDD congeners to a model of human AHR-LBD ranks their binding affinity similar to experimental ranking of their toxicity. Our study reveals key structural basis for prediction of toxicity and understanding the AHR signaling through diverse ligands. PMID:22659362

Lenticular cytoprotection. Part 1: the role of hypoxia inducible factors-1α and -2α and vascular endothelial growth factor in lens epithelial cell survival in hypoxia.

PubMed

Neelam, Sudha; Brooks, Morgan M; Cammarata, Patrick R

2013-01-01

The prosurvival signaling cascades that mediate the unique ability of human lens epithelial cells to survive in their naturally hypoxic environment are not well defined. Hypoxia induces the synthesis of the hypoxia inducible factor HIF-1α that in turn, plays a crucial role in modulating a downstream survival scheme, where vascular endothelial growth factor (VEGF) also plays a major role. To date, no published reports in the lens literature attest to the expression and functionality of HIF-2α and the role it might play in regulating VEGF expression. The aim of this study was to identify the functional expression of the hypoxia inducible factors HIF-1α and HIF-2α and establish their role in regulating VEGF expression. Furthermore, we demonstrate a link between sustained VEGF expression and the ability of the hypoxic human lens epithelial cell to thrive in low oxygen conditions and resist mitochondrial membrane permeability transition (also referred to as lenticular cytoprotection). Hypoxia inducible factor translation inhibitors were used to demonstrate the role of HIF-1α and HIF-2α and the simultaneous expression of both hypoxic inducible factors to determine their role in regulating VEGF expression. Axitinib, which inhibits lenticular cell autophosphorylation of its VEGF receptor, was employed to demonstrate a role for the VEGF-VEGFR2 receptor complex in regulating Bcl-2 expression. Specific antisera and western blot analysis were used to detect the protein levels of HIF-1α and HIF-2α, as well as the proapoptotic protein, BAX and the prosurvival protein, Bcl-2. VEGF levels were analyzed with enzyme-linked immunosorbent assay (ELISA). The potentiometric dye, 5,5',6,6'-tetrachloro1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide, was used to determine the effect of the inhibitors on mitochondrial membrane permeability transition. Cultured human lens epithelial cells (HLE-B3) maintained under hypoxic condition (1% oxygen) displayed consistent accumulation of VEGF throughout the 72 h incubation period. Using hypoxia inducible factor translation inhibitors targeting HIF-1α or HIF-2α, the specific inhibition of each protein did not diminish VEGF synthesis. The combined inhibition of HIF-1α and HIF-2α expression, using a double hypoxia inducible factor translation inhibitor, markedly decreased the level of VEGF. The inhibition of VEGF synthesis was associated with a profound deficiency in the level of the prosurvival protein, Bcl-2. Axitinib also prevented the VEGF-mediated expression of Bcl-2. The loss of VEGF coupled with the decrease in intracellular Bcl-2 correlated with marked mitochondrial depolarization, an early predictor of cellular apoptosis. Our data support a model in which the sustained synthesis of VEGF in human lens epithelial cells, maintained under hypoxic condition, is regulated by a compensatory inter-relationship between HIF-1α and HIF-2α. VEGF acts as a prosurvival factor in hypoxic lens epithelial cells by maintaining consistent expression of the prosurvival protein Bcl-2, which likely prevents the translocation of cytosolic BAX to the outer mitochondrial membrane, thus preventing the initiation of mitochondrial depolarization.

Roles of p300 and cyclic adenosine monophosphate response element binding protein in high glucose-induced hypoxia-inducible factor 1α inactivation under hypoxic conditions.

PubMed

Ding, Lingtao; Yang, Minlie; Zhao, Tianlan; Lv, Guozhong

2017-05-01

Given the high prevalence of diabetes and burn injuries worldwide, it is essential to dissect the underlying mechanism of delayed burn wound healing in diabetes patients, especially the high glucose-induced hypoxia-inducible factor 1 (HIF-1)-mediated transcription defects. Human umbilical vein endothelial cells were cultured with low or high concentrations of glucose. HIF-1α-induced vascular endothelial growth factor (VEGF) transcription was measured by luciferase assay. Immunofluorescence staining was carried out to visualize cyclic adenosine monophosphate response element binding protein (CREB) localization. Immunoprecipitation was carried out to characterize the association between HIF-1α/p300/CREB. To test whether p300, CREB or p300+CREB co-overexpression was sufficient to rescue the HIF-1-mediated transcription defect after high glucose exposure, p300, CREB or p300+CREB co-overexpression were engineered, and VEGF expression was quantified. Finally, in vitro angiogenesis assay was carried out to test whether the high glucose-induced angiogenesis defect is rescuable by p300 and CREB co-overexpression. Chronic high glucose treatment resulted in impaired HIF-1-induced VEGF transcription and CREB exclusion from the nucleus. P300 or CREB overexpression alone cannot rescue high glucose-induced HIF-1α transcription defects. In contrast, co-overexpression of p300 and CREB dramatically ameliorated high glucose-induced impairment of HIF-1-mediated VEGF transcription, as well as in vitro angiogenesis. Finally, we showed that co-overexpression of p300 and CREB rectifies the dissociation of HIF-1α-p300-CREB protein complex in chronic high glucose-treated cells. Both p300 and CREB are required for the function integrity of HIF-1α transcription machinery and subsequent angiogenesis, suggesting future studies to improve burn wound healing might be directed to optimization of the interaction between p300, CREB and HIF-1α. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility.

PubMed

Preston, R S; Philp, A; Claessens, T; Gijezen, L; Dydensborg, A B; Dunlop, E A; Harper, K T; Brinkhuizen, T; Menko, F H; Davies, D M; Land, S C; Pause, A; Baar, K; van Steensel, M A M; Tee, A R

2011-03-10

Under conditions of reduced tissue oxygenation, hypoxia-inducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von Hippel-Lindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt-Hogg-Dubé syndrome. For this, we analysed a Birt-Hogg-Dubé patient-derived renal tumour cell line (UOK257) that is devoid of the Birt-Hogg-Dubé protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1α or HIF2α protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1α and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt-Hogg-Dubé patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the 'Warburg effect'). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt-Hogg-Dubé-associated renal lesions. © 2011 Macmillan Publishers Limited

Molecular Mechanism Underlying the Action of Substituted Pro-Gly Dipeptide Noopept.

PubMed

Vakhitova, Y V; Sadovnikov, S V; Borisevich, S S; Ostrovskaya, R U; A Gudasheva, T; Seredenin, S B

2016-01-01

This study was performed in order to reveal the effect of Noopept (ethyl ester of N-phenylacetyl-Lprolylglycine, GVS-111) on the DNA-binding activity of transcriptional factors (TF) in HEK293 cells transiently transfected with luciferase reporter constructs containing sequences for CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Noopept (10 μM) was shown to increase the DNA-binding activity of HIF-1 only, while lacking the ability to affect that of CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, and HSF1. Noopept provoked an additional increase in the DNA-binding activity of HIF-1 when applied in conditions of CoCl2-induced HIF- 1 stabilization. The degree of this HIF-positive effect of Noopept was shown to be concentration-dependent. Piracetam (1 mM) failed to affect significantly any of the TF under study. The results of molecular docking showed that Noopept (L-isomer), as well as its metabolite, L-isomer of N-phenyl-acetylprolyl, unlike its pharmacologically ineffective D-isomer, is able to bind to the active site of prolyl hydroxylase 2. Taking into account the important role of the genes activated by HIF-1 in the formation of an adaptive response to hypoxia, data on the ability of Noopept to provoke a selective increase in the DNA-binding activity of HIF-1 explain the wide spectrum of neurochemical and pharmacological effects of Noopept revealed before. The obtained data allow one to propose the HIF-positive effect as the primary mechanism of the activity of this Pro-Gly-containing dipeptide.

HIF-1α represses the expression of the angiogenesis inhibitor thrombospondin-2.

PubMed

MacLauchlan, Susan C; Calabro, Nicole E; Huang, Yan; Krishna, Meenakshi; Bancroft, Tara; Sharma, Tanuj; Yu, Jun; Sessa, William C; Giordano, Frank; Kyriakides, Themis R

2018-01-01

Thrombospondin-2 (TSP2) is a potent inhibitor of angiogenesis whose expression is dynamically regulated following injury. In the present study, it is shown that HIF-1α represses TSP2 transcription. Specifically, in vitro studies demonstrate that the prolyl hydroxylase inhibitor DMOG or hypoxia decrease TSP2 expression in fibroblasts. This effect is shown to be via a transcriptional mechanism as hypoxia does not alter TSP2 mRNA stability and this effect requires the TSP2 promoter. In addition, the documented repressive effect of nitric oxide (NO) on TSP2 is shown to be non-canonical and involves stabilization of hypoxia inducible factor-1a (HIF-1α). The regulation of TSP2 by hypoxia is supported by the in vivo observation that TSP2 has spatiotemporal expression distinct from regions of hypoxia in gastrocnemius muscle following murine hindlimb ischemia (HLI). A role for TSP2 regulation by HIF-1α is supported by the dysregulation of TSP2 expression in SM22α-cre HIF-1α KO mice following HLI. Indeed, there is a reduction in blood flow recovery in the SM22a-cre HIF-1α KO mice compared to littermate controls following HLI surgery, associated with impaired recovery and increased TSP2 levels. Moreover, SM22α-cre HIF-1α KO smooth muscle cells mice have increased TSP2 mRNA levels that persist in hypoxia. These findings identify a novel, ischemia-induced pro-angiogenic mechanism involving the transcriptional repression of TSP2 by HIF-1α. Copyright © 2017. Published by Elsevier B.V.

Hypoxia-inducible factor 1–mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions

PubMed Central

Zhang, Feng-Lin; Shen, Guo-Min; Liu, Xiao-Ling; Wang, Fang; Zhao, Ying-Ze; Zhang, Jun-Wu

2012-01-01

Abstract Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type–specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34+ haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl2 induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. PMID:22050843

Transcription factor HIF1A: downstream targets, associated pathways, polymorphic hypoxia response element (HRE) sites, and initiative for standardization of reporting in scientific literature.

PubMed

Slemc, Lucija; Kunej, Tanja

2016-11-01

Hypoxia-inducible factor-1α (HIF-1α) has crucial role in adapting cells to hypoxia through expression regulation of many genes. Identification of HIF-1α target genes (HIF-1α-TGs) is important for understanding the adapting mechanism. The aim of the present study was to collect known HIF-1α-TGs and identify their associated pathways. Targets and associated genomics data were retrieved using PubMed, WoS ( http://apps.webofknowledge.com/ ), HGNC ( http://www.genenames.org/ ), NCBI ( http://www.ncbi.nlm.nih.gov/ ), Ensemblv.84 ( http://www.ensembl.org/index.html ), DAVID Bioinformatics Resources ( https://david.ncifcrf.gov /), and Disease Ontology database ( http://disease-ontology.org/ ). From 51 papers, we collected 98 HIF-1α TGs found to be associated with 20 pathways, including metabolism of carbohydrates and pathways in cancer. Reanalysis of genomic coordinates of published HREs (hypoxia response elements) revealed six polymorphisms within HRE sites (HRE-SNPs): ABCG2, ACE, CA9, and CP. Due to large heterogeneity of results presentation in scientific literature, we also propose a first step towards reporting standardization of HIF-1α-target interactions consisting of ten relevant data types. Suggested minimal checklist for reporting will enable faster development of a complete catalog of HIF-1α-TGs, data sharing, bioinformatics analyses, and setting novel more targeted hypotheses. The proposed format for data standardization is not yet complete but presents a baseline for further optimization of the protocol with additional details, for example, regarding the experimental validation.

Serum 8-OHdG and HIF-1α levels: do they affect the development of malignancy in patients with hypoactive thyroid nodules?

PubMed

Ece, Harman; Mehmet, Erdogan; Cigir, Biray Avci; Yavuz, Dodurga; Muammer, Karadeniz; Cumhur, Gunduz; Mustafa, Harman; Sevki, Cetinkalp; Fusun, Saygılı; Gokhan, Ozgen Ahmet

2013-01-01

This study aimed to evaluate 8-OHdG and hypoxia-inducible factor 1 (HIF-1α) levels in patients with hypoactive thyroid nodules (toxic multi-nodular goiter, Graves' disease, and Hashimoto's thyroiditis), as these parameters may be related to oxidative stress and the pathogenesis of cancer. The study included patients diagnosed with Graves' disease (n = 20), toxic multinodular goiter (n = 20), and Hashimoto thyroiditis (n = 20), and 20 healthy controls. HIF-1α levels were measured in blood samples and 8-OHdG levels were measured in urine - both via ELISA. HIF-1α and 8-OHdG levels were significantly higher in the patient groups than in the control group (p < 0.05). In the Hashimoto's thyroiditis patients a correlation was observed between 8-OHdG and thyroglobulin antibodies (p = 0.03). A significant relation was found between 8-OHdG and HIF-1α in the patient group (p < 0.01). Carcinoma was detected in 7 of 43 female patients, but not in any of the male patients. No difference was observed in 8-OHdG or HIF-1α levels between the patients with and without papillary carcinoma (p > 0.05). There was no significant difference in 8-OHdG or HIF-1α levels between the patients with biopsy results that were benign, malignant, and non-diagnostic (p > 0.05). Serum HIF-1α and urine 8-OHdG levels were significantly higher in the patients with thyroid diseases; however, a relationship with cancer was not observed.

HIF-1 Alpha and Placental Growth Factor in Pregnancies Complicated With Preeclampsia: A Qualitative and Quantitative Analysis.

PubMed

Rath, Gayatri; Aggarwal, Ruby; Jawanjal, Poonam; Tripathi, Richa; Batra, Aruna

2016-01-01

The pathophysiology of preeclampsia is not clearly understood worldwide. Hypoxia inducible factor 1α (HIF-1α) is thought to be the preliminary factor for the hypoxic conditions prevailing in preeclampsia, which causes imbalance in the expression of angiogenic proteins. A proangiogenic protein, placental growth factor (PIGF), is reported to be dysregulated in preeclampsia. Therefore, this study focuses on the investigation of HIF-1α and PIGF in preeclamptic conditions and a possible molecular association between them. Placental tissue (n = 45 + 45) and serum samples (n = 80 + 80) of preeclamptic patients and healthy control were collected and processed for the analysis of HIF-1α and PIGF by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). In preeclamptic group, the significant nuclear and cytoplasmic expression of HIF-1α was noticed in syncytiotrophoblast (P = 0.0001) but in control placenta, it was localized to cytoplasm (P = 0.0001). The intensity of PIGF expression was lower in syncytiotrophoblast cytoplasm (P = 0.0001) in preeclamptic cases as compared with control. Also, the significant upregulated concentration of HIF-1α and downregulated PIGF was observed in serum samples of preeclamptic woman (P = 0.0001). Thus, there was a significant direct negative correlation between HIF-1α and PIGF both at tissue and serum level (P < 0.01). The direct inverse association between HIF-1α and PIGF in serum and placental tissues may be responsible for the low oxidative stress and endothelial dysfunction, leading to the pathogenesis of preeclampsia. © 2014 Wiley Periodicals, Inc.

Sevoflurane postconditioning improves myocardial mitochondrial respiratory function and reduces myocardial ischemia-reperfusion injury by up-regulating HIF-1

PubMed Central

Yang, Long; Xie, Peng; Wu, Jianjiang; Yu, Jin; Yu, Tian; Wang, Haiying; Wang, Jiang; Xia, Zhengyuan; Zheng, Hong

2016-01-01

Background: Sevoflurane postconditioning (SPostC) can exert myocardial protective effects similar to ischemic preconditioning. However, the exact myocardial protection mechanism by SPostC is unclear. Studies indicate that hypoxia-inducible factor-1 (HIF-1) maintains cellular respiration homeostasis by regulating mitochondrial respiratory chain enzyme activity under hypoxic conditions. This study investigated whether SPostC could regulate the expression of myocardial HIF-1α and to improve mitochondrial respiratory function, thereby relieving myocardial ischemia-reperfusion injury in rats. Methods: The myocardial ischemia-reperfusion rat model was established using the Langendorff isolated heart perfusion apparatus. Additionally, postconditioning was performed using sevoflurane alone or in combination with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). The changes in hemodynamic parameters, HIF-1α protein expression levels, mitochondrial respiratory function and enzyme activity, mitochondrial reactive oxygen species (ROS) production rates, and mitochondrial ultrastructure were measured or observed. Results: Compared to the ischemia-reperfusion (I/R) group, HIF-1α expression in the SPostC group was significantly up-regulated. Additionally, cardiac function indicators, mitochondrial state 3 respiratory rate, respiratory control ratio (RCR), cytochrome C oxidase (CcO), NADH oxidase (NADHO), and succinate oxidase (SUCO) activities, mitochondrial ROS production rate, and mitochondrial ultrastructure were significantly better than those in the I/R group. However, these advantages were completely reversed by the HIF-1α specific inhibitor 2ME2 (P<0.05). Conclusion: The myocardial protective function of SPostC might be associated with the improvement of mitochondrial respiratory function after up-regulation of HIF-1α expression. PMID:27830025

Sevoflurane postconditioning improves myocardial mitochondrial respiratory function and reduces myocardial ischemia-reperfusion injury by up-regulating HIF-1.

PubMed

Yang, Long; Xie, Peng; Wu, Jianjiang; Yu, Jin; Yu, Tian; Wang, Haiying; Wang, Jiang; Xia, Zhengyuan; Zheng, Hong

2016-01-01

Sevoflurane postconditioning (SPostC) can exert myocardial protective effects similar to ischemic preconditioning. However, the exact myocardial protection mechanism by SPostC is unclear. Studies indicate that hypoxia-inducible factor-1 (HIF-1) maintains cellular respiration homeostasis by regulating mitochondrial respiratory chain enzyme activity under hypoxic conditions. This study investigated whether SPostC could regulate the expression of myocardial HIF-1α and to improve mitochondrial respiratory function, thereby relieving myocardial ischemia-reperfusion injury in rats. The myocardial ischemia-reperfusion rat model was established using the Langendorff isolated heart perfusion apparatus. Additionally, postconditioning was performed using sevoflurane alone or in combination with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). The changes in hemodynamic parameters, HIF-1α protein expression levels, mitochondrial respiratory function and enzyme activity, mitochondrial reactive oxygen species (ROS) production rates, and mitochondrial ultrastructure were measured or observed. Compared to the ischemia-reperfusion (I/R) group, HIF-1α expression in the SPostC group was significantly up-regulated. Additionally, cardiac function indicators, mitochondrial state 3 respiratory rate, respiratory control ratio (RCR), cytochrome C oxidase (C c O), NADH oxidase (NADHO), and succinate oxidase (SUCO) activities, mitochondrial ROS production rate, and mitochondrial ultrastructure were significantly better than those in the I/R group. However, these advantages were completely reversed by the HIF-1α specific inhibitor 2ME2 ( P <0.05). The myocardial protective function of SPostC might be associated with the improvement of mitochondrial respiratory function after up-regulation of HIF-1α expression.

Ptpmt1 induced by HIF-2α regulates the proliferation and glucose metabolism in erythroleukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qin-Qin; Qinghai Provincial People's Hospital, Xining; Xiao, Feng-Jun

Hypoxia provokes metabolism misbalance, mitochondrial dysfunction and oxidative stress in both human and animal cells. However, the mechanisms which hypoxia causes mitochondrial dysfunction and energy metabolism misbalance still remain unclear. In this study, we presented evidence that mitochondrial phosphatase Ptpmt1 is a hypoxia response molecule that regulates cell proliferation, survival and glucose metabolism in human erythroleukemia TF-1 cells. Exposure to hypoxia or DFO treatment results in upregulation of HIF1-α, HIF-2α and Ptpmt1. Only inhibition of HIF-2α by shRNA transduction reduces Ptpmt1 expression in TF-1 cells under hypoxia. Ptpmt1 inhibitor suppresses the growth and induces apoptosis of TF-1 cells. Furthermore, we demonstrated that Ptpmt1more » inhibition reduces the Glut1 and Glut3 expression and decreases the glucose consumption in TF-1 cells. In additional, Ptpmt1 knockdown also results in the mitochondrial dysfunction determined by JC1 staining. These results delineate a key role for HIF-2α-induced Ptpmt1 upregulation in proliferation, survival and glucose metabolism of erythroleukemia cells. It is indicated that Ptpmt1 plays important roles in hypoxia-induced cell metabolism and mitochondrial dysfunction. - Highlights: • Hypoxia induces upregulation of HIF-1α, HIF-2α and Ptpmt1; HIF-2a induces Ptpmt1 upregulation in TF-1 cells. • PTPMT-1 inhibition reduces growth and induces apoptosis of TF-1 cells. • PTPMT1 inhibition downregulates Glut-1, Glut-3 expression and reduces glucose consumption.« less

Evidence for the Slow Reaction of Hypoxia-Inducible Factor Prolyl Hydroxylase 2 with Oxygen

PubMed Central

Flashman, Emily; Hoffart, Lee M.; Hamed, Refaat B.; Bollinger, J. Martin; Krebs, Carsten; Schofield, Christopher J.

2010-01-01

SUMMARY The response of animals to hypoxia is mediated by the hypoxia-inducible transcription factor (HIF). Human HIF is regulated by four Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases: Prolyl hydroxylase domain enzymes (PHDs or EGLNs) 1–3 catalyse hydroxylation of two prolyl-residues in HIF, triggering its degradation by the proteasome. Factor inhibiting HIF (FIH) catalyses hydroxylation of an asparagine-residue in HIF, inhibiting its transcriptional activity. Collectively, the HIF hydroxylases negatively regulate HIF in response to increasing oxygen concentration. Prolyl hydroxylase domain 2 (PHD2) is the most important oxygen sensor in human cells; however the underlying kinetic basis of the oxygen sensing function of PHD2 is unclear. We report analyses of the reaction of PHD2 with oxygen. Chemical quench/mass spectrometry experiments showed that reaction of a complex of PHD2, Fe(II), 2OG and the C-terminal oxygen-dependent degradation domain of HIF-α (CODD) with oxygen to form hydroxylated CODD and succinate is much slower (~100 fold) than for other similarly studied 2OG oxygenases. Stopped flow/UV-visible spectroscopy experiments showed that the reaction produces a relatively stable species absorbing at 320nm; Mössbauer spectroscopic experiments implied that this species is likely not a Fe(IV)=O intermediate, as observed for other 2OG oxygenases. Overall the results suggest that, at least compared to other studied 2OG oxygenases, PHD2 reacts relatively slowly with oxygen, a property that may be associated with its function as an oxygen sensor. PMID:20840591

[HIF-2α/Notch3 pathway mediates CoCl2-induced migration and invasion in human breast cancer MCF-7 cells].

PubMed

Wang, Jian-Guo; Yuan, Lei

2016-12-25

The aim of this study is to investigate the effects of hypoxia inducible factor-2α (HIF-2α) and Notch3 on CoCl 2 -induced migration and invasion of human breast cancer cell line MCF-7. MCF-7 cells were exposed to normoxia (21% O 2 ) or chemical hypoxia (21% O 2 plus CoCl 2 ). Short hairpin RNA (shRNA) was used to knock down HIF-2α and Notch3 in MCF-7 cells. The mRNA expression levels of HIF-2α, Notch3 and Hey1 were measured by RT-PCR. Western blot was performed to determine the protein expression levels of HIF-2α, Notch3, Hey1, Snail and E-cadherin. CoCl 2 treatment resulted in higher protein expression levels of HIF-2α, Notch3, Hey1, Snail (P < 0.05) and lower levels of E-cadherin (P < 0.05), and promoted migration and invasion of MCF-7 cells (P < 0.05). shRNA-HIF-2α suppressed CoCl 2 -induced mRNA expression of Notch3 and Hey1. Notch3 knockdown down-regulated Snail and up-regulated E-cadherin at protein level under simulated hypoxia (P < 0.05), and inhibited CoCl 2 -induced migration and invasion of MCF-7 cells (P < 0.05). In conclusion, our data provide evidence that HIF-2α may promote the migration and invasion of MCF-7 cells under chemical hypoxic conditions by potentiating Notch3 pathway.

Xenon Preconditioning Protects against Renal Ischemic-Reperfusion Injury via HIF-1α Activation

PubMed Central

Ma, Daqing; Lim, Ta; Xu, Jing; Tang, Haidy; Wan, Yanjie; Zhao, Hailin; Hossain, Mahmuda; Maxwell, Patrick H.; Maze, Mervyn

2009-01-01

The mortality rate from acute kidney injury after major cardiovascular operations can be as high as 60%, and no therapies have been proved to prevent acute kidney injury in this setting. Here, we show that preconditioning with the anesthetic gas xenon activates hypoxia-inducible factor 1α (HIF-1α) and its downstream effectors erythropoietin and vascular endothelial growth factor in a time-dependent manner in the kidneys of adult mice. Xenon increased the efficiency of HIF-1α translation via modulation of the mammalian target of rapamycin pathway. In a model of renal ischemia-reperfusion injury, xenon provided morphologic and functional renoprotection; hydrodynamic injection of HIF-1α small interfering RNA demonstrated that this protection is HIF-1α dependent. These results suggest that xenon preconditioning is a natural inducer of HIF-1α and that administration of xenon before renal ischemia can prevent acute renal failure. If these data are confirmed in the clinical setting, then preconditioning with xenon may be beneficial before procedures that temporarily interrupt renal perfusion. PMID:19144758

Hypoxia-Dependent Modification of Collagen Networks Promotes Sarcoma Metastasis

PubMed Central

Eisinger-Mathason, T.S. Karin; Zhang, Minsi; Qiu, Qiong; Skuli, Nicolas; Nakazawa, Michael S.; Karakasheva, Tatiana; Mucaj, Vera; Shay, Jessica E.S.; Stangenberg, Lars; Sadri, Navid; Puré, Ellen; Yoon, Sam S.; Kirsch, David G.; Simon, M. Celeste

2013-01-01

Intratumoral hypoxia and expression of Hypoxia Inducible Factor 1α (HIF1α) correlate with metastasis and poor survival in sarcoma patients. We demonstrate here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF1α enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF1α or PLOD2 expression disrupts collagen modification, cell migration and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSLKrasG12D/+; Trp53fl/fl murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF1α-deficient tumors, and analysis of human sarcomas reveal elevated HIF1α and PLOD2 expression in metastatic primary lesions. Pharmacological inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF1α controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination. PMID:23906982

Cooperation of HIF- and NCAM-mediated mechanisms in cell viability of hippocampal cultures after oxygen-glucose deprivation.

PubMed

Lushnikova, Iryna; Nikandrova, Yelyzaveta; Skibo, Galyna

2017-10-01

Neurodegenerative diseases of different genesis are the result of cellular damages including those caused by oxygen and glucose deficit. Neuronal survival or death in brain pathologies depends on a variety of interrelated molecular mechanisms. A key role in modulation of neuron viability belongs to HIF (hypoxia-inducible factor) and NCAM (neural cell adhesion molecules) signaling pathways. In this work, we used organotypic and dissociated hippocampal cultures to analyze cell viability and HIF-1α immunopositive (HIF-1α + ) signal after 30 min oxygen-glucose deprivation (OGD) followed by 24 h of reoxygenation in the presence of FGL (synthetic NCAM-derived mimetic peptide). According to LDH- and MTS-assay of cell viability, FGL showed a neuroprotective effect, which was attributed to the association with FGFR. We showed that these effects correlated with changes of the HIF-1α + level suggesting the communications of HIF and NCAM signaling pathways. These data extend our knowledge of neurodegeneration mechanisms and open additional potential for the development of neuroprotection strategies. © 2017 International Federation for Cell Biology.

Septin 9 isoform 1 (SEPT9_i1) specifically interacts with importin-α7 to drive hypoxia-inducible factor (HIF)-1α nuclear translocation.

PubMed

Tazat, Keren; Schindler, Susanne; Deppeing, Reinhard; Mabjeesh, Nicola J

2018-05-09

We have shown previously that septin 9 isoform 1 (SEPT9_i1) protein associates with hypoxia-inducible factor (HIF)-1α to augment HIF-1 transcriptional activity by driving its importin-α-mediated nuclear translocation. Using in vitro and in vivo binding assays we identified that HIF-1α interacts with importin-α5 and importin-α7 in prostate cancer cells but only importin-α7 interacts with SEPT9_i1. The interaction with importin-α7 was dependent on the first 25 amino acids of SEPT9_i1 that are unique compared to other members of the mammalian septin family. Depletion of endogenous importin-α7 reduced nuclear HIF-1α levels in the nucleus. Our results provide evidence that there are importin-α specificities in the cytosolic/nuclear translocation process of HIF-1α protein, which may act differently under certain pathophysiological circumstances where SEPT9_i1 is overexpressed. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

HIF Activation Against CVD in CKD: Novel Treatment Opportunities.

PubMed

Tanaka, Tetsuhiro; Eckardt, Kai-Uwe

2018-05-01

Cardiovascular disease is a common and serious complication in patients with chronic kidney disease (CKD). One of the fundamental functions of the cardiovascular system is oxygen delivery, therefore cardiovascular disease inherently is linked to insufficient tissue oxygenation. Advances in our knowledge of cellular oxygen sensing by a family of prolyl hydroxylases (PHDs) and their role in regulating hypoxia-inducible factors (HIFs) have led to the discovery of PHD inhibitors as HIF stabilizers. Several small-molecule PHD inhibitors are currently in clinical trials for the treatment of anemia in CKD. An additional advantage of PHD inhibition may be found in the potential impact on cardiovascular consequences associated with CKD. Several preclinical studies have suggested a potential benefit of HIF activation in myocardial infarction, cardiac remodeling, atherosclerosis, and peripheral artery disease. Ameliorating glucose and lipid metabolism and lowering blood pressure may also contribute to cardiovascular protection. On the other hand, the broad spectrum of HIF-dependent functions also may include unwanted side effects. Clinical application of PHD inhibitors therefore necessitates careful evaluation of the net systemic effect of HIF activation. Copyright © 2018 Elsevier Inc. All rights reserved.

Myeloid hypoxia-inducible factor 1α prevents airway allergy in mice through macrophage-mediated immunoregulation.

PubMed

Toussaint, M; Fievez, L; Drion, P-V; Cataldo, D; Bureau, F; Lekeux, P; Desmet, C J

2013-05-01

Hypoxia-inducible factor (HIF) has important roles in promoting pro-inflammatory and bactericidal functions in myeloid cells. Conditional genetic ablation of its major subunit Hif1α in the myeloid lineage consequently results in decreased inflammatory responses in classical models of acute inflammation in mice. By contrast, we report here that mice conditionally deficient for Hif1α in myeloid cells display enhanced sensitivity to the development of airway allergy to experimental allergens and house-dust mite antigens. We support that upon allergen exposure, MyD88-dependent upregulation of Hif1α boosts the expression of the immunosuppressive cytokine interleukin (IL)-10 by lung interstitial macrophages (IMs). Hif1α-dependent IL-10 secretion is required for IMs to block allergen-induced dendritic cell activation and consequently for preventing the development of allergen-specific T-helper cell responses upon allergen exposure. Thus, this study supports that, in addition to its known pro-inflammatory activities, myeloid Hif1α possesses immunoregulatory functions implicated in the prevention of airway allergy.

Resting metabolic rate and heat increment of feeding in juvenile South American fur seals (Arctocephalus australis).

PubMed

Dassis, M; Rodríguez, D H; Ieno, E N; Denuncio, P E; Loureiro, J; Davis, R W

2014-02-01

Bio-energetic models used to characterize an animal's energy budget require the accurate estimate of different variables such as the resting metabolic rate (RMR) and the heat increment of feeding (HIF). In this study, we estimated the in air RMR of wild juvenile South American fur seals (SAFS; Arctocephalus australis) temporarily held in captivity by measuring oxygen consumption while at rest in a postabsorptive condition. HIF, which is an increase in metabolic rate associated with digestion, assimilation and nutrient interconversion, was estimated as the difference in resting metabolic rate between the postabsorptive condition and the first 3.5h postprandial. As data were hierarchically structured, linear mixed effect models were used to compare RMR measures under both physiological conditions. Results indicated a significant increase (61%) for the postprandial RMR compared to the postabsorptive condition, estimated at 17.93±1.84 and 11.15±1.91mL O2 min(-1)kg(-1), respectively. These values constitute the first estimation of RMR and HIF in this species, and should be considered in the energy budgets for juvenile SAFS foraging at-sea. Copyright © 2013 Elsevier Inc. All rights reserved.

[Current strategies in the treatment of renal-cell cancer: targeted therapies].

PubMed

Trigo, José Manuel; Bellmunt, Joaquim

2008-03-22

Renal-cell carcinoma represents 95% of all renal tumours. The Von Hippel-Lindau (VHL) tumor-suppressor gene is mutated or silenced in most clear cell renal carcinomas. pVHL loss results in the stabilization of the heterodimeric transcription factor hypoxia-inducible factor (HIF) and enhanced transactivation of HIF target genes. HIF itself has been difficult to inhibit with drug-like molecules although a number of agents that indirectly inhibit HIF, including mTOR (mammalian target of rapamycin) inhibitors, have been identified. Moreover, a number of drugs have been developed that target HIF-responsive gene products, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), implicated in tumor angiogenesis. Many of these targeted therapies, especially sunitinib, have demonstrated significant activity in kidney cancer clinical trials and represent a substantive advance in the treatment of this disease.

Metabolic Adaptation to Chronic Inhibition of Mitochondrial Protein Synthesis in Acute Myeloid Leukemia Cells

PubMed Central

Jhas, Bozhena; Sriskanthadevan, Shrivani; Skrtic, Marko; Sukhai, Mahadeo A.; Voisin, Veronique; Jitkova, Yulia; Gronda, Marcela; Hurren, Rose; Laister, Rob C.; Bader, Gary D.; Minden, Mark D.; Schimmer, Aaron D.

2013-01-01

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG) was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1β transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress. PMID:23520503

Metabolic adaptation to chronic inhibition of mitochondrial protein synthesis in acute myeloid leukemia cells.

PubMed

Jhas, Bozhena; Sriskanthadevan, Shrivani; Skrtic, Marko; Sukhai, Mahadeo A; Voisin, Veronique; Jitkova, Yulia; Gronda, Marcela; Hurren, Rose; Laister, Rob C; Bader, Gary D; Minden, Mark D; Schimmer, Aaron D

2013-01-01

Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG) was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1β transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress.

A Compact High-Brightness Heavy-Ion Injector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Westenskow, G A; Grote, D P; Halaxa, E

2005-05-11

To provide a compact high-brightness heavy-ion beam source for Heavy Ion Fusion (HIF) accelerators, we have been experimenting with merging multi-beamlets in an injector which uses an RF plasma source. In an 80-kV 20-microsecond experiment, the RF plasma source has produced up to 5 mA of Ar{sup +} in a single beamlet. An extraction current density of 100 mA/cm{sup 2} was achieved, and the thermal temperature of the ions was below 1 eV. We have tested at full voltage gradient the first 4 gaps of an injector design. Einzel lens were used to focus the beamlets while reducing the beamletmore » to beamlet space charge interaction. We were able to reach greater than 100 kV/cm in the first four gaps. We also performed experiments on a converging 119 multi-beamlet source. Although the source has the same optics as a full 1.6 MV injector system, these test were carried out at 400 kV due to the test stand HV limit. We have measured the beam's emittance after the beamlets are merged and passed through an electrostatic quadrupole (ESQ). Our goal is to confirm the emittance growth and to demonstrate the technical feasibility of building a driver-scale HIF injector.« less

Overexpression of hypoxia-inducible factor-1 alpha improves vasculogenesis-related functions of endothelial progenitor cells.

PubMed

Kütscher, Christian; Lampert, Florian M; Kunze, Mirjam; Markfeld-Erol, Filiz; Stark, G Björn; Finkenzeller, Günter

2016-05-01

Postnatal vasculogenesis is mediated by mobilization of endothelial progenitor cells (EPCs) from bone marrow and homing to ischemic tissues. This feature emphasizes this cell type for cell-based therapies aiming at the improvement of neovascularization in tissue engineering applications and regenerative medicine. In animal models, it was demonstrated that implantation of EPCs from cord blood (cbEPCs) led to the formation of a complex functional neovasculature, whereas EPCs isolated from adult peripheral blood (pbEPCs) showed a limited vasculogenic potential, which may be attributed to age-related dysfunction. Recently, it was demonstrated that activation of hypoxia-inducible factor-1α (Hif-1α) improves cell functions of progenitor cells of mesenchymal and endothelial origin. Thus, we hypothesized that overexpression of Hif-1α may improve the vasculogenesis-related phenotype of pbEPCs. In the present study, we overexpressed Hif-1α in pbEPCs and cbEPCs by using recombinant adenoviruses and investigated effects on stem cell- and vasculogenesis-related cell parameters. Overexpression of Hif-1α enhanced proliferation, invasion, cell survival and in vitro capillary sprout formation of both EPC populations. Migration was increased in cbEPCs upon Hif-1α overexpression, but not in pbEPCs. Cellular senescence was decreased in pbEPCs, while remained in cbEPCs, which showed, as expected, intrinsically a dramatically lower senescent phenotype in relation to pbEPCs. Similarly, the colony-formation capacity was much higher in cbEPCs in comparison to pbEPCs and was further increased by Hif-1α overexpression, whereas Hif-1α transduction exerted no significant influence on colony formation of pbEPCs. In summary, our experiments illustrated multifarious effects of Hif-1α overexpression on stem cell and vasculogenic parameters. Therefore, Hif-1α overexpression may represent a therapeutic option to improve cellular functions of adult as well as postnatal EPCs. Copyright © 2016. Published by Elsevier Inc.

Immunohistochemical detection of HIF-1alpha and CAIX in advanced head-and-neck cancer. Prognostic role and correlation with tumor markers and tumor oxygenation parameters.

PubMed

Kappler, Matthias; Taubert, Helge; Holzhausen, Hans-Jürgen; Reddemann, Rolf; Rot, Swetlann; Becker, Axel; Kuhnt, Thomas; Dellas, Kathrin; Dunst, Jürgen; Vordermark, Dirk; Hänsgen, Gabriele; Bache, Matthias

2008-08-01

Tumor hypoxia has an impact on the outcome of cancer patients treated with radiotherapy. The validity of endogenous markers such as hypoxia-inducible factor-1alpha (HIF-1alpha) and carbonic anhydrase isozyme IX (CAIX) to detect therapeutically relevant Levels of hypoxia within tumors is controversially discussed. Furthermore, the association of these hypoxia markers with tumor markers or tumor oxygenation parameters is of importance for understanding the relationship between the different factors. Tumortissue sections of 34 patients with advanced head-and-neck cancertreated with radio(chemo)therapy were assessed by immunohistochemistry for the expression of HIF-1alpha and CAIX. The relationships of both markers with tumor oxygenation parameters, molecular factors like P53, OPN, VEGF, VHL, survivin, and Ki67 levels, and clinical parameters were studied. Bivariate analysis showed a significant correlation of HIF-1alpha expression with high P53 and high OPN expression, high serum VEGF Levels, and low VHL and low Ki67 expression. The CAIX expression was inversely correlated with pH value and directly correlated with T-stage. However, no correlation was found between HIF-1alpha and CAIX expression. Neither in a univariate Cox proportional hazard regression nor in a Kaplan-Meier analysis did expression of HIF-1alpha or CAIX have a significant impact on clinical outcome. However, in a Kaplan-Meier analysis, the combination of both factors showed that patients with intratumoral overexpression of either HIF-1alpha or CAIX or both markers died on average 2 years earlier than patients whose tumors had low expression of both factors (p < 0.05). Expression of HIF-1alpha and CAIX was correlated with different tumor parameters. Only combined HIF-1alpha and CAIX expression was significantly predictive of patients' overall survival.

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas

Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers includingmore » HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.« less

Chondrogenic differentiation of growth factor-stimulated precursor cells in cartilage repair tissue is associated with increased HIF-1alpha activity.

PubMed

Gelse, K; Mühle, C; Knaup, K; Swoboda, B; Wiesener, M; Hennig, F; Olk, A; Schneider, H

2008-12-01

To investigate the chondrogenic potential of growth factor-stimulated periosteal cells with respect to the activity of Hypoxia-inducible Factor 1alpha (HIF-1alpha). Scaffold-bound autologous periosteal cells, which had been activated by Insulin-like Growth Factor 1 (IGF-1) or Bone Morphogenetic Protein 2 (BMP-2) gene transfer using both adeno-associated virus (AAV) and adenoviral (Ad) vectors, were applied to chondral lesions in the knee joints of miniature pigs. Six weeks after transplantation, the repair tissues were investigated for collagen type I and type II content as well as for HIF-1alpha expression. The functional role of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling on BMP-2/IGF-1-induced HIF-1alpha expression was assessed in vitro by employing specific inhibitors. Unstimulated periosteal cells formed a fibrous extracellular matrix in the superficial zone and a fibrocartilaginous matrix in deep zones of the repair tissue. This zonal difference was reflected by the absence of HIF-1alpha staining in superficial areas, but moderate HIF-1alpha expression in deep zones. In contrast, Ad/AAVBMP-2-stimulated periosteal cells, and to a lesser degree Ad/AAVIGF-1-infected cells, adopted a chondrocyte-like phenotype with strong intracellular HIF-1alpha staining throughout all zones of the repair tissue and formed a hyaline-like matrix. In vitro, BMP-2 and IGF-1 supplementation increased HIF-1alpha protein levels in periosteal cells, which was based on posttranscriptional mechanisms rather than de novo mRNA synthesis, involving predominantly the MEK/ERK pathway. This pilot experimental study on a relatively small number of animals indicated that chondrogenesis by precursor cells is facilitated in deeper hypoxic zones of cartilage repair tissue and is stimulated by growth factors which enhance HIF-1alpha activity.

N-Acetylcysteine and Allopurinol Confer Synergy in Attenuating Myocardial Ischemia Injury via Restoring HIF-1α/HO-1 Signaling in Diabetic Rats

PubMed Central

Mao, Xiaowen; Wang, Tingting; Liu, Yanan; Irwin, Michael G.; Ou, Jing-song; Liao, Xiao-long; Gao, Xia; Xu, Yuan; Ng, Kwok F. J.; Vanhoutte, Paul M.; Xia, Zhengyuan

2013-01-01

Objectives To determine whether or not the antioxidants N-acetylcysteine (NAC) and allopurinol (ALP) confer synergistic cardioprotection against myocardial ischemia/reperfusion (MI/R) injury by stabilizing hypoxia inducible factor 1α (HIF-1α)/heme oxygenase 1 (HO-1) signaling in diabetic myocardium. Methods Control or diabetic [streptozotocin (STZ)-induced] Sprague Dawley rats received vehicle or NAC, ALP or their combination for four weeks starting one week after STZ injection. The animals were then subjected to thirty minutes of coronary artery occlusion followed by two hours reperfusion in the absence or presence of the selective HO-1 inhibitor, tin protoporphyrin-IX (SnPP-IX) or the HIF-1α inhibitor 2-Methoxyestradiol (2ME2). Cardiomyocytes exposed to high glucose were subjected to hypoxia/re-oxygenation in the presence or absence of HIF-1α and HO-1 achieved by gene knock-down with related siRNAs. Results Myocardial and plasma levels of 15-F2t-isoprostane, an index of oxidative stress, were significantly increased in diabetic rats while cardiac HO-1 protein and activity were reduced; this was accompanied with reduced cardiac protein levels of HIF-1α, and increased post-ischemic myocardial infarct size and cellular injury. NAC and ALP given alone and in particular their combination normalized cardiac levels of HO-1 and HIF-1α protein expression and prevented the increase in 15-F2t-isoprostane, resulting in significantly attenuated post-ischemic myocardial infarction. NAC and ALP also attenuated high glucose-induced post-hypoxic cardiomyocyte death in vitro. However, all the above protective effects of NAC and ALP were cancelled either by inhibition of HO-1 or HIF-1α with SnPP-IX and 2ME2 in vivo or by HO-1 or HIF-1α gene knock-down in vitro. Conclusion NAC and ALP confer synergistic cardioprotection in diabetes via restoration of cardiac HIF-1α and HO-1 signaling. PMID:23874823

The association of genetic polymorphisms of hypoxia inducible factor-1 alpha and vascular endothelial growth factor with increased risk of chronic obstructive pulmonary disease: A case-control study.

PubMed

Yu, Zhen-Gang; Wang, Bing-Zhe; Cheng, Zhao-Zhong

2017-09-01

Accumulated data over the years have suggested that hypoxia inducible factor-1 alpha (HIF-1α) and its downstream vascular endothelial growth factor (VEGF) gene may be linked with chronic obstructive pulmonary disease (COPD). This study aims to investigate the association of HIF-1α and VEGF genetic polymorphisms and their correlated risks with COPD. COPD patients (case group) and healthy individuals (control group) were recruited. DNA was extracted to detect HIF-1α and VEGF genetic polymorphisms. Basal lung volume and forced expiratory capacity in 1st second (FEV1)/forced vital capacity (FVC) and FEV 1 /predicted value (pred)% were calculated. Genotype and allele distributions in HIF-1α and VEGF genes were analyzed. Kaplan-Meier curves and logistic regression model were used for analysis of survival and COPD risk factors. Haplotypes for HIF-1α rs11549465 and rs11549467 were analyzed. FEV 1 /FVC and FEV1/pred% in the case group were lower than the control group. Frequencies of HIF-1α rs11549465 CT + TT genotype and T allele, and rs11549467 GA + AA genotype and A allele were higher in the case group than the control group. Patients with rs11549465 CT + TT had higher COPD risk than those with the CC genotype. Patients with rs11549467 GA + AA showed higher COPD risk and lower FEV 1 /FVC and FEV 1 /pred% than those with the GG genotype. Patients with HIF-1α TA haplotype showed higher COPD risk than those with the CG haplotype. Survival rate of patients with HIF-1α rs11549467 GG genotype was higher than those with the GA + AA genotype. HIF-1α rs11549467 polymorphism may be associated with COPD risk. Copyright © 2017. Published by Elsevier Taiwan.

Hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat (FG-4592) protects against cisplatin-induced acute kidney injury.

PubMed

Yang, Yunwen; Yu, Xiaowen; Zhang, Yue; Ding, Guixia; Zhu, Chunhua; Huang, Songming; Jia, Zhanjun; Zhang, Aihua

2018-04-16

Renal hypoxia occurs in acute kidney injury (AKI) of various etiologies. Activation of hypoxia-inducible transcription factor (HIF) has been identified as an important mechanism of cellular adaptation to low oxygen. Preconditional HIF activation protects against AKI, suggesting a new approach in AKI treatment. HIF is degraded under normoxic conditions mediated by oxygen-dependent hydroxylation of specific prolyl residues of the regulative α-subunits by HIF prolyl hydroxylases (PHD). FG-4592 is a novel, orally active, small-molecule HIF PHD inhibitor for the treatment of anemia in patients with chronic kidney disease (CKD). The current study aimed to evaluate the effect of FG-4592 (Roxadustat) on cis -diamminedichloroplatinum (cisplatin)-induced kidney injury. In mice, pretreatment with FG-4592 markedly ameliorated cisplatin-induced kidney injury as shown by the improved renal function (blood urea nitrogen (BUN), serum creatinine (Scr), and cystatin C) and kidney morphology (periodic acid-Schiff (PAS) staining) in line with a robust blockade of renal tubular injury markers of kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Meanwhile, the renal apoptosis and inflammation induced by cisplatin were also strikingly attenuated in FG-4592-treated mice. Along with the protective effects shown above, FG-4592 pretreatment strongly enhanced HIF-1α in tubular cells, as well as the expressions of HIF target genes. FG-4592 alone did not affect the renal function and morphology in mice. In vitro , FG-4592 treatment significantly up-regulated HIF-1α and protected the tubular cells against cisplatin-induced apoptosis. In summary, FG-4592 treatment remarkably ameliorated the cisplatin-induced kidney injury possibly through the stabilization of HIF. Thus, besides the role in treating CKD anemia, the clinical use of FG-4592 also could be extended to AKI. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The Role of Factor Inhibiting HIF (FIH-1) in Inhibiting HIF-1 Transcriptional Activity in Glioblastoma Multiforme

PubMed Central

Liu, Fengming; Wu, Gang; Schroeder, Mark A.; Lau, Julie S.; Mukhopadhyay, Debabrata; Jiang, Shi-Wen; O'Neill, Brian Patrick; Datta, Kaustubh; Li, Jinping

2014-01-01

Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the Hypoxia-Inducible-Factor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that Factor Inhibiting HIF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis. PMID:24465898

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE PAGES

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini; ...

2016-10-26

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

Alterations of hypoxia-inducible factor-1 alpha in the hippocampus of mice acutely and repeatedly exposed to hypoxia.

PubMed

Shao, Guo; Gao, Cui-Ying; Lu, Guo-Wei

2005-01-01

This work aims at investigating the effects of hypoxic preconditioning on hypoxia-inducible factor-1 alpha (HIF-1alpha) expression in the hippocampus of mice during acute and repeated hypoxic exposures. The mice were randomly divided into three groups and exposed, respectively, to hypoxia for 4 runs (group H4), 1 run (group H1), and 0 run (group H0). Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation were used to examine the HIF-1alpha responses in the mouse hippocampus following exposure to hypoxia. The tolerance of mice to hypoxia increased significantly following acute and repetitive exposure to autoprogressive hypoxia. Total mRNA, total protein, and nuclear protein were extracted from the hippocampus for RT-PCR, Western blot, and EMSA, respectively. The HIF-1alpha mRNA levels were found to be increased in group H1 and decreased in group H4. The HIF-1alpha protein levels and HIF-1 DNA-binding activities were increased in group H1 and markedly increased in group H4. One of the HIF-1 target genes, vascular endothelial growth factor, increased in group H4. HIF-1 activation is thought to be involved in the protection of the brain of hypoxic preconditioned mice. Copyright 2005 S. Karger AG, Basel

Analysis of Haemophilus influenzae serotype f isolated from three Japanese children with invasive H. influenzae infection.

PubMed

Hoshino, Tadashi; Hachisu, Yushi; Kikuchi, Takashi; Tokutake, Shoko; Okui, Hideyuki; Kutsuna, Satoru; Fukasawa, Chie; Murayama, Kei; Oohara, Asami; Shimizu, Hiroyuki; Ito, Midori; Takahashi, Yoshiko; Ishiwada, Naruhiko

2015-04-01

In Japan, publicly subsidized Haemophilus influenzae serotype b vaccines became available in 2011; consequently, the incidence of invasive H. influenzae infection in paediatric patients of less than 5 years of age decreased dramatically. In 2013, the first case of H. influenzae serotype f (Hif) meningitis in a Japanese infant was reported, and another case of Hif meningitis in a Japanese infant was observed in 2013. We experienced a fatal paediatric case of Hif bacteraemia in 2004; therefore, we conducted an analysis of the three Hif strains isolated from these three Japanese children with invasive Hif infections. All three strains were β-lactamase-non-producing, ampicillin-sensitive strains, with MICs of 1 µg ml(-1) or less. However, one of the three strains showed slightly elevated MICs for ampicillin (1 µg ml(-1)), cefotaxime (0.25 µg ml(-1)) and meropenem (0.13 µg ml(-1)). A molecular analysis by multilocus sequence typing identified all three strains as sequence type (ST) 124, which is a predominant invasive Hif strain in many countries. SmaI-digested PFGE showed variable DNA fragmentation patterns among the strains, suggesting that some highly virulent strains have originated from a single ST124 clone and caused invasive Hif infections in Japan. Additional studies are needed to determine the factors that have led to the clonal expansion of virulent ST124 strains. © 2015 The Authors.

Hypoxia-Inducible Factor 1-Regulated Lysyl Oxidase Is Involved in Staphylococcus aureus Abscess Formation

PubMed Central

Beerlage, Christiane; Greb, Jessica; Kretschmer, Dorothee; Assaggaf, Mohammad; Trackman, Philip C.; Hansmann, Martin-Leo; Bonin, Michael; Eble, Johannes A.; Peschel, Andreas; Brüne, Bernhard

2013-01-01

Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor involved in the adaptation of mammals to hypoxia and plays a crucial role in cancer angiogenesis. Recent evidence suggests a leading role for HIF-1 in various inflammatory and infectious diseases. Here we describe the role of HIF-1 in Staphylococcus aureus infections by investigating the HIF-1-dependent host cell response. For this purpose, transcriptional profiling of HIF-1α-deficient HepG2 and control cells, both infected with Staphylococcus aureus, was performed. Four hours after infection, the expression of 190 genes, 24 of which were regulated via HIF-1, was influenced. LOX (encoding lysyl oxidase) was one of the upregulated genes with a potential impact on the course of S. aureus infection. LOX is an amine oxidase required for biosynthetic cross-linking of extracellular matrix components. LOX was upregulated in vitro in different cell cultures infected with S. aureus and also in vivo, in kidney abscesses of mice intravenously infected with S. aureus and in clinical skin samples from patients with S. aureus infections. Inhibition of LOX by β-aminopropionitrile (BAPN) did not affect the bacterial load in kidneys or blood but significantly influenced abscess morphology and collagenization. Our data provide evidence for a crucial role of HIF-1-regulated LOX in abscess formation. PMID:23649089

Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

PubMed Central

Han, Hyeong-jun; Kwon, Nayoung; Choi, Min-A; Jung, Kyung Oh; Piao, Juan-Yu; Ngo, Hoang Kieu Chi; Kim, Su-Jung; Kim, Do-Hee; Chung, June-Key; Cha, Young-Nam; Youn, Hyewon; Choi, Bu Young; Min, Sang-Hyun; Surh, Young-Joon

2016-01-01

Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target. PMID:26784107

Lenticular cytoprotection. Part 1: The role of hypoxia inducible factors-1α and -2α and vascular endothelial growth factor in lens epithelial cell survival in hypoxia

PubMed Central

Neelam, Sudha; Brooks, Morgan M.

2013-01-01

Purpose The prosurvival signaling cascades that mediate the unique ability of human lens epithelial cells to survive in their naturally hypoxic environment are not well defined. Hypoxia induces the synthesis of the hypoxia inducible factor HIF-1α that in turn, plays a crucial role in modulating a downstream survival scheme, where vascular endothelial growth factor (VEGF) also plays a major role. To date, no published reports in the lens literature attest to the expression and functionality of HIF-2α and the role it might play in regulating VEGF expression. The aim of this study was to identify the functional expression of the hypoxia inducible factors HIF-1α and HIF-2α and establish their role in regulating VEGF expression. Furthermore, we demonstrate a link between sustained VEGF expression and the ability of the hypoxic human lens epithelial cell to thrive in low oxygen conditions and resist mitochondrial membrane permeability transition (also referred to as lenticular cytoprotection). Methods Hypoxia inducible factor translation inhibitors were used to demonstrate the role of HIF-1α and HIF-2α and the simultaneous expression of both hypoxic inducible factors to determine their role in regulating VEGF expression. Axitinib, which inhibits lenticular cell autophosphorylation of its VEGF receptor, was employed to demonstrate a role for the VEGF–VEGFR2 receptor complex in regulating Bcl-2 expression. Specific antisera and western blot analysis were used to detect the protein levels of HIF-1α and HIF-2α, as well as the proapoptotic protein, BAX and the prosurvival protein, Bcl-2. VEGF levels were analyzed with enzyme-linked immunosorbent assay (ELISA). The potentiometric dye, 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide, was used to determine the effect of the inhibitors on mitochondrial membrane permeability transition. Results Cultured human lens epithelial cells (HLE-B3) maintained under hypoxic condition (1% oxygen) displayed consistent accumulation of VEGF throughout the 72 h incubation period. Using hypoxia inducible factor translation inhibitors targeting HIF-1α or HIF-2α, the specific inhibition of each protein did not diminish VEGF synthesis. The combined inhibition of HIF-1α and HIF-2α expression, using a double hypoxia inducible factor translation inhibitor, markedly decreased the level of VEGF. The inhibition of VEGF synthesis was associated with a profound deficiency in the level of the prosurvival protein, Bcl-2. Axitinib also prevented the VEGF-mediated expression of Bcl-2. The loss of VEGF coupled with the decrease in intracellular Bcl-2 correlated with marked mitochondrial depolarization, an early predictor of cellular apoptosis. Conclusions Our data support a model in which the sustained synthesis of VEGF in human lens epithelial cells, maintained under hypoxic condition, is regulated by a compensatory inter-relationship between HIF-1α and HIF-2α. VEGF acts as a prosurvival factor in hypoxic lens epithelial cells by maintaining consistent expression of the prosurvival protein Bcl-2, which likely prevents the translocation of cytosolic BAX to the outer mitochondrial membrane, thus preventing the initiation of mitochondrial depolarization. PMID:23335846

HIF-1α and GLUT-1 Expression in Atypical Endometrial Hyperplasia, Type I and II Endometrial Carcinoma: A Potential Role in Pathogenesis

PubMed Central

Abdou, Asmaa Gaber; Wahed, Moshira Mohammed Abdel; Kassem, Hend Abdou

2016-01-01

Introduction Hypoxia-Inducible Factor 1α (HIF-1α) is one of the major adaptive responses to hypoxia, regulating the activity of glucose transporter -1 (GLUT-1), responsible for glucose uptake. Aim To evaluate the immunohistochemical expression of both HIF-1α and GLUT-1 in type I and II endometrial carcinoma and their correlation with the available clinicopathologic variables in each type. Materials and Methods A retrospective study was conducted on archival blocks diagnosed from pathology department between April 2010 and August 2014 included 9 cases of atypical hyperplasia and 67 cases of endometrial carcinoma. Evaluation of both HIF-1α and GLUT-1 expression using standard immunohistochemical techniques performed on cut sections from selected paraffin embedded blocks. Statistical Analysis Descriptive analysis of the variables and statistical significances were calculated by non-parametric chi-square test using the Statistical Package for the Social Sciences version 12.0 (SPSS). Results HIF-1α was expressed in epithelial (88.9%, 52.2%, 61.2% and 50%) and stromal (33.3%, 74.6%. 71.4% and 83.3%) components of hyperplasia, total cases of EC, type I and II EC, respectively. GLUT-1 was expressed in the epithelial component of 88.9%, 98.5%, 98% and 100% of hyperplasia, total EC cases, type I and II EC, respectively. The necrosis related pattern of epithelial HIF-1α expression was in favour of type II (p=0.018) and grade III (p=0.038). HIF-1α H-score was associated with high apoptosis in both type I and total cases of EC (p=0.04). GLUT-1 H-score was negatively correlated with apoptotic count (p=0.04) and associated with high grade (p=0.003) and advanced stage in total EC (p=0.004). GLUT-1 H-score was correlated with the pattern of HIF-1α staining in all cases of EC (p= 0.04). Conclusion The role of HIF-1α in epithelial cells may differ from that of stromal cells in EC; however they augment the expression of each other supporting the crosstalk between them. The stepwise increase in H- score of GLUT-1 in the studied cases implies its potential role in carcinogenesis of EC. HIF-1α may promote GLUT-1 expression in EC especially surrounding areas of necrosis. The differences between type I and type II EC regarding HIF-1α and GLUT-1 expression may confirm the differences in their aetiopathogenesis. PMID:27437226

HIF-1α and GLUT-1 Expression in Atypical Endometrial Hyperplasia, Type I and II Endometrial Carcinoma: A Potential Role in Pathogenesis.

PubMed

Al-Sharaky, Dalia Rifaat; Abdou, Asmaa Gaber; Wahed, Moshira Mohammed Abdel; Kassem, Hend Abdou

2016-05-01

Hypoxia-Inducible Factor 1α (HIF-1α) is one of the major adaptive responses to hypoxia, regulating the activity of glucose transporter -1 (GLUT-1), responsible for glucose uptake. To evaluate the immunohistochemical expression of both HIF-1α and GLUT-1 in type I and II endometrial carcinoma and their correlation with the available clinicopathologic variables in each type. A retrospective study was conducted on archival blocks diagnosed from pathology department between April 2010 and August 2014 included 9 cases of atypical hyperplasia and 67 cases of endometrial carcinoma. Evaluation of both HIF-1α and GLUT-1 expression using standard immunohistochemical techniques performed on cut sections from selected paraffin embedded blocks. Descriptive analysis of the variables and statistical significances were calculated by non-parametric chi-square test using the Statistical Package for the Social Sciences version 12.0 (SPSS). HIF-1α was expressed in epithelial (88.9%, 52.2%, 61.2% and 50%) and stromal (33.3%, 74.6%. 71.4% and 83.3%) components of hyperplasia, total cases of EC, type I and II EC, respectively. GLUT-1 was expressed in the epithelial component of 88.9%, 98.5%, 98% and 100% of hyperplasia, total EC cases, type I and II EC, respectively. The necrosis related pattern of epithelial HIF-1α expression was in favour of type II (p=0.018) and grade III (p=0.038). HIF-1α H-score was associated with high apoptosis in both type I and total cases of EC (p=0.04). GLUT-1 H-score was negatively correlated with apoptotic count (p=0.04) and associated with high grade (p=0.003) and advanced stage in total EC (p=0.004). GLUT-1 H-score was correlated with the pattern of HIF-1α staining in all cases of EC (p= 0.04). The role of HIF-1α in epithelial cells may differ from that of stromal cells in EC; however they augment the expression of each other supporting the crosstalk between them. The stepwise increase in H- score of GLUT-1 in the studied cases implies its potential role in carcinogenesis of EC. HIF-1α may promote GLUT-1 expression in EC especially surrounding areas of necrosis. The differences between type I and type II EC regarding HIF-1α and GLUT-1 expression may confirm the differences in their aetiopathogenesis.

Molecular Mechanism Underlying the Action of Substituted Pro-Gly Dipeptide Noopept

PubMed Central

Vakhitova, Y. V.; Sadovnikov, S. V.; Borisevich, S. S.; Ostrovskaya, R. U.; A.Gudasheva, T.; Seredenin, S. B.

2016-01-01

This study was performed in order to reveal the effect of Noopept (ethyl ester of N-phenylacetyl-Lprolylglycine, GVS-111) on the DNA-binding activity of transcriptional factors (TF) in HEK293 cells transiently transfected with luciferase reporter constructs containing sequences for CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF-1. Noopept (10 μM) was shown to increase the DNA-binding activity of HIF-1 only, while lacking the ability to affect that of CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, and HSF1. Noopept provoked an additional increase in the DNA-binding activity of HIF-1 when applied in conditions of CoCl2-induced HIF- 1 stabilization. The degree of this HIF-positive effect of Noopept was shown to be concentration-dependent. Piracetam (1 mM) failed to affect significantly any of the TF under study. The results of molecular docking showed that Noopept (L-isomer), as well as its metabolite, L-isomer of N-phenyl-acetylprolyl, unlike its pharmacologically ineffective D-isomer, is able to bind to the active site of prolyl hydroxylase 2. Taking into account the important role of the genes activated by HIF-1 in the formation of an adaptive response to hypoxia, data on the ability of Noopept to provoke a selective increase in the DNA-binding activity of HIF-1 explain the wide spectrum of neurochemical and pharmacological effects of Noopept revealed before. The obtained data allow one to propose the HIF-positive effect as the primary mechanism of the activity of this Pro-Gly-containing dipeptide. PMID:27099787

Cyclin-dependent kinase inhibitor, P276-00, inhibits HIF-1α and induces G2/M arrest under hypoxia in prostate cancer cells.

PubMed

Manohar, S M; Padgaonkar, A A; Jalota-Badhwar, A; Rao, S V; Joshi, K S

2012-03-01

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to oxygen deprivation and controls genes involved in glycolysis, angiogenesis, migration and invasion. Overexpression of HIF-1α has been demonstrated in many common human cancers. Luciferase reporter gene assay under hypoxia and normoxia was used to demonstrate transcriptional inhibition of HIF-1 by P276-00. Detailed studies such as western blotting, reverse-transcriptase-PCR and immunofluorescence were carried out to elucidate its mechanism of action. Cytotoxic potential of P276-00 under normoxia and hypoxia was determined on prostate cancer cells using CCK-8 assay, and cell-cycle analysis was carried out using flow cytometry. Antiangiogenic activity of P276-00 was demonstrated by migration assay and tube-formation assay. Efficacy study of P276-00 was performed in a PC-3 xenograft model. P276-00 inhibits transcriptional activation of HIF-1 under hypoxia. It suppressed hypoxia-mediated nuclear HIF-1α expression, as well as phosphorylation of Akt and 4E-BP1 and abrogated expression of HIF-1-inducible gene viz. vascular endothelial growth factor. Under hypoxia, P276-00 did not exhibit enhanced cytotoxic activity in prostate cancer cells but arrested them in the G2/M phase of the cell cycle. The tubular formation of human umbilical vein endothelial cells and migration of prostate cancer cells were also inhibited by P276-00 in vitro. In addition, it demonstrated significant in vivo efficacy in the PC-3 xenograft model. Given its low toxicity profile, its demonstrated antitumor activity and its potential to inhibit the HIF-1 pathway, P276-00 should be considered as antiangiogenic chemotherapy for prostate cancer.

STAT3, p-STAT3 and HIF-1α are associated with vasculogenic mimicry and impact on survival in gastric adenocarcinoma

PubMed Central

SONG, YAN-YAN; SUN, LI-DAN; LIU, MIN-LI; LIU, ZHONG-LIANG; CHEN, FEI; ZHANG, YING-ZHE; ZHENG, YAN; ZHANG, JIAN-PING

2014-01-01

Vasculogenic mimicry (VM) formation is important for invasion and metastasis of tumor cells in gastric adenocarcinoma (GAC). The present study aimed to investigate the association between signal transducer and activator of transcription-3 (STAT3), phosphor-STAT3 (p-STAT3), hypoxia-inducible factor-1α (HIF-1α) and VM formation in GAC, and discuss their clinical significance and correlation with the prognosis of patients with GAC. The expression levels of STAT3, p-STAT3, HIF-1α and VM were assessed in 60 cases of patients with GAC and 20 cases of patients with gastritis on tissue microarrays by immunohistochemical methods. The expression levels of STAT3, p-STAT3, HIF-1α and VM were higher in patients with GAC (particularly in poorly differentiated GAC) than in those with gastritis (P<0.05). The expression levels of STAT3, p-STAT3 and HIF-1α were higher in VM tissues compared with non-VM tissues (P<0.05). Positive correlations existed between STAT3, p-STAT3, HIF-1α and VM expression (P<0.05). The expression levels of STAT3, p-STAT3 and HIF-1α, VM, status of lymph node metastasis and tumor differentiation degree were associated with the overall survival time of patients with GAC (P<0.05). However, only p-STAT3 and VM expression were identified as the independent risk factors of GAC OS when analyzed with multivariate analysis. p-STAT3 and VM play a significant role in indicating the prognosis of patients with GAC. STAT3 activation may play a positive role in VM formation of GAC by the STAT3-p-STAT3-HIF-1α-VM effect axis. PMID:24959290

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression

PubMed Central

Westerlund, Isabelle; Shi, Yao; Toskas, Konstantinos; Fell, Stuart M.; Li, Shuijie; Surova, Olga; Södersten, Erik; Kogner, Per; Nyman, Ulrika; Schlisio, Susanne; Holmberg, Johan

2017-01-01

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma. PMID:28696319

Honokiol inhibits pathological retinal neovascularization in oxygen-induced retinopathy mouse model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vavilala, Divya Teja; O’Bryhim, Bliss E.; Ponnaluri, V.K. Chaithanya

2013-09-06

Highlights: •Aberrant activation of HIF pathway is the underlying cause of ischemic neovascularization. •Honokiol has better therapeutic index as a HIF inhibitor than digoxin and doxorubicin. •Daily IP injection of honokiol in OIR mouse model reduced retinal neovascularization. •Honokiol also prevents vaso-obliteration, the characteristic feature of the OIR model. •Honokiol enhanced physiological revascularization of the retinal vascular plexuses. -- Abstract: Aberrant activation of the hypoxia inducible factor (HIF) pathway is the underlying cause of retinal neovascularization, one of the most common causes of blindness worldwide. The HIF pathway also plays critical roles during tumor angiogenesis and cancer stem cell transformation.more » We have recently shown that honokiol is a potent inhibitor of the HIF pathway in a number of cancer and retinal pigment epithelial cell lines. Here we evaluate the safety and efficacy of honokiol, digoxin, and doxorubicin, three recently identified HIF inhibitors from natural sources. Our studies show that honokiol has a better safety to efficacy profile as a HIF inhibitor than digoxin and doxorubicin. Further, we show for the first time that daily intraperitoneal injection of honokiol starting at postnatal day (P) 12 in an oxygen-induced retinopathy (OIR) mouse model significantly reduced retinal neovascularization at P17. Administration of honokiol also prevents the oxygen-induced central retinal vaso-obliteration, characteristic feature of the OIR model. Additionally, honokiol enhanced physiological revascularization of the retinal vascular plexuses. Since honokiol suppresses multiple pathways activated by HIF, in addition to the VEGF signaling, it may provide advantages over current treatments utilizing specific VEGF antagonists for ocular neovascular diseases and cancers.« less

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression.

PubMed

Westerlund, Isabelle; Shi, Yao; Toskas, Konstantinos; Fell, Stuart M; Li, Shuijie; Surova, Olga; Södersten, Erik; Kogner, Per; Nyman, Ulrika; Schlisio, Susanne; Holmberg, Johan

2017-07-25

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.

Positive feedback loop and synergistic effects between hypoxia-inducible factor-2α and stearoyl-CoA desaturase-1 promote tumorigenesis in clear cell renal cell carcinoma.

PubMed

Zhang, Yujian; Wang, Hui; Zhang, Jin; Lv, Jianwei; Huang, Yiran

2013-04-01

Adapting to hypoxic stress is pivotal in tumor progression and determining tumor malignancy. The transcriptional factor hypoxia-inducible factor (HIF) is crucial in modulating tumorous hypoxic responses through altering cell energy metabolism, which includes the modification of glucose and lipid metabolism-associated gene expression. Stearoyl-CoA desaturase-1 (SCD1) is the main isoform of SCDs, the rate-limiting enzymes in the biosynthesis of monounsaturated fatty acids from saturated fatty acids, which is extensively activated in cancer progression. In this study, we found that SCD1 and HIF-2α were overexpressed in human clear cell renal cell carcinoma (ccRCC) tissues and ccRCC cell lines, and were upregulated in the 786-0 ccRCC cell line under hypoxia. Knockdown of SCD1 or HIF-2α impacted the other's expression. Enhancing SCD1 resulted in HIF-2α upregulation, which could be blocked by inhibiting the PI3K/Akt pathway. Deficiency of SCD1 or HIF-2α in 768-0 cells led to apoptosis, less colony formation ability, and decreased cell migration. More obvious effects were observed in 786-0 cells with double SCD1 and HIF-2α knockdown. These results indicate a PI3K/Akt-mediated loop between SCD1 and HIF-2α that mutually enhances their protein levels. Both SCD1 and HIF-2α are critical to promoting tumorigenesis by synergistically acting on maintaining cell survival, triggering cell migration, and enhancing the colony formation ability of cancer cells. © 2013 Japanese Cancer Association.

Diallyl trisulfide exerts cardioprotection against myocardial ischemia-reperfusion injury in diabetic state, role of AMPK-mediated AKT/GSK-3β/HIF-1α activation

PubMed Central

Yu, Liming; Di, Wencheng; Dong, Xue; Li, Zhi; Xue, Xiaodong; Zhang, Jian; Wang, Qi; Xiao, Xiong; Han, Jinsong; Yang, Yang; Wang, Huishan

2017-01-01

Diallyl trisulfide (DATS), the major active ingredient in garlic, has been reported to confer cardioprotective effects. However, its effect on myocardial ischemia-reperfusion (MI/R) injury in diabetic state and the underlying mechanism are still unknown. We hypothesize that DATS reduces MI/R injury in diabetic state via AMPK-mediated AKT/GSK-3β/HIF-1α activation. Streptozotocin-induced diabetic rats received MI/R surgery with or without DATS (20mg/kg) treatment in the presence or absence of Compound C (Com.C, an AMPK inhibitor, 0.25mg/kg) or LY294002 (a PI3K inhibitor, 5mg/kg). We found that DATS significantly improved heart function and reduced myocardial apoptosis. Additionally, in cultured H9c2 cells, DATS (10μM) also attenuated simulated ischemia-reperfusion injury. We found that AMPK and AKT/GSK-3β/HIF-1α signaling were down-regulated under diabetic condition, while DATS markedly increased the phosphorylation of AMPK, ACC, AKT and GSK-3β as well as HIF-1α expression in MI/R-injured myocardium. However, these protective actions were all blunted by Com.C administration. Additionally, LY294002 abolished the stimulatory effect of DATS on AKT/GSK-3β/HIF-1α signaling without affecting AMPK signaling. While 2-methoxyestradiol (a HIF-1α inhibitor) reduced HIF-1α expression without affecting AKT/GSK-3β signaling. Taken together, these data showed that DATS protected against MI/R injury in diabetic state by attenuating cellular apoptosis via AMPK-mediated AKT/GSK-3β/HIF-1α signaling. Its cardioprotective effect deserves further study. PMID:29088824

Hydrogen sulphide induces HIF-1α and Nrf2 in THP-1 macrophages.

PubMed

Lohninger, Lilian; Tomasova, Lenka; Praschberger, Monika; Hintersteininger, Michael; Erker, Thomas; Gmeiner, Bernhard M K; Laggner, Hilde

2015-05-01

The transcription factor HIF-1α regulates the adaptive response of cells to hypoxia and oxidative stress. In addition, an important regulatory role for HIF-1α in immune reactions and inflammation is suggested. The present study attempts to investigate the effect of the gaseous signalling molecule hydrogen sulphide (H2S) on HIF-1α in THP-1 macrophages using the slow H2S releasing donor GYY4137. We found that H2S induced HIF-1α protein accumulation in THP-1 macrophages in a concentration-dependent manner. Western blot analysis of cell fractions showed that HIF-1α protein translocates into the nucleus and leads to an increase of its target protein glucose transporter-1 (GLUT-1). Activation of nuclear factor-κB (NF-κB), as well as secretion of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced in the presence of H2S. These findings indicate that HIF-1α accumulation due to H2S was not triggered by the NF-κB pathway. The antioxidant pathway Nrf2/HO-1 (nuclear factor erythroid 2-related factor 2/heme oxygenase-1) was activated by H2S. Inhibition of the p38 mitogen-activated protein kinase (MAPK) reversed H2S mediated effects, suggesting that the p38 MAPK pathway may be involved in H2S induced HIF-1α/Nrf2 signalling pathways. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Cobalt Chloride Upregulates Impaired HIF-1α Expression to Restore Sevoflurane Post-conditioning-Dependent Myocardial Protection in Diabetic Rats.

PubMed

Wu, Jianjiang; Yang, Long; Xie, Peng; Yu, Jin; Yu, Tian; Wang, Haiying; Maimaitili, Yiliyaer; Wang, Jiang; Ma, Haiping; Yang, Yining; Zheng, Hong

2017-01-01

Previous studies from our group have demonstrated that sevoflurane post-conditioning (SPC) protects against myocardial ischemia reperfusion injury via elevating the intranuclear expression of hypoxia inducible factor-1 alpha (HIF-1α). However, diabetic SPC is associated with decreased myocardial protection and disruption of the HIF-1 signaling pathway. Previous studies have demonstrated that cobalt chloride (CoCl 2 ) can upregulate HIF-1α expression under diabetic conditions, but whether myocardial protection by SPC can be restored afterward remains unclear. We established a rat model of type 2 diabetes and a Langendorff isolated heart model of ischemia-reperfusion injury. Prior to reperfusion, 2.4% sevoflurane was used as a post-conditioning treatment. The diabetic rats were treated with CoCl 2 24 h before the experiment. At the end of reperfusion, tests were performed to assess myocardial function, infarct size, mitochondrial morphology, nitric oxide (NO), Mitochondrial reactive oxygen species (ROS), mitochondrial respiratory function and enzyme activity, HIF-1α, vascular endothelial growth factor (VEGF) and endothelial NO synthase (eNOS) protein levels. In addition, myocardial protection by SPC was monitored after the blood glucose levels were lowered by insulin. The diabetic state was associated with deficient SPC protection and decreased HIF-1α expression. After treating the diabetic rats with CoCl 2 , SPC significantly upregulated the expression of HIF-1α, VEGF and eNOS, which markedly improved cardiac function, NO, mitochondrial respiratory function, and enzyme activity and decreased the infarction areas and ROS. In addition, these effects were not influenced by blood glucose levels. This study proved that CoCl 2 activates the HIF-1α signaling pathway, which restores SPC-dependent myocardial protection under diabetic conditions, and the protective effects of SPC were independent of blood glucose levels.

High glucose concentrations attenuate hypoxia-inducible factor-1{alpha} expression and signaling in non-tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dehne, Nathalie, E-mail: dehne@biochem.uni-frankfurt.de; Hintereder, Gudrun, E-mail: Gudrun.Hintereder@kgu.de; Bruene, Bernhard, E-mail: bruene@pathobiochemie1.de

2010-04-15

Hypoxia-inducible factor (HIF) is the major transcription factor mediating adaption to hypoxia e.g. by enhancing glycolysis. In tumor cells, high glucose concentrations are known to increase HIF-1{alpha} expression even under normoxia, presumably by enhancing the concentration of tricarboxylic acid cycle intermediates, while reactions of non-tumor cells are not well defined. Therefore, we analyzed cellular responses to different glucose concentrations in respect to HIF activation comparing tumor to non-tumor cells. Using cells derived from non-tumor origin, we show that HIF-1{alpha} accumulation was higher under low compared to high glucose concentrations. Low glucose allowed mRNA expression of HIF-1 target genes like adrenomedullin.more » Transfection of C{sub 2}C{sub 12} cells with a HIF-1{alpha} oxygen-dependent degradation domaine-GFP fusion protein revealed that prolyl hydroxylase (PHD) activity is impaired at low glucose concentrations, thus stabilizing the fusion protein. Mechanistic considerations suggested that neither O{sub 2} redistribution nor an altered redox state explains impaired PHD activity in the absence of glucose. In order to affect PHD activity, glucose needs to be metabolized. Amino acids present in the medium also diminished HIF-1{alpha} expression, while the addition of fatty acids did not. This suggests that glucose or amino acid metabolism increases oxoglutarate concentrations, which enhances PHD activity in non-tumor cells. Tumor cells deprived of glutamine showed HIF-1{alpha} accumulation in the absence of glucose, proposing that enhanced glutaminolysis observed in many tumors enables these cells to compensate reduced oxoglutarate production in the absence of glucose.« less

[The effect of hypoxia preconditioning no binding activity of HIF-1 on the HRE with EPO in the hippocampus of mice].

PubMed

Shao, Guo; Zhou, Wei-Hua; Gao, Cui-Ying; Zhang, Ran; Lu, Guo-Wei

2007-02-01

To observe change of binding activity of HIF-1 with erythropoietin (EPO) hypoxia response element (HRE) in the hippocampus of mice preconditioned to hypoxia and explore relationship between the changes and the preconditioning. The hippocampus was removed from mice exposed to hypoxia for 0 run (control group), 1 run (H1 group) and 4 runs(H4 group). Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP)and real time PCR were used to detect the change of activity of HIF-1 on HRE of EPO. Both in vitro and in vivo binding tests showed that the HIF-1 DNA-binding activities were increased in group H1 and markedly increased in group H4. The increase of HIF-1 and HRE of EPO binding activities is thought be involved in hypoxic preconditioning.

Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Inducible Factor-Dependent Extension of the Replicative Life Span during Hypoxia▿

PubMed Central

Bell, Eric L.; Klimova, Tatyana A.; Eisenbart, James; Schumacker, Paul T.; Chandel, Navdeep S.

2007-01-01

Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span. PMID:17562866

Hypoxia Inducible Factor-Stabilizing Bioactive Glasses for Directing Mesenchymal Stem Cell Behavior

PubMed Central

Azevedo, Maria M.; Tsigkou, Olga; Nair, Rekha; Jones, Julian R.; Jell, Gavin

2015-01-01

Oxygen tension is a known regulator of mesenchymal stem cell (MSC) plasticity, differentiation, proliferation, and recruitment to sites of injury. Materials capable of affecting the MSC oxygen-sensing pathway, independently of the environmental oxygen pressure, are therefore of immense interest to the tissue engineering (TE) and regenerative medicine community. In this study, we describe the evaluation of the effect of hypoxia inducible factor (HIF)-stabilizing bioactive glasses (BGs) on human MSCs. The dissolution products from these hypoxia-mimicking BGs stabilized HIF-1α in a concentration-dependent manner, altered cell proliferation and metabolism, and upregulated a number of genes involved in the hypoxic response (HIF1A, HIF2A, and VHL), MSC survival (SAG and BCL2), extracellular matrix remodeling (MMP1), and angiogenesis (VEGF and PDGF). These HIF-stabilizing materials can therefore be used to improve MSC survival and enhance regeneration in a number of TE strategies. PMID:25167933

Effects of the HIF1 inhibitor, echinomycin, on growth and NOTCH signalling in leukaemia cells.

PubMed

Yonekura, Satoru; Itoh, Mai; Okuhashi, Yuki; Takahashi, Yusuke; Ono, Aya; Nara, Nobuo; Tohda, Shuji

2013-08-01

To examine the effects of echinomycin, a compound that inhibits DNA-binding activity of hypoxia-inducible factor-1 (HIF1), on leukaemia cell growth. Three acute myeloid leukaemia cell lines and three T-lymphoblastic leukaemia cell lines were cultured with echinomycin. Cell growth, mRNA and protein expression levels were examined by WST-1 assay, reverse-transcription polymerase chain reaction and immunoblotting, respectively. HIF1α protein was expressed in all cell lines under normoxia. Treatment with echinomycin suppressed cell growth and induced apoptosis in association with decreased mRNA expression of HIF1 targets, glucose transporter-1 (GLUT1) and B-cell CLL/lymphoma-2 (BCL2). Echinomycin also suppressed the protein expression of NOTCH1, cleaved NOTCH1, v-myc myelocytomatosis viral oncogene homolog (MYC), v-akt murine thymoma viral oncogene homolog-1 (AKT), phosphorylated AKT, mechanistic target of rapamycin (mTOR), and phosphorylated mTOR and increased that of cleaved caspase-3 in some cell lines. Echinomycin suppresses leukaemia cell growth in association with reduced NOTCH1 expression. This is the first report to show that HIF inhibitor treatment suppresses NOTCH1 signalling. HIF inhibitors could be novel candidates for a molecular-targeted therapy against leukaemia.

ROS mediated EGFR/MEK/ERK/HIF-1α Loop Regulates Glucose metabolism in pancreatic cancer.

PubMed

Wang, Gang; Li, Yifeng; Yang, Zeyu; Xu, Weina; Yang, Yifan; Tan, Xiaodong

2018-06-12

To investigate the glycometabolism associated mechanism in invasion and metastasis of pancreatic cancer, We screened out genes involved in anaerobic glycolysis headed by HIF-1α,using pre-established a pair of pancreatic cancer cell lines. In this study, we further detected the glucose metabolism state not only in the cells but all also in two groups of patients with different SUVmax on 18 F-FDG PET/CT. The data suggests that ROS mediated EGFR/MEK/ERK/HIF-1α loop is activated in high glucose metabolic samples both in vitro and in vivo: The increasing of HIF-1α expression is controlled by activation of EGFR/MEK/ERK pathway in hypoxia condition, HIF-1α inhibits excessive release of ROS, the reduction of ROS further activates EGFR to form a positive feedback loop. This difference is closely related to invasion and metastasis capacity of pancreatic cancer, and can be rescued by separate or combined inhibition of EGFR or HIF-1α in various degree. These results indicate a new clue to develop therapy of pancreatic cancer by regulating the glucose metabolism. Copyright © 2018 Elsevier Inc. All rights reserved.

The role of hypoxia and HIF1α in the regulation of STAR-mediated steroidogenesis in granulosa cells.

PubMed

Kowalewski, Mariusz Pawel; Gram, Aykut; Boos, Alois

2015-02-05

The adaptive responses to hypoxia are mediated by hypoxia-inducible factor 1 alpha (HIF1α). Its role, however, in regulating steroidogenesis remains poorly understood. We examined the role of hypoxia and HIF1α in regulating steroid acute regulatory protein (STAR) expression and steroidogenesis in immortalized (KK1) mouse granulosa cells under progressively lowering O2 concentrations (20%, 15%, 10%, 5%, 1%). Basal and dbcAMP-stimulated progesterone synthesis was decreased under severe hypoxia (1% and 5% O2). The partial hypoxia revealed opposing effects, with a significant increase in steroidogenic response at 10% O2 in dbcAMP-treated cells: Star-promoter activity, mRNA and protein expression were increased. The hypoxia-stimulated STAR expression was PKA-dependent. Binding of HIF1α to the Star-promoter was potentiated under partial hypoxia. Inhibition of the transcriptional activity or expression of HIF1α suppressed STAR-expression. HIF1α appears to be a positive regulator of basal and stimulated STAR-expression, which under partial hypoxia is capable of increasing the steroidogenic capacity of granulosa cells. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

HIF1α-dependent glycolysis promotes macrophage functional activities in protecting against bacterial and fungal infection.

PubMed

Li, Chunxiao; Wang, Yu; Li, Yan; Yu, Qing; Jin, Xi; Wang, Xiao; Jia, Anna; Hu, Ying; Han, Linian; Wang, Jian; Yang, Hui; Yan, Dapeng; Bi, Yujing; Liu, Guangwei

2018-02-26

Macrophages are important innate immune defense system cells in the fight against bacterial and fungal pathogenic infections. They exhibit significant plasticity, particularly with their ability to undergo functional differentiation. Additionally, HIF1α is critically involved in the functional differentiation of macrophages during inflammation. However, the role of macrophage HIF1α in protecting against different pathogenic infections remains unclear. In this study, we investigated and compared the roles of HIF1α in different macrophage functional effects of bacterial and fungal infections in vitro and in vivo. We found that bacterial and fungal infections produced similar effects on macrophage functional differentiation. HIF1α deficiency inhibited pro-inflammatory macrophage functional activities when cells were stimulated with LPS or curdlan in vitro or when mice were infected with L. monocytogenes or C. albicans in vivo, thus decreasing pro-inflammatory TNFα and IL-6 secretion associated with pathogenic microorganism survival. Alteration of glycolytic pathway activation was required for the functional differentiation of pro-inflammatory macrophages in protecting against bacterial and fungal infections. Thus, the HIF1α-dependent glycolytic pathway is essential for pro-inflammatory macrophage functional differentiation in protecting against bacterial and fungal infections.

Adaptation to HIF-1 deficiency by upregulation of the AMP/ATP ratio and phosphofructokinase activation in hepatomas.

PubMed

Golinska, Monika; Troy, Helen; Chung, Yuen-Li; McSheehy, Paul M; Mayr, Manuel; Yin, Xiaoke; Ly, Lucy; Williams, Kaye J; Airley, Rachel E; Harris, Adrian L; Latigo, John; Perumal, Meg; Aboagye, Eric O; Perrett, David; Stubbs, Marion; Griffiths, John R

2011-05-25

HIF-1 deficiency has marked effects on tumour glycolysis and growth. We therefore investigated the consequences of HIF-1 deficiency in mice, using the well established Hepa-1 wild-type (WT) and HIF-1β-deficient (c4) model. These mechanisms could be clinically relevant, since HIF-1 is now a therapeutic target. Hepa-1 WT and c4 tumours grown in vivo were analysed by 18FDG-PET and 19FDG Magnetic Resonance Spectroscopy for glucose uptake; by HPLC for adenine nucleotides; by immunohistochemistry for GLUTs; by immunoblotting and by DIGE followed by tandem mass spectrometry for protein expression; and by classical enzymatic methods for enzyme activity. HIF-1β deficient Hepa-1 c4 tumours grew significantly more slowly than WT tumours, and (as expected) showed significantly lower expression of many glycolytic enzymes. However, HIF-1β deficiency caused no significant change in the rate of glucose uptake in c4 tumours compared to WT when assessed in vivo by measuring fluoro-deoxyglucose (FDG) uptake. Immunohistochemistry demonstrated less GLUT-1 in c4 tumours, whereas GLUT-2 (liver type) was similar to WT. Factors that might upregulate glucose uptake independently of HIF-1 (phospho-Akt, c-Myc) were shown to have either lower or similar expression in c4 compared to WT tumours. However the AMP/ATP ratio was 4.5 fold higher (p < 0.01) in c4 tumours, and phosphofructokinase-1 (PFK-1) activity, measured at prevailing cellular ATP and AMP concentrations, was up to two-fold higher in homogenates of the deficient c4 cells and tumours compared to WT (p < 0.001), suggesting that allosteric PFK activation could explain their normal level of glycolysis. Phospho AMP-Kinase was also higher in the c4 tumours. Despite their defective HIF-1 and consequent down-regulation of glycolytic enzyme expression, Hepa-1 c4 tumours maintain glucose uptake and glycolysis because the resulting low [ATP] high [AMP] allosterically activate PFK-1. This mechanism of resistance would keep glycolysis functioning and also result in activation of AMP-Kinase and growth inhibition; it may have major implications for the therapeutic activity of HIF inhibitors in vivo. Interestingly, this control mechanism does not involve transcriptional control or proteomics, but rather the classical activation and inhibition mechanisms of glycolytic enzymes.

Edaravone Improves Septic Cardiac Function by Inducing an HIF-1α/HO-1 Pathway

PubMed Central

He, Chao; Zhang, Wei; Li, Suobei; Ruan, Wei; Xu, Junmei

2018-01-01

Septic myocardial dysfunction remains prevalent and raises mortality rate in patients with sepsis. During sepsis, tissues undergo tremendous oxidative stress which contributes critically to organ dysfunction. Edaravone, a potent radical scavenger, has been proved beneficial in ischemic injuries involving hypoxia-inducible factor- (HIF-) 1, a key regulator of a prominent antioxidative protein heme oxygenase- (HO-) 1. However, its effect in septic myocardial dysfunction remains unclarified. We hypothesized that edaravone may prevent septic myocardial dysfunction by inducing the HIF-1/HO-1 pathway. Rats were subjected to cecal ligation and puncture (CLP) with or without edaravone infusion at three doses (50, 100, or 200 mg/kg, resp.) before CLP and intraperitoneal injection of the HIF-1α antagonist, ME (15 mg/kg), after CLP. After CLP, rats had cardiac dysfunction, which was associated with deformed myocardium, augmented lipid peroxidation, and increased myocardial apoptosis and inflammation, along with decreased activities of catalase, HIF-1α, and HO-1 in the myocardium. Edaravone pretreatment dose-dependently reversed the changes, of which high dose most effectively improved cardiac function and survival rate of septic rats. However, inhibition of HIF-1α by ME demolished the beneficial effects of edaravone at high dose, reducing the survival rate of the septic rats without treatments. Taken together, edaravone, by inducing the HIF-1α/HO-1 pathway, suppressed oxidative stress and protected the heart against septic myocardial injury and dysfunction. PMID:29765498

Overexpression of Hif-1α in Mesenchymal Stem Cells Affects Cell-Autonomous Angiogenic and Osteogenic Parameters.

PubMed

Lampert, F M; Kütscher, C; Stark, G B; Finkenzeller, G

2016-03-01

Reconstruction of large bone defects still represents a major medical challenge. In recent years tissue engineering has developed techniques based on adult mesenchymal stem cells (MSCs) that could represent an attractive therapeutical option to treat large bone defects in the future. It has been demonstrated in various animal models that ex vivo expanded MSCs are capable of promoting the regeneration of skeletal defects after implantation. However, for the efficient regeneration of bone in tissue engineering applications, a rapid vascularization of implanted grafts is essential to ensure the survival of cells in the early post-implantational phase. A promising strategy to enhance vascularization of MSC-containing implants could consist of overexpression of the angiogenic master transcription factor Hypoxia-inducible factor 1 (Hif-1) in the MSCs in order to induce angiogenesis and support osteogenesis. In the present study, we overexpressed Hif-1α in MSCs by using recombinant adenoviruses and investigated cell-autonomous effects. Overexpression of Hif-1α enhanced proliferation, migration, cell survival and expression of pro-angiogenic genes. Other parameters such as expression of the osteogenic markers BMP-2 and RunX2 were decreased. Hif-1α overexpression had no effect on invasion, senescence and osteogenic differentiation of MSCs. Our experiments revealed multifarious effects of Hif-1α overexpression on cell-autonomous parameters. Therefore, Hif-1α overexpression may represent a therapeutic option to improve cellular functions of MSCs to treat critical sized bone defects. © 2015 Wiley Periodicals, Inc.

Muc1 is protective during kidney ischemia-reperfusion injury

PubMed Central

Pastor-Soler, Núria M.; Sutton, Timothy A.; Mang, Henry E.; Kinlough, Carol L.; Gendler, Sandra J.; Madsen, Cathy S.; Bastacky, Sheldon I.; Ho, Jacqueline; Al-bataineh, Mohammad M.; Hallows, Kenneth R.; Singh, Sucha; Monga, Satdarshan P.; Kobayashi, Hanako; Haase, Volker H.

2015-01-01

Ischemia-reperfusion injury (IRI) due to hypotension is a common cause of human acute kidney injury (AKI). Hypoxia-inducible transcription factors (HIFs) orchestrate a protective response in renal endothelial and epithelial cells in AKI models. As human mucin 1 (MUC1) is induced by hypoxia and enhances HIF-1 activity in cultured epithelial cells, we asked whether mouse mucin 1 (Muc1) regulates HIF-1 activity in kidney tissue during IRI. Whereas Muc1 was localized on the apical surface of the thick ascending limb, distal convoluted tubule, and collecting duct in the kidneys of sham-treated mice, Muc1 appeared in the cytoplasm and nucleus of all tubular epithelia during IRI. Muc1 was induced during IRI, and Muc1 transcripts and protein were also present in recovering proximal tubule cells. Kidney damage was worse and recovery was blocked during IRI in Muc1 knockout mice compared with congenic control mice. Muc1 knockout mice had reduced levels of HIF-1α, reduced or aberrant induction of HIF-1 target genes involved in the shift of glucose metabolism to glycolysis, and prolonged activation of AMP-activated protein kinase, indicating metabolic stress. Muc1 clearly plays a significant role in enhancing the HIF protective pathway during ischemic insult and recovery in kidney epithelia, providing a new target for developing therapies to treat AKI. Moreover, our data support a role specifically for HIF-1 in epithelial protection of the kidney during IRI as Muc1 is present only in tubule epithelial cells. PMID:25925251

Mechanism Governing Human Kappa-Opioid Receptor Expression under Desferrioxamine-Induced Hypoxic Mimic Condition in Neuronal NMB Cells

PubMed Central

Babcock, Jennifer; Herrera, Alberto; Coricor, George; Karch, Christopher; Liu, Alexander H.; Rivera-Gines, Aida; Ko, Jane L.

2017-01-01

Cellular adaptation to hypoxia is a protective mechanism for neurons and relevant to cancer. Treatment with desferrioxamine (DFO) to induce hypoxia reduced the viability of human neuronal NMB cells. Surviving/attached cells exhibited profound increases of expression of the human kappa-opioid receptor (hKOR) and hypoxia inducible factor-1α (HIF-1α). The functional relationship between hKOR and HIF-1α was investigated using RT-PCR, Western blot, luciferase reporter, mutagenesis, siRNA and receptor-ligand binding assays. In surviving neurons, DFO increased HIF-1α expression and its amount in the nucleus. DFO also dramatically increased hKOR expression. Two (designated as HIFC and D) out of four potential HIF response elements of the hKOR gene (HIFA–D) synergistically mediated the DFO response. Mutation of both elements completely abolished the DFO-induced effect. The CD11 plasmid (containing HIFC and D with an 11 bp spacing) produced greater augmentation than that of the CD17 plasmid (HIFC and D with a 17 bp-spacing), suggesting that a proper topological interaction of these elements synergistically enhanced the promoter activity. HIF-1α siRNA knocked down the increase of endogenous HIF-1α messages and diminished the DFO-induced increase of hKOR expression. Increased hKOR expression resulted in the up-regulation of hKOR protein. In conclusion, the adaptation of neuronal hKOR under hypoxia was governed by HIF-1, revealing a new mechanism of hKOR regulation. PMID:28117678

[Phase changes in energy metabolism during periodic hypoxia].

PubMed

Portnichenko, V I; Nosar', V I; Portnichenko, A G; Drevitskaia, T I; Sidorenko, A M; Man'kovskaia, I N

2012-01-01

Male Wistar rats were exposed to periodic hypobaric hypoxia (PHH), by "lifting" in barochamber at "altitude" 5600 m for 1 h every 3 days (6 séances). The dynamics of changes in oxygen consumption (VO2), and body temperature (Tm), as well as in HIF-1alpha and HIF-3alpha gene expression, and mitochondrial respiration in the ventricles of the heart was studied. On the basis of the data we identified four phases of the physiological changes. The first phase, hypometabolic (1-3 séances), is characterized by decrease in VO2 and Tm, induction of HIF-1alpha and HIF-3alpha with delayed transient stimulation of metabolism in response to each séance of hypoxia. In heart mitochondria, V3 and V4 are increased, but V3/V4 and ADP/O are reduced. During the second phase, transitional (3-4 séances), there is reorganization of metabolism and decrease its hypoxic reactivity. The third phase, hypermetabolic (4-5 séances), is characterized by intensification of metabolism and compensation of hypoxic disorders. The fourth phase (after 5 séance) - is a state of metabolic adaptation with normalization of VO2 and Tm, expression of HIF-1alpha and HIF-3alpha, mitochondrial respiration, increased NAD-dependent oxidation of carbohydrate and lipid substrates. Thus, during PHH consequent rebuilding of processes of oxygen transport, tissue respiration and thermogenesis occurs, mediated by induction of the HIF subunits.

STAT3 precedes HIF1α transcriptional responses to oxygen and oxygen and glucose deprivation in human brain pericytes.

PubMed

Carlsson, Robert; Özen, Ilknur; Barbariga, Marco; Gaceb, Abderahim; Roth, Michaela; Paul, Gesine

2018-01-01

Brain pericytes are important to maintain vascular integrity of the neurovascular unit under both physiological and ischemic conditions. Ischemic stroke is known to induce an inflammatory and hypoxic response due to the lack of oxygen and glucose in the brain tissue. How this early response to ischemia is molecularly regulated in pericytes is largely unknown and may be of importance for future therapeutic targets. Here we evaluate the transcriptional responses in in vitro cultured human brain pericytes after oxygen and/or glucose deprivation. Hypoxia has been widely known to stabilise the transcription factor hypoxia inducible factor 1-alpha (HIF1α) and mediate the induction of hypoxic transcriptional programs after ischemia. However, we find that the transcription factors Jun Proto-Oncogene (c-JUN), Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B-Cells (NFκB) and signal transducer and activator of transcription 3 (STAT3) bind genes regulated after 2hours (hs) of omitted glucose and oxygen before HIF1α. Potent HIF1α responses require 6hs of hypoxia to substantiate transcriptional regulation comparable to either c-JUN or STAT3. Phosphorylated STAT3 protein is at its highest after 5 min of oxygen and glucose (OGD) deprivation, whereas maximum HIF1α stabilisation requires 120 min. We show that STAT3 regulates angiogenic and metabolic pathways before HIF1α, suggesting that HIF1α is not the initiating trans-acting factor in the response of pericytes to ischemia.

Glycolysis determines dichotomous regulation of T cell subsets in hypoxia

PubMed Central

Xu, Yang; Zhang, Ming; Savoldo, Barbara; Metelitsa, Leonid S.; Rodgers, John; Yustein, Jason T.; Neilson, Joel R.

2016-01-01

Hypoxia occurs in many pathological conditions, including chronic inflammation and tumors, and is considered to be an inhibitor of T cell function. However, robust T cell responses occur at many hypoxic inflammatory sites, suggesting that functions of some subsets are stimulated under low oxygen conditions. Here, we investigated how hypoxic conditions influence human T cell functions and found that, in contrast to naive and central memory T cells (TN and TCM), hypoxia enhances the proliferation, viability, and cytotoxic action of effector memory T cells (TEM). Enhanced TEM expansion in hypoxia corresponded to high hypoxia-inducible factor 1α (HIF1α) expression and glycolytic activity compared with that observed in TN and TCM. We determined that the glycolytic enzyme GAPDH negatively regulates HIF1A expression by binding to adenylate-uridylate–rich elements in the 3′-UTR region of HIF1A mRNA in glycolytically inactive TN and TCM. Conversely, active glycolysis with decreased GAPDH availability in TEM resulted in elevated HIF1α expression. Furthermore, GAPDH overexpression reduced HIF1α expression and impaired proliferation and survival of T cells in hypoxia, indicating that high glycolytic metabolism drives increases in HIF1α to enhance TEM function during hypoxia. This work demonstrates that glycolytic metabolism regulates the translation of HIF1A to determine T cell responses to hypoxia and implicates GAPDH as a potential mechanism for controlling T cell function in peripheral tissue. PMID:27294526

Expression of hypoxia-inducible factor-1α, vascular endothelial growth factor and prolyl hydroxylase domain protein 2 in cutaneous squamous cell carcinoma and precursor lesions and their relationship with histological stages and clinical features.

PubMed

An, Xiangjie; Xu, Guangfen; Yang, Liu; Wang, Yuejue; Li, Yan; McHepange, Uwesu O; Shen, Guanxin; Tu, Yating; Tao, Juan

2014-01-01

The hypoxia-inducible factor-1 (HIF-1α) pathway is associated with tumor growth, angiogenesis and metastasis in various carcinomas. Little is known regarding the role of the HIF-1α signaling pathway in cutaneous squamous cell carcinoma (SCC). We investigated the expression of HIF-1α, vascular endothelial growth factor (VEGF) and the HIF negative regulator, prolyl hydroxylase domain protein 2 (PHD2), in cutaneous SCC, Bowen's disease, seborrheic keratosis (SK) and normal skin by immunohistochemistry and in situ hybridization. Additionally, we explored the relationships between these factors and the clinical and histological characteristics of each disease. Our study indicated that the expression of HIF-1α and VEGF was significantly higher (P < 0.05) in cutaneous SCC than in Bowen's disease, SK or normal skin. In contrast, PHD2 showed significantly higher expression in normal skin compared with SK, Bowen's disease and cutaneous SCC (P < 0.05). Grade II-IV cutaneous SCC had higher expression levels of nuclear HIF-1α and cytoplasm VEGF protein but less nuclear PHD2 protein than grade Ι cutaneous SCC (P < 0.05). Overexpression of HIF-1α and VEGF, as well as the decreased expression of PHD2, may play important roles in the development of cutaneous SCC. © 2013 Japanese Dermatological Association.

HIF-1α regulates epithelial inflammation by cell autonomous NFκB activation and paracrine stromal remodeling

PubMed Central

Scortegagna, Marzia; Cataisson, Christophe; Martin, Rebecca J.; Hicklin, Daniel J.; Schreiber, Robert D.; Yuspa, Stuart H.

2008-01-01

Hypoxia inducible factor-1 (HIF-1) is a master regulatory transcription factor controlling multiple cell-autonomous and non–cell-autonomous processes, such as metabolism, angiogenesis, matrix invasion, and cancer metastasis. Here we used a new line of transgenic mice with constitutive gain of HIF-1 function in basal keratinocytes and demonstrated a signaling pathway from HIF-1 to nuclear factor κ B (NFκB) activation to enhanced epithelial chemokine and cytokine elaboration. This pathway was responsible for a phenotypically silent accumulation of stromal inflammatory cells and a marked inflammatory hypersensitivity to a single 12-O-tetradecanoylphorbol-13-acetate (TPA) challenge. HIF-1–induced NFκB activation was composed of 2 elements, IκB hyperphosphorylation and phosphorylation of Ser276 on p65, enhancing p65 nuclear localization and transcriptional activity, respectively. NFκB transcriptional targets macrophage inflammatory protein-2 (MIP-2/CXCL2/3), keratinocyte chemokine (KC/CXCL1), and tumor necrosis factor [alfa] (TNFα) were constitutively up-regulated and further increased after TPA challenge both in cultured keratinocytes and in transgenic mice. Whole animal KC, MIP-2, or TNFα immunodepletion each abrogated TPA-induced inflammation, whereas blockade of either VEGF or placenta growth factor (PlGF) signaling did not affect transgenic inflammatory hyper-responsiveness. Thus, epithelial HIF-1 gain of function remodels the local environment by cell-autonomous NFκB-mediated chemokine and cytokine secretion, which may be another mechanism by which HIF-1 facilitates either inflammatory diseases or malignant progression. PMID:18199827

Reactive oxygen species (ROS) and the heat stress response of Daphnia pulex: ROS-mediated activation of hypoxia-inducible factor 1 (HIF-1) and heat shock factor 1 (HSF-1) and the clustered expression of stress genes.

PubMed

Klumpen, Eva; Hoffschröer, Nadine; Zeis, Bettina; Gigengack, Ulrike; Dohmen, Elias; Paul, Rüdiger J

2017-01-01

Heat stress in ectotherms involves direct (e.g. protein damage) and/or indirect effects (temperature-induced hypoxia and ROS formation), which cause activation of the transcription factors (TF) heat shock factor 1 (HSF-1) and/or hypoxia-inducible factor 1 (HIF-1). The present study focused on the links between stress (ROS) signals, nuclear (n) and cytoplasmic (c) HSF-1/HIF-1 levels, and stress gene expression on mRNA and protein levels (e.g. heat-shock protein 90, HSP90) upon acute heat and ROS (H 2 O 2 ) stress. Acute heat stress (30°C) evoked fluctuations in ROS level. Different feeding regimens, which affected the glutathione (GSH) level, allowed altering the frequency of ROS fluctuations. Other data showed fluctuation frequency to depend also on ROS production rate. The heat-induced slow or fast ROS fluctuations (at high or low GSH levels) evoked slow or fast fluctuations in the levels of nHIF-1α, nHSF-1 and gene products (mRNAs and protein), albeit after different time delays. Time delays to ROS fluctuations were, for example,shorter for nHIF-1α than for nHSF-1 fluctuations, and nHIF-1α fluctuations preceded and nHSF-1 fluctuations followed fluctuations in HSP90 mRNA level. Cytoplasmic TF levels either changed little (cHIF-1α) or showed a steady increase (cHSF-1). Applying acute H 2 O 2 stress (at 20°C) revealed effects on nHIF-1α and mRNA levels, but no significant effects on nHSF-1 level. Transcriptome data additionally showed coordinated fluctuations of mRNA levels upon acute heat stress, involving mRNAs for HSPs and other stress proteins, with all corresponding genes carrying DNA binding motifs for HIF-1 and HSF-1. This study provided evidence for promoting effects of ROS and HIF-1 on early haemoglobin, HIF-1α and HSP90 mRNA expressions upon heat or ROS stress. The increasing cHSF-1 level likely affected nHSF-1 level and later HSP90 mRNA expression. Heat stress evoked ROS fluctuations, with this stress signal forwarded via nHIF-1 and nHSF-1 fluctuations to stress gene expression. The frequency of ROS fluctuations seemed to integrate information about ROS productionrate and GSH antioxidant buffer capacity, resulting in stress protein expression of different speed. Results of this study suggest ROS as early (pre-damage) and protein defects as later (post-damage) stress signals to trigger heat stress responses. © 2016 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Xiaoli; Jia, Yanfei; Zu, Shanshan

By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and apoptosis of non-small cell lung cancer (NSCLC). Previous studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. However, the mechanisms through which α5-nAChRs may influence lung carcinogenesis are far from clear. In the present study, we investigated the roles of α5-nAChR in the nicotine-induced expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). Immunohistochemistry was used to detect the expression of α5-nAChR and HIF-1α in 60 specimens of lung cancer and para-carcinoma tissue. The correlations between the expression levels ofmore » α5-nAChR and HIF-1α and other clinicopathological data were analyzed. In a cell line that highly expressed α5-nAChR, the loss of α5-nAChR function by siRNA was used to study whether α5-nAChR is involved in the nicotine-induced expression of HIF-1α and VEGF through the activation of the ERK1/2 and PI3K/Akt signaling pathways. Cell growth was detected using the cell counting kit-8 (CCK-8). α5-nAChR (78.3%) and HIF-1α (88.3%) were both overexpressed in NSCLC, and their expression levels were found to be correlated with each other (P < 0.05). In the A549 cell line, α5-nAChR and HIF-1α were found to be expressed under normal conditions, and their expression levels were significantly increased in response to nicotine treatment. The silencing of α5-nAChR significantly inhibited the nicotine-induced cell proliferation compared with the control group and attenuated the nicotine-induced upregulation of HIF-1α and VEGF, and these effects required the cooperation of the ERK1/2 and PI3K/Akt signaling pathways. These results show that the α5-nAChR/HIF-1α/VEGF axis is involved in nicotine-induced tumor cell proliferation, which suggests that α5-nAChR may serve as a potential anticancer target in nicotine-associated lung cancer. - Highlights: • α5-nAChR and HIF-1α over-expressed in NSCLC and correlated with each other. • Nicotine induces cell proliferation and over-expressions of α5-nAChR, HIF-1α and VEGF in NSCLC. • Silence of α5-nAChR inhibited nicotine-induced A549 cell proliferation. • α5-nAChR involved in nicotine-induced HIF-1α and VEGF expression. • Down-regulation of α5-nAChR decreased the phosphorylation level of ERK1/2 and Akt.« less

[Treatment and mechanism of compound carraghenates suppository to rat acute rectal mucous injury].

PubMed

Wang, Zhen-jun; Zhao, Bo; Han, Wei; Tang, Xiu-ying; Yang, Xin-qing; Huang, Yan-ting

2005-05-01

To investigate the treatment and mechanism of compound carraghenates suppository to rat acute rectal mucous injury. The model of rat acute rectal mucous injury was established by 3% acetic acid. Two hundred and forty rats were divided equally into control and experimental group. The rats of experimental group were administrated with 20 mg carraghenates suppository via rectum twice a day, but rats of control group were not administrated with carraghenates suppository. Thirty rats in both groups were executed at different time points. The pathologic changes were observed and the rectal mucous injury was scored. Immunohistochemical staining was used to evaluate the effect of carraghenates suppository on expression of VEGF, iNOS, IL-8, MMP9, HIF-1 alpha and PCNA in the two groups. The scores of rectal mucous injury was lower, the pathologic changes such as hyperaemia, edema, destroy of glands were less severe, and tissue repair time was shorter in experimental group compared with those in the control group at 24 h, 78 h and 120 h after administration of carraghenates suppository. No obvious cicatrisation was observed in experimental group. Expression of VEGF and MMP9 was significantly lower in experimental group compared with those in the control group at 24 h after administration. Expression of VEGF, iNOS, IL-8, MMP9, HIF-1alpha and PCNA were statistically decreased in experimental group than those in the control group at 72 h, 120 h after administration. MVD in experimental group was statistically decreased than that in the control group. The compound carraghenates suppository can reduce the rectal mucous injury from 3% acetic acid, and accelerate the wound healing without obvious cicatrisation. The compound carraghenates suppository can reduce the expression of MMP9, VEGF, IL-8, PCNA, iNOS and HIF-1 alpha, which may play a role in its protective mechanism.

Effects of multi-environmental factors on physiological and biochemical responses of large yellow croaker, Larimichthys crocea.

PubMed

Wang, Qian-Feng; Shen, Wei-Liang; Liu, Cheng; Mu, Dan-Li; Wu, Xiong-Fei; Guo, Nian-Gang; Zhu, Jun-Quan

2017-10-01

Land-based recirculating aquaculture systems (RAS) and cage culture are important methods of Larimichthys crocea production. The effects of environmental factors on physiological and biochemical aspects of L. crocea require clarification. Temperature and salinity are controlled in RAS and directly affect L. crocea growth and survival. To explore optimal parameters, the oxygen consumption rate (R O ), ammonium excretion rate (R N ), and O/N ratio at different temperatures (8, 14, 20, 26, and 32 °C) and salinities (5, 15, 25, and 35‰) were determined. R O , R N , and O/N first increased and then decreased with elevated temperature and salinity, peaking at 26 °C and 25‰, respectively. This suggests that the metabolism of L. crocea was maximal at 26 °C and 25‰ salinity, which promote its growth and survival. Additionally, hypoxia affects cage culture, and has significant effects on enzymatic activities and stress-inducible gene expression. To accelerate the selective breeding of hypoxia-tolerant L. crocea in cage culture, we measured adenosine triphosphatase (ATPase), lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) activities, and hypoxia-inducing factor 1 (HIF-1) mRNA expression in the myocardium under hypoxia (2.5, 3.5, and 4.5 mg L -1 ). ATPase and SDH activities first decreased and then increased under hypoxia, whereas LDH activity and HIF-1α expression first increased and then decreased. Thus, under hypoxia, the myocardial mitochondria switched from being susceptible to being resistant to injury induced by energy metabolism, and respiration in L. crocea likely converted from aerobic to anaerobic during adaptation. Furthermore, the upregulation of HIF-1α mRNA suggests it has an active role in protection against anoxic damage. Copyright © 2017 Elsevier Ltd. All rights reserved.

CB2 cannabinoid receptors modulate HIF-1α and TIM-3 expression in a hypoxia-ischemia mouse model.

PubMed

Kossatz, Elk; Maldonado, Rafael; Robledo, Patricia

2016-12-01

The role of CB2 cannabinoid receptors (CB 2 R) in global brain lesions induced by hypoxia-ischemia (HI) insult is still unresolved. The aim of this study was to evaluate the involvement of CB 2 R in the behavioural and biochemical underpinnings related to brain damage induced by HI in adult mice, and the mechanisms involved. CB 2 R knockout (KO) mice and wild-type littermates (WT) underwent permanent ligation of the left common carotid artery and hypoxia. Behavioural measurements in the rotarod, beam walking, object recognition, open field, and Irwin tests were carried out 24h, 72h and 7 days. In KO mice, more extensive brain injury was observed. Behavioural deficits in the Irwin test were observed in both genotypes; while WT mice showed progressive recovery by day 7, KO mice did not. Only KO mice showed alterations in motor learning, coordination and balance, and did not recover over time. A higher expression of microglia and astrocytes was observed in several brain areas of lesioned WT and KO mice. The possible alteration of the inflammatory-related factors HIF-1α and TIM-3 was evaluated in these animals. In both genotypes, HIF-1α and TIM-3 expression was observed in lesioned areas associated with activated microglia. However, the expression levels of these proteins were exacerbated in KO mice in several lesioned and non-lesioned brain structures. Our results indicate that CB 2 R may have a crucial neuroprotective role following HI insult through the modulation of the inflammatory-related HIF-1α/TIM-3 signalling pathway in microglia. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

Complex role of HIF in cancer: the known, the unknown, and the unexpected

PubMed Central

Tiburcio, Patricia Denise; Choi, Hyunsung; Huang, L Eric

2014-01-01

Tumor hypoxia has long been recognized as a driving force of malignant progression and therapeutic resistance. The discovery of hypoxia-inducible transcription factors (HIFs) has greatly advanced our understanding of how cancer cells cope with hypoxic stress by maintaining bioenergetics through the stimulation of glycolysis. Until recently, however, it remained perplexing why proliferative cancer cells opt for aerobic glycolysis, an energy-inefficient process of glucose metabolism. Furthermore, the role of HIF in cancer has also become complex. In this review, we highlight recent groundbreaking findings in cancer metabolism, put forward plausible explanations to the complex role of HIF, and underscore remaining issues in cancer biology. PMID:27774467

Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase.

PubMed

Selak, Mary A; Armour, Sean M; MacKenzie, Elaine D; Boulahbel, Houda; Watson, David G; Mansfield, Kyle D; Pan, Yi; Simon, M Celeste; Thompson, Craig B; Gottlieb, Eyal

2005-01-01

Several mitochondrial proteins are tumor suppressors. These include succinate dehydrogenase (SDH) and fumarate hydratase, both enzymes of the tricarboxylic acid (TCA) cycle. However, to date, the mechanisms by which defects in the TCA cycle contribute to tumor formation have not been elucidated. Here we describe a mitochondrion-to-cytosol signaling pathway that links mitochondrial dysfunction to oncogenic events: succinate, which accumulates as a result of SDH inhibition, inhibits HIF-alpha prolyl hydroxylases in the cytosol, leading to stabilization and activation of HIF-1alpha. These results suggest a mechanistic link between SDH mutations and HIF-1alpha induction, providing an explanation for the highly vascular tumors that develop in the absence of VHL mutations.

Role of HIF-1α in skeletal development

PubMed Central

Wan, Chao; Shao, Jin; Gilbert, Shawn R.; Riddle, Ryan C.; Long, Fanxin; Johnson, Randall S.; Schipani, Ernestina; Clemens, Thomas L.

2011-01-01

Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration. Mesenchymal cells in the developing stroma elicit angiogenic signals to recruit new blood vessels into bone. Reciprocal signals, likely emanating from the incoming vascular endothelium, stimulate mesenchymal cell specification through additional interactions with cells within the vascular stem cell niche. The hypoxia-inducible factor-1 alpha (HIF-1) pathway has been identified as a key component in this process. We demonstrated that overexpression of HIF-1 in mature osteoblasts through disruption of the von Hippel-Lindau protein profoundly increases angiogenesis and osteogenesis; these processes appear to be coupled by cell nonautonomous mechanisms involving the action of vascular endothelial growth factor (VEGF) on the endothelial cells. The same occurred in the model of injury-mediated bone regeneration (distraction osteogenesis). Surprisingly, manipulation of HIF-1 does not influence angiogenesis of the skull bones, where earlier activation of HIF-1 in the condensing mesenchyme upregulates osterix during cranial bone formation. PMID:20392254

E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis.

PubMed

Jung, Cho-Rok; Hwang, Kyung-Sun; Yoo, Jinsang; Cho, Won-Kyung; Kim, Jin-Man; Kim, Woo Ho; Im, Dong-Soo

2006-07-01

The von Hippel-Lindau tumor suppressor, pVHL, forms part of an E3 ubiquitin ligase complex that targets specific substrates for degradation, including hypoxia-inducible factor-1alpha (HIF-1alpha), which is involved in tumor progression and angiogenesis. It remains unclear, however, how pVHL is destabilized. Here we show that E2-EPF ubiquitin carrier protein (UCP) associates with and targets pVHL for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1alpha. UCP is detected coincidently with HIF-1alpha in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma and colon cancer cells. UCP level correlates inversely with pVHL level in most tumor cell lines. In vitro and in vivo, forced expression of UCP boosts tumor-cell proliferation, invasion and metastasis through effects on the pVHL-HIF pathway. Our results suggest that UCP helps stabilize HIF-1alpha and may be a new molecular target for therapeutic intervention in human cancers.

HIF-2α is essential for carotid body development and function

PubMed Central

Cowburn, Andrew S; Torres-Torrelo, Hortensia; Ortega-Sáenz, Patricia; López-Barneo, José

2018-01-01

Mammalian adaptation to oxygen flux occurs at many levels, from shifts in cellular metabolism to physiological adaptations facilitated by the sympathetic nervous system and carotid body (CB). Interactions between differing forms of adaptive response to hypoxia, including transcriptional responses orchestrated by the Hypoxia Inducible transcription Factors (HIFs), are complex and clearly synergistic. We show here that there is an absolute developmental requirement for HIF-2α, one of the HIF isoforms, for growth and survival of oxygen sensitive glomus cells of the carotid body. The loss of these cells renders mice incapable of ventilatory responses to hypoxia, and this has striking effects on processes as diverse as arterial pressure regulation, exercise performance, and glucose homeostasis. We show that the expansion of the glomus cells is correlated with mTORC1 activation, and is functionally inhibited by rapamycin treatment. These findings demonstrate the central role played by HIF-2α in carotid body development, growth and function. PMID:29671738

The protein level of hypoxia-inducible factor-1alpha is increased in the plateau pika (Ochotona curzoniae) inhabiting high altitudes.

PubMed

Li, Hong-Ge; Ren, Yong-Ming; Guo, Song-Chang; Cheng, Long; Wang, De-Peng; Yang, Jie; Chang, Zhi-Jie; Zhao, Xin-Quan

2009-02-01

The plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant species living only at 3,000-5,000 m above sea-level on the Qinghai-Tibetan plateau. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates a variety of cellular and systemic adaptations to hypoxia. To investigate how the plateau pika adapts to a high-altitude hypoxic environment at the molecular level, we examined the expression pattern of the HIF-1alpha protein in the pika by Western blot and immunohistochemical analysis. We found that HIF-1alpha protein is expressed at a significantly high level in the pika, which is higher in most tissues (particularly in the lung, liver, spleen and kidney) of the plateau pika than that of mice living at sea-level. Importantly, we found that the protein levels of HIF-1alpha in the lung, liver, spleen and kidney of the pika were increased with increased habitat altitudes. We observed that the plateau pika HIF-1alpha localized to the nucleus of cells by an immunostaining analysis, and enhanced HRE-driven gene expression by luciferase reporter assays. Our study suggests that the HIF-1alpha protein levels are related to the adaptation of the plateau pika to the high-altitude hypoxic environment.

HIF-1-mediated production of exosomes during hypoxia is protective in renal tubular cells.

PubMed

Zhang, Wei; Zhou, Xiangjun; Yao, Qisheng; Liu, Yutao; Zhang, Hao; Dong, Zheng

2017-10-01

Exosomes are nano-sized vesicles produced and secreted by cells to mediate intercellular communication. The production and function of exosomes in kidney tissues and cells remain largely unclear. Hypoxia is a common pathophysiological condition in kidneys. This study was designed to characterize exosome production during hypoxia of rat renal proximal tubular cells (RPTCs), investigate the regulation by hypoxia-inducible factor-1 (HIF-1), and determine the effect of the exosomes on ATP-depletion-induced tubular cell injury. Hypoxia did not change the average sizes of exosomes secreted by RPTCs, but it significantly increased exosome production in a time-dependent manner. HIF-1 induction with dimethyloxalylglycine also promoted exosome secretion, whereas pharmacological and genetic suppression of HIF-1 abrogated the increase of exosome secretion under hypoxia. The exosomes from hypoxic RPTCs had inhibitory effects on apoptosis of RPTCs following ATP depletion. The protective effects were lost in the exosomes from HIF-1α knockdown cells. It is concluded that hypoxia stimulates exosome production and secretion in renal tubular cells. The exosomes from hypoxic cells are protective against renal tubular cell injury. HIF-1 mediates exosome production during hypoxia and contributes to the cytoprotective effect of the exosomes. Copyright © 2017 the American Physiological Society.

SN-38 Acts as a Radiosensitizer for Colorectal Cancer by Inhibiting the Radiation-induced Up-regulation of HIF-1α.

PubMed

Okuno, Takayuki; Kawai, Kazushige; Hata, Keisuke; Murono, Koji; Emoto, Shigenobu; Kaneko, Manabu; Sasaki, Kazuhito; Nishikawa, Takeshi; Tanaka, Toshiaki; Nozawa, Hiroaki

2018-06-01

Hypoxia offers resistance to therapy in human solid tumors. The aim of the study was to investigate whether SN-38, the active metabolite of irinotecan, acts as a radiosensitizer through inhibition of hypoxia-inducible factor (HIF)-1α in the human colorectal cancer (CRC) cells. HT29 and SW480 cells were cultured with SN-38 (0-4 μM) immediately after irradiation (0-8 Gy). HIF-1α expression was assessed using flow-cytometry and western blot analysis. Cell proliferation was evaluated by the calcein assay. Apoptosis and cell cycle were determined by flow-cytometry. Radiation up-regulated HIF-1α, and SN-38 inhibited the radiation-induced HIF-1α. The combination of radiation and SN-38 inhibited cell proliferation more than radiation alone; treatment with SN-38 after radiation exposure did not increase the number of apoptotic cells, whereas, it enhanced the S and G 2 /M cell-cycle arrest and decreased the population of cells in G 1 Conclusion: SN-38 inhibits the radiation-induced up-regulation of HIF-1α and acts as a radiosensitizer by inducing cell-cycle arrest in CRC cells. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

[Expression of HIF1-alpha on myocardium and lung in rats model of asphyxia death].

PubMed

Zhang, Geng-qian; Zhou, Bin; Du, Bing; Yang, Zhi-hui; Zhang, Bei-lei; Zhu, Yin-hua; Zhang, Lin

2006-12-01

To investigate the expression of HIF1-alpha in heart and lung tissue died from asphyxia. The rats model of asphyxia death was constructed by hanging, different asphyxia groups and control group sets were made according the postmortem time (0,2,6,24 h), immunohistochemistry and half-quantitative RT-PCR methods were used to investigate expression of HIF1-alpha and mRNA changes on heart and lung tissue. The positive staining of HIF1-alpha could be observed in the myocardium and lung tissue. Significant differences were found between the groups of asphyxia and their corresponding control group. HIF1-alpha expression was found in all the asphyxia groups while it was only expressed in the control groups of 2 h, 6 h and 24 h. Nucleic positive staining could be detected in all the asphyxia groups but none was found in the control groups. RT-PCR showed that the expression of mRNA between 0 h asphyxia group and 0 h control group were equal in both cardic muscle and lung, but elevated expression in groups of 2,6,24h compared to their control groups. The nuclear positive staining of HIF1-alpha in heart and lung can be a special character of suffocation death.

Chamber transport for heavy ion fusion

NASA Astrophysics Data System (ADS)

Olson, Craig L.

2014-01-01

A brief review is given of research on chamber transport for HIF (heavy ion fusion) dating from the first HIF Workshop in 1976 to the present. Chamber transport modes are categorized into ballistic transport modes and channel-like modes. Four major HIF reactor studies are summarized (HIBALL-II, HYLIFE-II, Prometheus-H, OSIRIS), with emphasis on the chamber transport environment. In general, many beams are used to provide the required symmetry and to permit focusing to the required small spots. Target parameters are then discussed, with a summary of the individual heavy ion beam parameters required for HIF. The beam parameters are then classified as to their line charge density and perveance, with special emphasis on the perveance limits for radial space charge spreading, for the space charge limiting current, and for the magnetic (Alfven) limiting current. The major experiments on ballistic transport (SFFE, Sabre beamlets, GAMBLE II, NTX, NDCX) are summarized, with specific reference to the axial electron trapping limit for charge neutralization. The major experiments on channel-like transport (GAMBLE II channel, GAMBLE II self-pinch, LBNL channels, GSI channels) are discussed. The status of current research on HIF chamber transport is summarized, and the value of future NDCX-II transport experiments for the future of HIF is noted.

Activation of Hypoxia-Inducible Factors Prevents Diabetic Nephropathy

PubMed Central

Nordquist, Lina; Friederich-Persson, Malou; Fasching, Angelica; Liss, Per; Shoji, Kumi; Nangaku, Masaomi; Hansell, Peter

2015-01-01

Hyperglycemia results in increased oxygen consumption and decreased oxygen tension in the kidney. We tested the hypothesis that activation of hypoxia-inducible factors (HIFs) protects against diabetes-induced alterations in oxygen metabolism and kidney function. Experimental groups consisted of control and streptozotocin-induced diabetic rats treated with or without chronic cobalt chloride to activate HIFs. We elucidated the involvement of oxidative stress by studying the effects of acute administration of the superoxide dismutase mimetic tempol. Compared with controls, diabetic rats displayed tissue hypoxia throughout the kidney, glomerular hyperfiltration, increased oxygen consumption, increased total mitochondrial leak respiration, and decreased tubular sodium transport efficiency. Diabetic kidneys showed proteinuria and tubulointerstitial damage. Cobalt chloride activated HIFs, prevented the diabetes-induced alterations in oxygen metabolism, mitochondrial leak respiration, and kidney function, and reduced proteinuria and tubulointerstitial damage. The beneficial effects of tempol were less pronounced after activation of HIFs, indicating improved oxidative stress status. In conclusion, activation of HIFs prevents diabetes-induced alteration in kidney oxygen metabolism by normalizing glomerular filtration, which reduces tubular electrolyte load, preventing mitochondrial leak respiration and improving tubular transport efficiency. These improvements could be related to reduced oxidative stress and account for the reduced proteinuria and tubulointerstitial damage. Thus, pharmacologic activation of the HIF system may prevent development of diabetic nephropathy. PMID:25183809

Iron overload promotes erythroid apoptosis through regulating HIF-1a/ROS signaling pathway in patients with myelodysplastic syndrome.

PubMed

Zheng, Qing-Qing; Zhao, You-Shan; Guo, Juan; Zhao, Si-da; Song, Lu-Xi; Fei, Cheng-Ming; Zhang, Zheng; Li, Xiao; Chang, Chun-Kang

2017-07-01

Erythroid apoptosis increases significantly in myelodysplastic syndrome (MDS) patients with iron overload, but the underlying mechanism is not fully clear. In this study, we aim to explore the effect of HIF-1a/ROS on erythroid apoptosis in MDS patients with iron overload. We found that iron overload injured cellular functions through up-regulating ROS levels in MDS/AML cells, including inhibited cell viability, increased cell apoptosis and blocked cell cycle at G0/G1 phase. Interestingly, overexpression of hypoxia inducible factor-1a (HIF-1a), which was under-expressed in iron overload models, reduced ROS levels and attenuated cell damage caused by iron overload in MDS/AML cells. And gene knockdown of HIF-1a got the similar results as iron overload in MDS/AML cells. Furthermore, iron overload caused high erythroid apoptosis was closely related with ROS in MDS patients. Importantly, the HIF-1a protein levels of erythrocytes elevated obviously after incubation with desferrioxamine (DFO) from MDS patients with iron overload, accompanied by ROS levels inhibited and erythroid apoptosis reduced. Taken together, our findings determine that the HIF-1a/ROS signaling pathway plays a key role in promoting erythroid apoptosis in MDS patients with iron overload. Copyright © 2017 Elsevier Ltd. All rights reserved.

Involvement of HIF-1α-regulated miR-21, acting via the Akt/NF-κB pathway, in malignant transformation of HBE cells induced by cigarette smoke extract.

PubMed

Lu, Lu; Xu, Hui; Yang, Ping; Xue, Junchao; Chen, Chao; Sun, Qian; Yang, Qianlei; Lu, Jiachun; Shi, Aimin; Liu, Qizhan

2018-06-01

Although the relationship between cigarette smoke and lung cancer has been widely studied, the molecular mechanism for cigarette smoke-induced lung cancer remains largely unclear. The present study investigated the roles of hypoxia-inducible factor (HIF)-1α and miR-21 in the malignant transformation of human bronchial epithelial (HBE) cells induced by cigarette smoke extract (CSE). In case of acute and chronic treatment of HBE cells, CSE increased the levels of HIF-1α, p-Akt, p-NF-κB, and miR-21 and decreased PTEN levels. The increased miR-21 levels induced by CSE were prevented by down-regulation of HIF-1α. Further, elevated miR-21 suppressed PTEN levels, which decreased the levels of p-Akt and p-NF-κB. However, those changes were attenuated in cells co-transfected with HIF-1α siRNA and an miR-21 mimic. Silencing of HIF-1α or NF-κB decreased colony formation and the invasion and migration capacities of CSE-transformed HBE cells; however, up-regulation of miR-21 reversed these effects. These results indicate that the oncogenic capacity of HIF-1α in regulation of miR-21-inhibited PTEN in a manner dependent on the Akt/NF-κB pathway, a process that is involved in the CSE-induced malignant transformation of HBE cells. Thus, the present research has established a new mechanism for cigarette smoke-induced lung carcinogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

Targeting Hypoxia-Inducible Factor-1α/Pyruvate Dehydrogenase Kinase 1 Axis by Dichloroacetate Suppresses Bleomycin-induced Pulmonary Fibrosis.

PubMed

Goodwin, Justin; Choi, Hyunsung; Hsieh, Meng-Hsiung; Neugent, Michael L; Ahn, Jung-Mo; Hayenga, Heather N; Singh, Pankaj K; Shackelford, David B; Lee, In-Kyu; Shulaev, Vladimir; Dhar, Shanta; Takeda, Norihiko; Kim, Jung-Whan

2018-02-01

Hypoxia has long been implicated in the pathogenesis of fibrotic diseases. Aberrantly activated myofibroblasts are the primary pathological driver of fibrotic progression, yet how various microenvironmental influences, such as hypoxia, contribute to their sustained activation and differentiation is poorly understood. As a defining feature of hypoxia is its impact on cellular metabolism, we sought to investigate how hypoxia-induced metabolic reprogramming affects myofibroblast differentiation and fibrotic progression, and to test the preclinical efficacy of targeting glycolytic metabolism for the treatment of pulmonary fibrosis. Bleomycin-induced pulmonary fibrotic progression was evaluated in two independent, fibroblast-specific, promoter-driven, hypoxia-inducible factor (Hif) 1A knockout mouse models and in glycolytic inhibitor, dichloroacetate-treated mice. Genetic and pharmacological approaches were used to explicate the role of metabolic reprogramming in myofibroblast differentiation. Hypoxia significantly enhanced transforming growth factor-β-induced myofibroblast differentiation through HIF-1α, whereas overexpression of the critical HIF-1α-mediated glycolytic switch, pyruvate dehydrogenase kinase 1 (PDK1) was sufficient to activate glycolysis and potentiate myofibroblast differentiation, even in the absence of HIF-1α. Inhibition of the HIF-1α/PDK1 axis by genomic deletion of Hif1A or pharmacological inhibition of PDK1 significantly attenuated bleomycin-induced pulmonary fibrosis. Our findings suggest that HIF-1α/PDK1-mediated glycolytic reprogramming is a critical metabolic alteration that acts to promote myofibroblast differentiation and fibrotic progression, and demonstrate that targeting glycolytic metabolism may prove to be a potential therapeutic strategy for the treatment of pulmonary fibrosis.

Estrogen stabilizes hypoxia inducible factor 1 α through G protein coupled estrogen receptor 1 in eutopic endometrium of endometriosis

PubMed Central

Zhang, Ling; Xiong, Wenqian; Li, Na; Liu, Hengwei; He, Haitang; Du, Yu; Zhang, Zhibing; Liu, Yi

2016-01-01

Objective To investigate whether G protein-coupled estrogen receptor (GPER, also known as GPR30 and GPER1) stabilizes Hypoxia inducible factor 1α (HIF-1α) in eutopic endometrium (EuEM) of endometriosis? Design Immunohistochemical analysis and experimental in vitro study. Setting University hospital Patient(s) Patients with or without endometriosis Intervention(s) The EuEM and normal control endometrium (CoEM) were obtained by curettage. Primary cultured endometrial stromal cells (ESCs) were treated with 17β-estrogen (E2), G1 or G15. Main Outcome Measure(s) The EuEM and CoEM were collected for immunohistochemistry. Western blot, PCR, Elisa, and dual luciferase experiments were used to detect expression of GPER, HIF-1α, VEGF, and MMP9 in ESCs. E2 and G1 were used as agonists of GPER while G15 as an antagonist. Migration of ESCs and endothelial tube formation of HUVECs cultured in medium collected from ESCs were measured. Results Protein levels of GPER and HIF-1α were higher in EuEM than in CoEM. HIF-1α protein levels but not HIF-1α mRNA levels increased concurrently with GPER after E2 and G1 treatment. Furthermore, expression and activity of VEGF and MMP9 increased under E2 and G1 stimulation. However these effects disappeared when GPER was blocked. Conclusion GPER stabilizes HIF-1α thus promotes HIF-1α induced vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9) in ESCs, which plays critical roles in endometriosis. PMID:27939762

Carbamylated erythropoietin ameliorates the metabolic stress induced in vivo by severe chronic hypoxia

PubMed Central

Fantacci, Monica; Bianciardi, Paola; Caretti, Anna; Coleman, Thomas R.; Cerami, Anthony; Brines, Michael; Samaja, Michele

2006-01-01

Ischemia and chronic hypoxia (CH) trigger a variety of adverse effects arising from metabolic stress that injures cells. In response to reduced O2, hypoxia-inducible factor 1α (HIF-1α) activates erythropoietin (Epo) as well as many other target genes that counteract the effects of O2 deficiency. Epo produced by the kidney stimulates erythrocyte production, leading to decreased HIF-1α production by improved tissue O2 delivery. However, Epo is produced by many other tissues, and it is currently unclear to what extent, if any, locally produced Epo modulates HIF-1α expression. Derivatives of Epo that possess tissue-protective activities but do not stimulate erythropoiesis [e.g., carbamylated Epo (CEpo)] are useful tools with which to determine whether exogenous Epo modulates HIF-1α in the absence of changes in hemoglobin concentration. We compared the effects of CH (6.5% O2 for 10 days) with or without CEpo administered by daily s.c. injection (10 μg/kg of body weight). CEpo administration did not alter the survival rate, weight loss, or increased hemoglobin concentration associated with CH. Therefore, CEpo does not directly suppress HIF-mediated erythropoiesis. CEpo does, however, prevent CH-induced neuronal increases of HIF-1α and Epo receptor-associated immunoreactivity (a measure of stress) while reducing the apoptotic index. In contrast, the myocardium did not exhibit increased HIF-1α expression during CH, although CEpo did reduce the apoptotic index. These observations therefore demonstrate that CEpo administration reduces the metabolic stress caused by severe CH, resulting in improved cellular survival independent of erythrocyte production. PMID:17090665

Magnolol suppresses hypoxia-induced angiogenesis via inhibition of HIF-1α/VEGF signaling pathway in human bladder cancer cells.

PubMed

Chen, Meng-Chuan; Lee, Chi-Feng; Huang, Wen-Hsin; Chou, Tz-Chong

2013-05-01

The hypoxic environment in tumors is an important factor causing tumor angiogenesis by activating the key transcription factor, hypoxia-inducible factors-1α (HIF-1α). Magnolol isolated from Magnolia officinalis has been reported to exhibit an anticancer activity via elevation of apoptosis. However, whether magnolol inhibits tumor angiogenesis remains unknown. In the present study, we demonstrated that magnolol significantly inhibited angiogenesis in vitro and in vivo evidenced by the attenuation of hypoxia and vascular endothelial growth factor (VEGF)-induced tube formation of human umbilical vascular endothelial cells, vasculature generation in chicken chorioallantoic membrane and Matrigel plug. In hypoxic human bladder cancer cells (T24), treatment with magnolol inhibited hypoxia-stimulated H2O2 formation, HIF-1α induction including mRNA, protein expression, and transcriptional activity as well as VEGF secretion. Additionally, the enhanced degradation of HIF-1α protein via enhancing prolyl hydroxylase activity and the decreased newly-synthesized HIF-1α protein in hypoxic T24 cells may involve the reduction of HIF-1α protein accumulation by magnolol. Interestingly, magnolol also acts as a VEGFR2 antagonist, and subsequently attenuates the down-stream AKT/mTOR/p70S6K/4E-BP-1 kinase activation both in hypoxic T24 cells and tumor tissues. As expected, administration of magnolol greatly attenuated tumor growth, angiogenesis and the protein expression of HIF-1α, VEGF, CD31, a marker of endothelial cells, and carbonic anhydrase IX, an endogenous marker for hypoxia, in the T24 xenograft mouse model. Collectively, these findings strongly indicate that the anti-agngiogenic activity of magnolol is, at least in part, mediated by suppressing HIF-1α/VEGF-dependent pathways, and suggest that magnolol may be a potential drug for human bladder cancer therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

The two glycolytic markers GLUT1 and MCT1 correlate with tumor grade and survival in clear-cell renal cell carcinoma

PubMed Central

Dadone, Bérengère; Durand, Matthieu; Borchiellini, Delphine; Amiel, Jean; Pouyssegur, Jacques; Rioux-Leclercq, Nathalie; Pages, Gilles; Burel-Vandenbos, Fanny; Mazure, Nathalie M.

2018-01-01

Background Clear-cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer. Although ccRCC is characterized by common recurrent genetic abnormalities, including inactivation of the von Hippel-Lindau (vhl) tumor suppressor gene resulting in stabilization of hypoxia-inducible factors (HIFs), the tumor aggressiveness and outcome of ccRCC is variable. New biomarkers are thus required to improve ccRCC diagnosis, prognosis and therapeutic options. This work aims to investigate the expression of HIF and proteins involved in metabolism and pH regulation. Their correlation to histoprognostic parameters and survival was analyzed. Methods ccRCC of 45 patients were analyzed. HIF-1α, HIF-2α, HAF, GLUT1, MCT1, MCT4, CAIX and CAXII expression was assessed by immunohistochemistry in a semi-quantitative and qualitative manner. The GLUT1, MCT1, MCT4, CAIX and CAXII mRNA levels were analyzed in an independent cohort of 43 patients. Results A significant correlation was observed between increased GLUT1, MCT1, CAXII protein expression and a high Fuhrman grade in ccRCC patients. Moreover, while HIF-1α, HIF-2α and HAF expression was heterogenous within tumors, we observed and confirmed that HIF-2α co-localized with HAF. We confirmed, in an independent cohort, that GLUT1, MCT1 and CAXII mRNA levels correlated with the Fuhrman grade. Moreover, we demonstrated that the high mRNA level of both MCT1 and GLUT1 correlated with poor prognosis. Conclusions This study demonstrates for the first time a link between the aggressiveness of high- Fuhrman grade ccRCC and metabolic reprogramming. It also confirms the role of HIF-2α and HAF in tumor invasiveness. Finally, these results demonstrate that MCT1 and GLUT1 are strong prognostic markers and promising therapeutic targets. PMID:29481555

Inhibition of HIF-1α decreases expression of pro-inflammatory IL-6 and TNF-α in diabetic retinopathy.

PubMed

Gao, Xiuhua; Li, Yonghua; Wang, Hongxia; Li, Chuanbao; Ding, Jianguang

2017-12-01

Recent studies demonstrate that pro-inflammatory cytokines (PICs, i.e. IL-1β, IL-6 and TNF-α) in retinal tissues are likely involved in the development of diabetic retinopathy (DR). In this report, we particularly examined contributions of hypoxia inducible factor subtype 1α (HIF-1α) to the expression of PICs and their receptors in diabetic retina. Streptozotocin (STZ) was systemically injected to induce hyperglycaemia in rats. ELISA and Western blot analysis were employed to determine the levels of HIF-1α and PICs as well as PIC receptors in retinal tissues of control rats and STZ rats. The levels of retinal HIF-1α were significantly increased in STZ rats 4-10 weeks after induction of hyperglycaemia as compared with control animals. With increasing HIF-1α retinal PICs including IL-1β, IL-6 and TNF-α, their respective receptors, namely IL-1R, IL-6R and TNFR1, were also elevated in STZ rats. Moreover, inhibition of HIF-1α by injection of 2-methoxyestradiol (2-MET) significantly decreased the amplified expression IL-6, TNF-α, IL-6R and TNFR1 in diabetic retina, but did not modify IL-1β pathway. In addition, we examined protein expression of Caspase-3 indicating cell apoptosis in the retina of STZ rats after infusing 2-MET, demonstrating that 2-MET attenuated an increase in Caspase-3 evoked by STZ. Hypoxia inducible factor subtype 1α (HIF-1α) activated in diabetic retina is likely to play a role in regulating pathophysiological process via IL-6 and TNF-α mechanism. This has pharmacological implications to target specific HIF-1α, IL-6 and TNF-α signalling pathway for dysfunction and vulnerability related to DR. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Pneumococcal infection of respiratory cells exposed to welding fumes; Role of oxidative stress and HIF-1 alpha.

PubMed

Grigg, Jonathan; Miyashita, Lisa; Suri, Reetika

2017-01-01

Welders are more susceptible to pneumococcal pneumonia. The mechanisms are yet unclear. Pneumococci co-opt the platelet activating factor receptor (PAFR) to infect respiratory epithelial cells. We previously reported that exposure of respiratory cells to welding fumes (WF), upregulates PAFR-dependent pneumococcal infection. The signaling pathway for this response is unknown, however, in intestinal cells, hypoxia-inducible factor-1 α (HIF 1α) is reported to mediate PAFR-dependent infection. We sought to assess whether oxidative stress plays a role in susceptibility to pneumococcal infection via the platelet activating factor receptor. We also sought to evaluate the suitability of nasal epithelial PAFR expression in welders as a biomarker of susceptibility to infection. Finally, we investigated the generalisability of the effect of welding fumes on pneumococcal infection and growth using a variety of different welding fume samples. Nasal epithelial PAFR expression in welders and controls was analysed by flow cytometry. WF were collected using standard methodology. The effect of WF on respiratory cell reactive oxygen species production, HIF-1α expression, and pneumococcal infection was determined using flow cytometry, HIF-1α knockdown and overexpression, and pneumococcal infection assays. We found that nasal PAFR expression is significantly increased in welders compared with controls and that WF significantly increased reactive oxygen species production, HIF-1α and PAFR expression, and pneumococcal infection of respiratory cells. In unstimulated cells, HIF-1α knockdown decreased PAFR expression and HIF-1α overexpression increased PAFR expression. However, in knockdown cells pneumococcal infection was paradoxically increased and in overexpressing cells infection was unaffected. Nasal epithelial PAFR expression may be used as a biomarker of susceptibility to pneumococcal infection in order to target individuals, particularly those at high risk such as welders, for the pneumococcal vaccine. Expression of HIF-1α in unexposed respiratory cells inhibits basal pneumococcal infection via PAFR-independent mechanisms.

The two glycolytic markers GLUT1 and MCT1 correlate with tumor grade and survival in clear-cell renal cell carcinoma.

PubMed

Ambrosetti, Damien; Dufies, Maeva; Dadone, Bérengère; Durand, Matthieu; Borchiellini, Delphine; Amiel, Jean; Pouyssegur, Jacques; Rioux-Leclercq, Nathalie; Pages, Gilles; Burel-Vandenbos, Fanny; Mazure, Nathalie M

2018-01-01

Clear-cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer. Although ccRCC is characterized by common recurrent genetic abnormalities, including inactivation of the von Hippel-Lindau (vhl) tumor suppressor gene resulting in stabilization of hypoxia-inducible factors (HIFs), the tumor aggressiveness and outcome of ccRCC is variable. New biomarkers are thus required to improve ccRCC diagnosis, prognosis and therapeutic options. This work aims to investigate the expression of HIF and proteins involved in metabolism and pH regulation. Their correlation to histoprognostic parameters and survival was analyzed. ccRCC of 45 patients were analyzed. HIF-1α, HIF-2α, HAF, GLUT1, MCT1, MCT4, CAIX and CAXII expression was assessed by immunohistochemistry in a semi-quantitative and qualitative manner. The GLUT1, MCT1, MCT4, CAIX and CAXII mRNA levels were analyzed in an independent cohort of 43 patients. A significant correlation was observed between increased GLUT1, MCT1, CAXII protein expression and a high Fuhrman grade in ccRCC patients. Moreover, while HIF-1α, HIF-2α and HAF expression was heterogenous within tumors, we observed and confirmed that HIF-2α co-localized with HAF. We confirmed, in an independent cohort, that GLUT1, MCT1 and CAXII mRNA levels correlated with the Fuhrman grade. Moreover, we demonstrated that the high mRNA level of both MCT1 and GLUT1 correlated with poor prognosis. This study demonstrates for the first time a link between the aggressiveness of high- Fuhrman grade ccRCC and metabolic reprogramming. It also confirms the role of HIF-2α and HAF in tumor invasiveness. Finally, these results demonstrate that MCT1 and GLUT1 are strong prognostic markers and promising therapeutic targets.

Apparent diffusion coefficients in prostate cancer: correlation with molecular markers Ki-67, HIF-1α and VEGF.

PubMed

Ma, Teng; Yang, Shaolin; Jing, Haiyan; Cong, Lin; Cao, Zhixin; Liu, Zhiling; Huang, Zhaoqin

2018-03-01

Prostate cancer (PCa) is the second most common cancer in men. The Gleason score (GS) and biomarkers play important roles in the diagnosis and treatment of patients with PCa. The purpose of this study was to investigate the relationship between the apparent diffusion coefficient (ADC) and the molecular markers Ki-67, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in PCa. Thirty-nine patients with 39 lesions, who had been diagnosed with PCa, were enrolled in this study. All patients underwent diffusion-weighted magnetic resonance imaging (DW-MRI) (b = 800 s/mm 2 ). The expression of Ki-67, HIF-1α and VEGF was assessed by immunohistochemistry. Statistical analysis was applied to analyze the association between ADC and prostate-specific antigen (PSA), GS and the expression of Ki-67, HIF-1α and VEGF. The group differences in ADC among different grades of Ki-67, HIF-1α and VEGF were also analyzed. The mean ± standard deviation of ADC was (0.76 ± 0.27) × 10 -3  mm 2 /s. ADC correlated negatively with PSA and GS (p < 0.05). The Ki-67 staining index (SI), HIF-1α expression and VEGF expression in PCa were correlated inversely with ADC, controlling for age (r = -0.332, p < 0.05; r = -0.662, p < 0.0005; and r = -0.714, p < 0.0005, respectively). ADC showed a significant difference among different grades of Ki-67 (F = 9.164, p = 0.005), HIF-1α (F = 40.333, p < 0.0005) and VEGF (F = 22.048, p < 0.0005). In conclusion, ADC was correlated with PSA, GS, and Ki-67, HIF-1α and VEGF expression in patients with PCa. ADC may be used to evaluate tumor proliferation, hypoxia and angiogenesis in PCa. Copyright © 2018 John Wiley & Sons, Ltd.

Enhanced Aerobic Glycolysis by S-Nitrosoglutathione via HIF-1α Associated GLUT1/Aldolase A Axis in Human Endothelial Cells.

PubMed

Yan, Jieping; Huang, Xin; Zhu, Danyan; Lou, Yijia

2017-08-01

S-nitrosoglutathione (GSNO)-induced apoptosis is associated with reactive oxygen species and loss of mitochondrial Omi/HtrA2 in human endothelial cells (ECs). But its upstream regulation is still not elucidated. Here, we demonstrate that hypoxia induced factor-1α (HIF-1α)-linked aerobic glycolysis is associated with mitochondrial abnormality by treatment of human EC-derived EA.hy926 cells with GSNO (500 µM) for 6 h. GSNO exposure increased the levels of Aldolase A and glucose transporter-1 (GLUT1) mRNAs and proteins. And selectively enhanced aldolase A activity to form glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, which subsequently increased intracellular levels of methylglyoxal and reactive oxygen species in parallel. Using the biotin switch assay, we found that GSNO increased the S-nitrosylating levels of total protein and HIF-1α. Knockdown of HIF-1α with siRNA attenuated its target aldolase A and GLUT1 expression but not VEGF. In contrast, nitrosylation scanvenger dithiothreitol could decrease all the protein levels. It suggested that aerobic glycolytic flux was more dependent on HIF-1α level, and that HIF-1α S-nitrosylation was crucial for its target expression under the normoxic condition. Moreover, GSNO-induced PI3 K (phosphoinositide 3-kinase)/Akt phosphorylation might contribute to HIF-1α stabilization and nucleus translocation, thereby aiding aldolase A and GLUT1 mRNAs upregulation. Taken together, higher concentration GSNO promotes glycolytic flux enhancement and methylglyoxal formation via HIF-1α S-nitrosylation. These findings reveal the mechanism of enhanced glycolysis-associated mitochondrial dysfunction in ECs by GSNO exposure under normoxic and non-hyperglycemic condition. And offer the early potential targets for vascular pathophysiological evaluation. J. Cell. Biochem. 118: 2443-2453, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

A gain-of-function mutation in Tnni2 impeded bone development through increasing Hif3a expression in DA2B mice.

PubMed

Zhu, Xiaoquan; Wang, Fengchao; Zhao, Yanyang; Yang, Peng; Chen, Jun; Sun, Hanzi; Liu, Lei; Li, Wenjun; Pan, Lin; Guo, Yanru; Kou, Zhaohui; Zhang, Yu; Zhou, Cheng; He, Jiang; Zhang, Xue; Li, Jianxin; Han, Weitian; Li, Jian; Liu, Guanghui; Gao, Shaorong; Yang, Ze

2014-10-01

Distal arthrogryposis type 2B (DA2B) is an important genetic disorder in humans. However, the mechanisms governing this disease are not clearly understood. In this study, we generated knock-in mice carrying a DA2B mutation (K175del) in troponin I type 2 (skeletal, fast) (TNNI2), which encodes a fast-twitch skeletal muscle protein. Tnni2K175del mice (referred to as DA2B mice) showed typical DA2B phenotypes, including limb abnormality and small body size. However, the current knowledge concerning TNNI2 could not explain the small body phenotype of DA2B mice. We found that Tnni2 was expressed in the osteoblasts and chondrocytes of long bone growth plates. Expression profile analysis using radii and ulnae demonstrated that Hif3a expression was significantly increased in the Tnni2K175del mice. Chromatin immunoprecipitation assays indicated that both wild-type and mutant tnni2 protein can bind to the Hif3a promoter using mouse primary osteoblasts. Moreover, we showed that the mutant tnni2 protein had a higher capacity to transactivate Hif3a than the wild-type protein. The increased amount of hif3a resulted in impairment of angiogenesis, delay in endochondral ossification, and decrease in chondrocyte differentiation and osteoblast proliferation, suggesting that hif3a counteracted hif1a-induced Vegf expression in DA2B mice. Together, our data indicated that Tnni2K175del mutation led to abnormally increased hif3a and decreased vegf in bone, which explain, at least in part, the small body size of Tnni2K175del mice. Furthermore, our findings revealed a new function of tnni2 in the regulation of bone development, and the study of gain-of-function mutation in Tnni2 in transgenic mice opens a new avenue to understand the pathological mechanism of human DA2B disorder.

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

PubMed

Günter, Julia; Ruiz-Serrano, Amalia; Pickel, Christina; Wenger, Roland H; Scholz, Carsten C

2017-07-15

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level. Copyright © 2017 Elsevier Inc. All rights reserved.

Cobalt Chloride Upregulates Impaired HIF-1α Expression to Restore Sevoflurane Post-conditioning-Dependent Myocardial Protection in Diabetic Rats

PubMed Central

Wu, Jianjiang; Yang, Long; Xie, Peng; Yu, Jin; Yu, Tian; Wang, Haiying; Maimaitili, Yiliyaer; Wang, Jiang; Ma, Haiping; Yang, Yining; Zheng, Hong

2017-01-01

Previous studies from our group have demonstrated that sevoflurane post-conditioning (SPC) protects against myocardial ischemia reperfusion injury via elevating the intranuclear expression of hypoxia inducible factor-1 alpha (HIF-1α). However, diabetic SPC is associated with decreased myocardial protection and disruption of the HIF-1 signaling pathway. Previous studies have demonstrated that cobalt chloride (CoCl2) can upregulate HIF-1α expression under diabetic conditions, but whether myocardial protection by SPC can be restored afterward remains unclear. We established a rat model of type 2 diabetes and a Langendorff isolated heart model of ischemia-reperfusion injury. Prior to reperfusion, 2.4% sevoflurane was used as a post-conditioning treatment. The diabetic rats were treated with CoCl2 24 h before the experiment. At the end of reperfusion, tests were performed to assess myocardial function, infarct size, mitochondrial morphology, nitric oxide (NO), Mitochondrial reactive oxygen species (ROS), mitochondrial respiratory function and enzyme activity, HIF-1α, vascular endothelial growth factor (VEGF) and endothelial NO synthase (eNOS) protein levels. In addition, myocardial protection by SPC was monitored after the blood glucose levels were lowered by insulin. The diabetic state was associated with deficient SPC protection and decreased HIF-1α expression. After treating the diabetic rats with CoCl2, SPC significantly upregulated the expression of HIF-1α, VEGF and eNOS, which markedly improved cardiac function, NO, mitochondrial respiratory function, and enzyme activity and decreased the infarction areas and ROS. In addition, these effects were not influenced by blood glucose levels. This study proved that CoCl2activates the HIF-1α signaling pathway, which restores SPC-dependent myocardial protection under diabetic conditions, and the protective effects of SPC were independent of blood glucose levels. PMID:28659817

Induction of hypoxia-inducible factor-1alpha and activation of caspase-3 in hypoxia-reoxygenated bone marrow stroma is negatively regulated by the delayed production of substance P.

PubMed

Qian, J; Ramroop, K; McLeod, A; Bandari, P; Livingston, D H; Harrison, J S; Rameshwar, P

2001-10-15

The bone marrow (BM), which is the major site of immune cell development in the adult, responds to different stimuli such as inflammation and hemorrhagic shock. Substance P (SP) is the major peptide encoded by the immune/hemopoietic modulator gene, preprotachykinin-1 (PPT-I). Differential gene expression using a microarray showed that SP reduced hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA levels in BM stroma. Because long-term hypoxia induced the expression of PPT-I in BM mononuclear cells, we used timeline studies to determine whether PPT-I is central to the biologic responses of BM stroma subjected to 30-min hypoxia (pO(2) = 35 mm Hg) followed by reoxygenation. HIF-1alpha mRNA and protein levels were increased up to 12 h. At this time, beta-PPT-I mRNA was detected with the release of SP at 16 h. SP release correlated with down-regulation of HIF-1alpha to baseline. A direct role for SP in HIF-1alpha expression was demonstrated as follows: 1) transient knockout of beta-PPT-I showed an increase in HIF-1alpha expression up to 48 h of reoxygenation; and 2) HIF-1alpha expression remained baseline during reoxygenation when stroma was subjected to hypoxia in the presence of SP. Reoxygenation activated the PPT-I promoter with concomitant nuclear translocation of HIF-1alpha that can bind to the respective consensus sequences within the PPT-I promoter. SP reversed active caspase-3, an indicator of apoptosis and erythropoiesis, to homeostasis level after reoxygenation of hypoxic stroma. The results show that during reoxgenation the PPT-I gene acts as a negative regulator on the expression of HIF-1alpha and active caspase-3 in BM stroma subjected to reoxygenation.

Prolyl hydroxylase domain 2 deficiency promotes skeletal muscle fiber-type transition via a calcineurin/NFATc1-dependent pathway.

PubMed

Shin, Junchul; Nunomiya, Aki; Kitajima, Yasuo; Dan, Takashi; Miyata, Toshio; Nagatomi, Ryoichi

2016-01-01

Hypoxia exposure is known to induce an alteration in skeletal muscle fiber-type distribution mediated by hypoxia-inducible factor (HIF)-α. The downstream pathway of HIF-α leading to fiber-type shift, however, has not been elucidated. The calcineurin pathway is one of the pathways responsible for slow muscle fiber transition. Because calcineurin pathway is activated by vascular endothelial growth factor (VEGF), one of the factors induced by HIF-1α, we hypothesized that the stabilization of HIF-1α may lead to slow muscle fiber transition via the activation of calcineurin pathway in skeletal muscles. To induce HIF-1α stabilization, we used a loss of function strategy to abrogate Prolyl hydroxylase domain protein (PHD) 2 responsible for HIF-1α hydroxylation making HIF-1α susceptible to ubiquitin dependent degradation by proteasome. The purpose of this study was therefore to examine the effect of HIF-1α stabilization in PHD2 conditional knockout mouse on skeletal muscle fiber-type transition and to elucidate the involvement of calcineurin pathway on muscle fiber-type transition. PHD2 deficiency resulted in an increased capillary density in skeletal muscles due to the induction of vascular endothelial growth factor. It also elicited an alteration of skeletal muscle phenotype toward the type I fibers in both of the soleus (35.8 % in the control mice vs. 46.7 % in the PHD2-deficient mice, p < 0.01) and the gastrocnemius muscle (0.94 vs. 1.89 %, p < 0.01), and the increased proportion of type I fibers appeared to correspond to the area of increased capillary density. In addition, calcineurin and nuclear factor of activated T cell (NFATc1) protein levels were increased in both the gastrocnemius and soleus muscles, suggesting that the calcineurin/NFATc1 pathway was responsible for the type I fiber transition regardless of PGC-1α, which responded minimally to PHD2 deficiency. Indeed, we found that tacrolimus (FK-506), a calcineurin inhibitor, successfully suppressed slow fiber-type formation in PHD2-deficient mice. Taken together, stabilized HIF-1α induced by PHD2 conditional knockout resulted in the transition of muscle fibers toward a slow fiber type via a calcineurin/NFATc1 signaling pathway. PHD2 conditional knockout mice may serve as a model for chronic HIF-1α stabilization as in mice exposed to low oxygen concentration.

Developmentally arrested structures preceding cerebellar tumors in von Hippel–Lindau disease

PubMed Central

Shively, Sharon B; Falke, Eric A; Li, Jie; Tran, Maxine G B; Thompson, Eli R; Maxwell, Patrick H; Roessler, Erich; Oldfield, Edward H; Lonser, Russell R; Vortmeyer, Alexander O

2011-01-01

There is increasing evidence that suggests that knockout of tumor-suppressor gene function causes developmental arrest and protraction of cellular differentiation. In the peripheral nervous system of patients with the tumor-suppressor gene disorder, von Hippel–Lindau disease, we have demonstrated developmentally arrested structural elements composed of hemangioblast progenitor cells. Some developmentally arrested structural elements progress to a frank tumor, hemangioblastoma. However, in von Hippel–Lindau disease, hemangioblastomas are frequently observed in the cerebellum, suggesting an origin in the central nervous system. We performed a structural and topographic analysis of cerebellar tissues obtained from von Hippel–Lindau disease patients to identify and characterize developmentally arrested structural elements in the central nervous system. We examined the entire cerebella of five tumor-free von Hippel–Lindau disease patients and of three non-von Hippel–Lindau disease controls. In all, 9 cerebellar developmentally arrested structural elements were detected and topographically mapped in 385 blocks of von Hippel–Lindau disease cerebella. No developmentally arrested structural elements were seen in 214 blocks from control cerebella. Developmentally arrested structural elements are composed of poorly differentiated cells that express hypoxia-inducible factor (HIF)2α, but not HIF1α or brachyury, and preferentially involve the molecular layer of the dorsum cerebelli. For the first time, we identify and characterize developmentally arrested structural elements in the central nervous system of von Hippel–Lindau patients. We provide evidence that developmentally arrested structural elements in the cerebellum are composed of developmentally arrested hemangioblast progenitor cells in the molecular layer of the dorsum cerebelli. PMID:21499240

Hypoxia in Invasion and Metastasis

DTIC Science & Technology

2007-08-01

hypoxia and activating HIF-1 downregulate the DNA mismatch repair proteins ( mlh1 and/or msh2), a group of important proteins for maintaining genetic...Investigate the hypoxia and activating HIF-1 downregulate the DNA mismatch repair proteins ( mlh1 and/or msh2) (Month 7-12) Methods: We performed a parallel...inducible factors from invasive tumor cells. Changes in the level of multiple hypoxia related factor (HIF-1) and DNA mismatch repair proteins ( MLH1 , MSH2

[Correlation between bacterial L-form infection, expression of HIF-1α/MMP-9 and vasculogenic mimicry in epithelial ovarian cancer].

PubMed

Yu, Lan; Wu, Shi-Wu; Zhou, Lei; Song, Wen-Qing

2012-12-25

The aim of the present study is to explore whether vasculogenic mimicry (VM) and bacterial L-form infection exist in human epithelial ovarian cancer (EOC) or not and to elucidate the correlation of L-form infection, expression of hypoxia inducible factor 1α (HIF-1α)/MMP-9 and VM. In 87 specimens of EOC and 20 specimens of ovarian benign epithelial tumor tissues, L-form infection was detected by Gram's staining, expression of HIF-1α/MMP-9 and VM were detected by immunohistochemical and histochemical staining. The results showed that the positive rates of HIF-1α and MMP-9 protein in EOC were 52.9% and 66.7%, while in benign epithelial tumor tissues, the positive rates were 10.0% and 10.0% respectively, and there were significant differences between them (P < 0.05). In EOC and benign epithelial tumor tissues, L-form infections ratios were 24.1% and 0, respectively, and the difference was also significant (P < 0.01). Expression of VM, HIF-1α and MMP-9 in EOC was significantly related to differentiation, abdominal implantation and lymph node metastasis and FIGO stage (P < 0.01). L-form infection had relationship with abdominal implantation, lymph node metastasis and FIGO stage (P < 0.01 or 0.05). The expression of HIF-1α had positive relationship with expression of MMP-9 and VM (r = 0.505, 0.585, respectively, P < 0.01); there was also a positive relationship between MMP-9 expression and VM (r = 0.625, P < 0.01). Overexpression of VM, HIF-1α and MMP-9 were related to poor prognosis: the survival rates were significantly lower in positive patients than those in negative patients (P < 0.05). And the group with L-form infection also had poor prognosis: the survival rates were lower than those in group without infection (P < 0.05). FIGO stage, expression of VM, HIF-1α and MMP-9 were independent prognosis factors of EOC (P < 0.05). The results suggest that L-form infection, the expression of HIF-1α, MMP-9 and VM in EOC are related to differentiation, lymph node metastasis, clinical stage and prognosis. Combined detection of these indexes has an important role in predicting the progression and prognosis of EOC.

Expression of hypoxia-inducible factor 1α mRNA in hearts and lungs of broiler chickens with ascites syndrome induced by excess salt in drinking water.

PubMed

Zhang, Jianjun; Feng, Xuejian; Zhao, Lihong; Wang, Wei; Gao, Mingyu; Wu, Boning; Qiao, Jian

2013-08-01

Hypoxia-inducible factor 1 (HIF-1) is a ubiquitously expressed heterodimeric transcription factor that mediates adaptive responses to hypoxia in all nucleated cells of metazoan organisms. Hypoxia-inducible factor 1α is involved in the pathogenesis of pulmonary hypertension in humans and animals, but whether HIF-1α is associated with the development of pulmonary hypertension syndrome (also known as ascites syndrome, AS) in broiler chickens has not been determined. In the present paper we addressed this issue by measuring the expression of HIF-1α mRNA in hearts and lungs of broiler chickens with AS induced by excess salt in drinking water. We conducted 2 experiments. The first experiment was used to observe the effects of excess salt on AS incidence. The results indicated that total incidence (20%) of AS in excess salt group (receiving 0.3% NaCl in drinking water) was much higher compared with the control group (receiving tap water) over a 43-d time course (P < 0.05). In the second experiment, we determined mean pulmonary arterial pressure (mPAP), ascites heart index (AHI), and expression of HIF-1α mRNA in lungs and hearts of broiler chickens after the excess salt treatment. Our results showed that excess salt induced pulmonary hypertension (indicated by higher mPAP) and right ventricular hypertrophy (greater ascites heart index) in broiler chickens. Meanwhile, the expression levels of HIF-1α mRNA in lungs and hearts were significantly increased at different time points in the excess salt group compared with the control group. Linear correlation analysis showed that the expression of HIF-1α mRNA in lungs was significantly positively correlated with mPAP (correlation coefficient = 0.79, P < 0.001), demonstrating that expression of HIF-1α mRNA was gradually increased in the excess salt group with the increase of pulmonary arterial pressure. In addition, the ascitic chickens showed significantly higher transcriptional levels of HIF-1α in hearts and lungs, compared with the age-matched healthy chickens, respectively. Our findings hinted that HIF-1α might be associated with the development of AS induced by excess salt in drinking water in broiler chickens.

The effects of icariin on the expression of HIF-1α, HSP-60 and HSP-70 in PC12 cells suffered from oxygen-glucose deprivation-induced injury.

PubMed

Mo, Zhen-Tao; Li, Wen-Na; Zhai, Yu-Rong; Gao, Shu-Ying

2017-12-01

The effects of icariin, a chief constituent of flavonoids from Epimedium brevicornum Maxim (Berberidaceae), on the levels of HIF-1α, HSP-60 and HSP-70 remain unknown. To explore the effects of icariin on the levels of HSP-60, HIF-1α and HSP-70 neuron-specific enolase (NSE) and cell viability. PC12 cells were treated with icariin (10 -7 , 10 -6 or 10 -5  mol/L) for 3 h (1 h before oxygen-glucose deprivation (OGD) plus 2 h OGD). HSP-60, HIF-1α, HSP-70 and NSE were measured using enzyme-linked immunosorbent assay (ELISA). Cell viability was determined by metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After 2 h OGD, levels of HIF-1α, HSP-60, HSP-70 and NSE were increased significantly (HIF-1α: 33.3 ± 1.9 ng/L, HSP-60: 199 ± 16 ng/L, HSP-70: 195 ± 17 ng/L, NSE: 1487 ± 125 ng/L), and cell viability was significantly decreased (0.26 ± 0.03), while icariin (10 -7 , 10 -6 , or 10 -5  mol/L) significantly reduced the contents of HIF-1α, HSP-60, HSP-70 and NSE (HIF-1α: 14.1 ± 1.4, 22.6 ± 1.8, 15.7 ± 2.1, HSP-60: 100 ± 12, 89 ± 6, 113 ± 11, HSP-70: 139 ± 9, 118 ± 7, 95 ± 9 and NSE: 1121 ± 80, 1019 ± 52, 731 ± 88), and improved cell viability (0.36 ± 0.03, 0.38 ± 0.04, 0.37 ± 0.03) in OGD-treated PC12 cells. These results indicate that the protective mechanisms of icariin against OGD-induced injury may be related to down-regulating the expression of HIF-1α, HSP-60 and HSP-70.

Peptide-matrix-mediated gene transfer of an oxygen-insensitive hypoxia-inducible factor-1alpha variant for local induction of angiogenesis.

PubMed

Trentin, Diana; Hall, Heike; Wechsler, Sandra; Hubbell, Jeffrey A

2006-02-21

Hypoxia-inducible factor (HIF) constitutes a target in therapeutic angiogenesis. HIF-1alpha functions as a sensor of hypoxia and induces expression of vascular endothelial growth factor (VEGF), which then induces angiogenesis. To explore the potential of HIF-1alpha gene therapy in stimulating wound healing, we delivered a gene encoding a stabilized form of HIF-1alpha, lacking the oxygen-sensitive degradation domain, namely HIF-1alpha deltaODD, by using a previously characterized peptide-based gene delivery vector in fibrin as a surgical matrix. The peptide vector consisted of multiple domains: (i) A cysteine-flanked lysine hexamer provided DNA interactions that were stable extracellularly but destabilized intracellularly after reduction of the formed disulfide bonds. This DNA-binding domain was fused to either (ii) a fibrin-binding peptide for entrapment within the matrix or (iii) a nuclear localization sequence for efficient nuclear targeting. The HIF-1alpha deltaODD gene was expressed and translocated to the nucleus under normoxic conditions, leading to up-regulation of vascular endothelial growth factor (VEGF)-A165 mRNA and protein levels in vitro. When the peptide-DNA nanoparticles entrapped in fibrin matrices were applied to full-thickness dermal wounds in the mouse (10 microg per wound in 30 microl of fibrin), angiogenesis was increased comparably strongly to that induced by VEGF-A165 protein (1.25 microg per wound in 30 microl of fibrin). However, the maturity of the vessels induced by HIF-1alpha deltaODD was significantly higher than that induced by VEGF-A165 protein, as shown by stabilization of the neovessels with smooth muscle. Nonviral, local administration of this potent angiogenesis-inducing gene by using this peptide vector represents a powerful approach in tissue engineering and therapeutic angiogenesis.

Circadian factors BMAL1 and RORα control HIF-1α transcriptional activity in nucleus pulposus cells: implications in maintenance of intervertebral disc health

PubMed Central

Suyama, Kaori; Silagi, Elizabeth S.; Choi, Hyowon; Sakabe, Kou; Mochida, Joji; Shapiro, Irving M.; Risbud, Makarand V.

2016-01-01

BMAL1 and RORα are major regulators of the circadian molecular oscillator. Since previous work in other cell types has shown cross talk between circadian rhythm genes and hypoxic signaling, we investigated the role of BMAL1 and RORα in controlling HIF-1-dependent transcriptional responses in NP cells that exist in the physiologically hypoxic intervertebral disc. HIF-1-dependent HRE reporter activity was further promoted by co-transfection with either BMAL1 or RORα. In addition, stable silencing of BMAL1 or inhibition of RORα activity resulted in decreased HRE activation. Inhibition of RORα also modulated HIF1α-TAD activity. Interestingly, immunoprecipitation studies showed no evidence of BMAL1, CLOCK or RORα binding to HIF-1α in NP cells. Noteworthy, stable silencing of BMAL1 as well as inhibition of RORα decreased expression of select HIF-1 target genes including VEGF, PFKFB3 and Eno1. To delineate if BMAL1 plays a role in maintenance of disc health, we studied the spinal phenotype of BMAL1-null mice. The lumbar discs of null mice evidenced decreased height, and several parameters associated with vertebral trabecular bone quality were also affected in nulls. In addition, null animals showed a higher ratio of cells to matrix in NP tissue and hyperplasia of the annulus fibrosus. Taken together, our results indicate that BMAL1 and RORα form a regulatory loop in the NP and control HIF-1 activity without direct interaction. Importantly, activities of these circadian rhythm molecules may play a role in the adaptation of NP cells to their unique niche. PMID:27049729

Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxiainducible factor-1alpha expression via Poly(ADP-ribose) polymerase -1

PubMed Central

Pan, Rong; Chen, Chen; Liu, Wenlan; Liu, Ke Jian

2013-01-01

Aim Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study testes the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Methods Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-hour hypoxic treatment. Results Although 3-hr hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc concentration dependent manner. Exposure of astrocytes to hypoxia for 3-hr remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Conclusions Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. PMID:23582235

HIF-1α stabilization reduces retinal degeneration in a mouse model of retinitis pigmentosa.

PubMed

Olivares-González, Lorena; Martínez-Fernández de la Cámara, Cristina; Hervás, David; Millán, José María; Rodrigo, Regina

2018-05-01

Retinitis pigmentosa (RP) is a group of inherited retinal dystrophies characterized by progressive and irreversible loss of vision due to rod and cone degeneration. Evidence suggests that an inappropriate oxygen level could contribute to its pathogenesis. Rod cell death could increase oxygen concentration, reduce hypoxia-inducible factor 1 (HIF-1α) and contribute to cone cell death. The purposes of this study were: 1) to analyze the temporal profile of HIF-1α, its downstream effectors VEGF, endothelin-1 (ET-1), iNOS, and glucose transporter 1 (GLUT1), and neuroinflammation in retinas of the murine model of rd10 ( retinal degeneration 10) mice with RP; 2) to study oxygen bioavailability in these retinas; and 3) to investigate how stabilizing HIF-1α proteins with dimethyloxaloglycine (DMOG), a prolyl hydroxylase inhibitor, affects retinal degeneration, neuroinflammation, and antioxidant response in rd10 mice. A generalized down-regulation of HIF-1α and its downstream targets was detected in parallel with reactive gliosis, suggesting high oxygen levels during retinal degeneration. At postnatal d 18, DMOG treatment reduced photoreceptor cell death and glial activation. In summary, retinas of rd10 mice seem to be exposed to a hyperoxic environment even at early stages of degeneration. HIF-1α stabilization could have a temporal neuroprotective effect on photoreceptor cell survival, glial activation, and antioxidant response at early stages of RP.-Olivares-González, L., Martínez-Fernández de la Cámara, C., Hervás, D., Millán, J. M., Rodrigo, R. HIF-1α stabilization reduces retinal degeneration in a mouse model of retinitis pigmentosa.

NEU3 sialidase role in activating HIF-1α in response to chronic hypoxia in cyanotic congenital heart patients.

PubMed

Piccoli, Marco; Conforti, Erika; Varrica, Alessandro; Ghiroldi, Andrea; Cirillo, Federica; Resmini, Giulia; Pluchinotta, Francesca; Tettamanti, Guido; Giamberti, Alessandro; Frigiola, Alessandro; Anastasia, Luigi

2017-03-01

Hypoxia is a common feature of many congenital heart defects (CHDs) and significantly contributes to their pathophysiology. Thus, understanding the mechanism underlying cell response to hypoxia is vital for the development of novel therapeutic strategies. Certainly, the hypoxia inducible factor (HIF) has been extensively investigated and it is now recognized as the master regulator of cell defense machinery counteracting hypoxic stress. Along this line, we recently discovered and reported a novel mechanism of HIF activation, which is mediated by sialidase NEU3. Thus, aim of this study was to test whether NEU3 played any role in the cardiac cell response to chronic hypoxia in congenital cyanotic patients. Right atrial appendage biopsies were obtained from pediatric patients with cyanotic/non-cyanotic CHDs and processed to obtain mRNA and proteins. Real-Time PCR and Western Blot were performed to analyze HIF-1α and its downstream targets expression, NEU3 expression, and the NEU3 mediated effects on the EGFR signaling cascade. Cyanotic patients showed increased levels of HIF-1α, NEU3, EGFR and their downstream targets, as compared to acyanotic controls. The same patients were also characterized by increased phosphorylation of the EGFR signaling cascade proteins. Moreover, we found that HIF-1α expression levels positively correlated with those recorded for NEU3 in both cyanotic and control patients. Sialidase NEU3 plays a central role in activating cell response to chronic hypoxia inducing the up-regulation of HIF-1α, and this represent a possible novel tool to treat several CHD pathologies. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Jumonji Domain Containing Protein 6: A Novel Oxygen Sensor in the Human Placenta.

PubMed

Alahari, Sruthi; Post, Martin; Caniggia, Isabella

2015-08-01

Persistent low oxygen is implicated in the pathogenesis of placental-associated pathologies such as preeclampsia, a serious disorder of pregnancy. Emerging evidence implicates a novel family of Jumonji C catalytic domain proteins as mediators of hypoxic gene expression. Here, we investigated the regulatory relationship between Jumonji C domain containing protein 6 (JMJD6) and hypoxia-inducible factor (HIF)1A in the human placenta at physiological and pathological conditions. JMJD6 expression inversely correlated with changes in oxygen tension during early placental development, ie, high at 7-9 weeks when-partial pressure of O2 is low and declining afterwards when-partial pressure of O2 increases. Moreover, JMJD6 protein was significantly elevated in early-onset preeclamptic placentae, localizing to the syncytiotrophoblast layer and syncytial knots. Exposure of primary isolated trophoblast cells, human villous explants, and JEG3 choriocarcinoma cells to low oxygen (3%) and sodium nitroprusside (inducer of oxidative stress) also resulted in elevated JMJD6 levels, which was abrogated by HIF1A knockdown. In normoxia, knockdown of JMJD6 in JEG3 cells stabilized HIF1A with a concomitant decrease in von Hippel-Lindau (VHL) tumor suppressor protein, a negative regulator of HIF1A stability. In contrast, overexpression of JMJD6 enhanced VHL expression and destabilized HIF1A. JMJD6 regulation of VHL stability did not involve the ubiquitin-proteasome system but likely occurred through lysyl hydroxylation and small ubiquitin-like modifier 1-dependent small ubiquitin-like modifierylation. In summary, our data signify a novel role for JMJD6 as an oxygen sensor in the human placenta, and alterations in the JMJD6-VHL-HIF1A feedback loop may indirectly contribute to elevated HIF1A found in preeclampsia.

Hypoxia promotes noncanonical autophagy in nucleus pulposus cells independent of MTOR and HIF1A signaling.

PubMed

Choi, Hyowon; Merceron, Christophe; Mangiavini, Laura; Seifert, Erin L; Schipani, Ernestina; Shapiro, Irving M; Risbud, Makarand V

2016-09-01

Nucleus pulposus (NP) cells reside in the avascular and hypoxic microenvironment of intervertebral discs. Importantly, many activities related to survival and function of NP cells are controlled by the HIF-family of transcription factors. We hypothesize that NP cells adapt to their hypoxic niche through modulation of macroautophagy/autophagy. In various cell types, hypoxia induces autophagy in a HIF1A-dependent fashion; however, little is known about hypoxic regulation of autophagy in NP cells. Hypoxia increases the number of autophagosomes as seen by TEM analysis and LC3-positive puncta in NP cells. Hypoxic induction of autophagy was also demonstrated by a significantly higher number of autophagosomes and smaller change in autolysosomes in NP cells expressing tandem-mCherry-EGFP-LC3B. Increased LC3-II levels were not accompanied by a concomitant increase in BECN1 or the ATG12-ATG5 complex. In addition, ULK1 phosphorylation at Ser757 and Ser777 responsive to MTOR and AMPK, respectively, was not affected in hypoxia. Interestingly, when MTOR activity was inhibited by rapamycin or Torin1, LC3-II levels did not change, suggesting a novel MTOR-independent regulation. Noteworthy, while silencing of HIF1A affected hypoxic induction of BNIP3, it did not affect LC3-II levels, indicating hypoxia-induced autophagy is HIF1-independent. Importantly, there was no change in the number of LC3-positive autophagosomes in NP-specific Hif1a null mice. Finally, inhibition of autophagic flux did not affect the glycolytic metabolism of NP cells, suggesting a possible nonmetabolic role of autophagy. Taken together, our study for the first time shows that NP cells regulate autophagy in a noncanonical fashion independent of MTOR and HIF1A signaling.

Microparticles released by vascular endothelial cells increase hypoxia inducible factor expression in human proximal tubular HK-2 cells.

PubMed

Fernandez-Martínez, Ana Belen; Torija, Ana Valdehita; Carracedo, Julia; Ramirez, Rafael; de Lucio-Cazaña, Francisco Javier

2014-08-01

Microparticles are produced by vesiculation of the cell plasma membrane and serve as vectors of cell-to-cell communication. Co-culture experiments have shown that hypoxia-inducible factor-α (HIF-α)-regulated-genes are up-regulated in human renal proximal tubular HK-2 cells by endothelial cell factors which might be transported inside endothelial microparticles (EMP). Here we aimed to study in HK-2 cells the effect of EMP, produced by activated endothelial cells, on HIF-α and HIF-α-regulated vascular endothelial growth factor-A (VEGF-A). EMP, at a concentration much lower than that found in plasma, increased the expression of HIF-α/VEGF-A in a COX-2/EP2 receptor dependent manner. Since the EMP/cells ratio was ∼1/1000, we hypothesized that paracrine mediators produced by HK-2 cells amplified the initial signal. This hypothesis was confirmed by two facts which also suggested that the mediators were conveyed by particles released by HK-2 cells: (i) HIF-α was up-regulated in HK-2 cells treated with the pellet obtained from the conditioned medium of the EMP-treated HK-2 cells. (ii) In transwell experiments, EMP-treated cells increased the expression of HIF-α in untreated HK-2 cells. Interestingly, we detected these cells, particles that were released by EMP-treated HK-2 cells. Depending on the pathological context, activation of HIF-α and VEGF-A signaling in renal tissue/cells may have either beneficial or harmful effects. Therefore, our results suggest that their presence in the urinary space of EMP produced by activated endothelial cells may influence the outcome of a number of renal diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

Estrogen stabilizes hypoxia-inducible factor 1α through G protein-coupled estrogen receptor 1 in eutopic endometrium of endometriosis.

PubMed

Zhang, Ling; Xiong, Wenqian; Li, Na; Liu, Hengwei; He, Haitang; Du, Yu; Zhang, Zhibing; Liu, Yi

2017-02-01

To investigate whether G protein-coupled estrogen receptor (GPER, also known as GPR30 and GPER1) stabilizes hypoxia-inducible factor 1α (HIF-1α) in eutopic endometrium (EuEM) of endometriosis. Immunohistochemical analysis and experimental in vitro study. University hospital. Patients with or without endometriosis. The EuEM and normal control endometrium (CoEM) were obtained by curettage. Primary cultured endometrial stromal cells (ESCs) were treated with 17β-E 2 , G1, or G15. The EuEM and CoEM were collected for immunohistochemistry. Western blot, polymerase chain reaction, ELISA, and dual luciferase experiments were used to detect expression of GPER, HIF-1α, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 9 (MMP9) in ESCs. Estradiol and G1 were used as agonists of GPER, G15 as an antagonist. Migration of ESCs and endothelial tube formation of human umbilical vein endothelial cells cultured in medium collected from ESCs were measured. Protein levels of GPER and HIF-1α were higher in EuEM than in CoEM. Protein levels of HIF-1α but not HIF-1α mRNA levels increased concurrently with GPER after E 2 and G1 treatment. Furthermore, expression and activity of VEGF and MMP9 increased under E 2 and G1 stimulation. However, these effects disappeared when GPER was blocked. G protein-coupled estrogen receptor stabilizes HIF-1α and thus promotes HIF-1α-induced VEGF and MMP9 in ESCs, which play critical roles in endometriosis. Copyright © 2016 American Society for Reproductive Medicine. All rights reserved.

First report of bilateral pheochromocytoma in the clinical spectrum of HIF2A-related polycythemia-paraganglioma syndrome.

PubMed

Taïeb, David; Yang, Chunzhang; Delenne, Blandine; Zhuang, Zhengping; Barlier, Anne; Sebag, Fréderic; Pacak, Karel

2013-05-01

Molecular genetic research has so far resulted in the identification of 10 well-characterized susceptibility genes for hereditary pheochromocytoma (PHEO) or paraganglioma (PGL). Recently, a new syndrome characterized by multiple PGLs and somatostatinomas associated with congenital polycythemia due to somatic mutations in HIF2A has been reported. The aim of the study was to define the genetic defect in a new case of bilateral PHEO and multiple PGLs associated with congenital polycythemia. A female patient presented with neonatal polycythemia (treated by phlebotomies, 1 session approximately every 4 mo), mildly enlarged cerebral ventricles, and bilateral PHEO and multiple PGLs. There was no family history of any neuroendocrine tumor or polycythemia. Surgical removal of the tumors only temporarily normalized plasma erythropoietin (Epo) levels and discontinued phlebotomies. No germline mutations were initially detected in the SDHB, SDHC, SDHD, VHL, and PHD2 genes, known to be associated with polycythemia. The PHEOs presented with a typical noradrenergic biochemical phenotype. A heterozygous missense mutation (c.1589C>T) was identified in exon 12 of HIF2A, resulting in an alanine 530 substitution in the HIF-2α protein with valine (A530V). This somatic mutation was detected in the tissue from 1 PHEO and 1 PGL, with no HIF2A germline mutation found. This mutation led to stabilization of HIF-2α and hence a gain-of-function phenotype, as in previously published studies. This case represents the first association of a somatic HIF2A gain-of-function mutation with PHEO and congenital polycythemia, and it alerts physicians to perform proper genetic screening in patients presenting with multiple norepinephrine-producing PHEOs and polycythemia. This report also extends the previous findings of a new syndrome of only multiple PGLs, somatostatinomas, and polycythemia to multiple PHEOs.

A Somatic HIF2α Mutation-Induced Multiple and Recurrent Pheochromocytoma/Paraganglioma with Polycythemia: Clinical Study with Literature Review.

PubMed

Liu, Qiuli; Wang, Yan; Tong, Dali; Liu, Gaolei; Yuan, Wenqiang; Zhang, Jun; Ye, Jin; Zhang, Yao; Yuan, Gang; Feng, Qingxing; Zhang, Dianzheng; Jiang, Jun

2017-03-01

A syndrome known as pheochromocytomas (PCC)/paragangliomas (PGL) and polycythemia resulted from gain-of-function mutation of hypoxia-inducible factor 2α (HIF2α) has been reported recently. However, clinical features of this syndrome vary from patient to patient. In our study, we described the clinical features of the patient within 15-year follow-up with a literature review. The patient presented with "red face" since childhood and was diagnosed with polycythemia and pheochromocytoma in 2000, and then, tumor was removed at his age of 27 (year 2000). However, 13 years later (2013), he was diagnosed with multiple paragangliomas. Moreover, 2 years later (2015), another two paragangaliomas were also confirmed. Genetic analysis of hereditary PCC/PGL-related genes was conducted. A somatic heterozygous missense mutation of HIF2α (c.1589C>T) was identified at exon 12, which is responsible for the elevated levels of HIF2α and erythropoietin (EPO) and subsequent development of paragangaliomas. However, this mutation was only found in the tumors from three different areas, not in the blood. So far, 13 cases of PCC/PGL with polycythemia have been reported. Among them, somatic mutations of HIF2α at exon 12 are responsible for 12 cases, and only 1 case was caused by germline mutation of HIF2α at exon 9. The HIF2α mutation-induced polycythemia with PCC/PGL is a rare syndrome with no treatment for cure. Comprehensive therapies for this disease include removal of the tumors and intermittent phlebotomies; administration of medications to control blood pressure and to prevent complications or death resulted from high concentration of red blood cell (RBC). Genetic test is strongly recommended for patients with early onset of polycythemia and multiple/recurrent PCC/PGL.

Regulation of hypoxia-inducible factor-1alpha by nitric oxide through mitochondria-dependent and -independent pathways.

PubMed Central

Mateo, Jesús; García-Lecea, Marta; Cadenas, Susana; Hernández, Carlos; Moncada, Salvador

2003-01-01

Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the alpha-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1alpha is unstable in cells maintained at 21% O(2), but is progressively stabilized as the O(2) concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 microM) stabilize HIF-1alpha at all O(2) concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (rho(0)-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1alpha stabilized by exposure of the cells to 3% O(2). This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1alpha by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance. PMID:14531732

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping

PubMed Central

Schörg, Alexandra; Santambrogio, Sara; Platt, James L.; Schödel, Johannes; Lindenmeyer, Maja T.; Cohen, Clemens D.; Schrödter, Katrin; Mole, David R.; Wenger, Roland H.; Hoogewijs, David

2015-01-01

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the −82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the −82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the −82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia. PMID:26007655

VHL deletion impairs mammary alveologenesis but is not sufficient for mammary tumorigenesis.

PubMed

Seagroves, Tiffany N; Peacock, Danielle L; Liao, Debbie; Schwab, Luciana P; Krueger, Robin; Handorf, Charles R; Haase, Volker H; Johnson, Randall S

2010-05-01

Overexpression of hypoxia inducible factor-1 (HIF-1)alpha, which is common in most solid tumors, correlates with poor prognosis and high metastatic risk in breast cancer patients. Because HIF-1alpha protein stability is tightly controlled by the tumor suppressor von Hippel-Lindau (VHL), deletion of VHL results in constitutive HIF-1alpha expression. To determine whether VHL plays a role in normal mammary gland development, and if HIF-1alpha overexpression is sufficient to initiate breast cancer, Vhl was conditionally deleted in the mammary epithelium using the Cre/loxP system. During first pregnancy, loss of Vhl resulted in decreased mammary epithelial cell proliferation and impaired alveolar differentiation; despite these phenotypes, lactation was sufficient to support pup growth. In contrast, in multiparous dams, Vhl(-/-) mammary glands exhibited a progressive loss of alveolar epithelium, culminating in lactation failure. Deletion of Vhl in the epithelium also impacted the mammary stroma, as there was increased microvessel density accompanied by hemorrhage and increased immune cell infiltration. However, deletion of Vhl was not sufficient to induce mammary tumorigenesis in dams bred continuously for up to 24 months of age. Moreover, co-deletion of Hif1a could not rescue the Vhl(-/-)-dependent phenotype as dams were unable to successfully lactate during the first lactation. These results suggest that additional VHL-regulated genes besides HIF1A function to maintain the proliferative and regenerative potential of the breast epithelium.

[Induction of hypoxia-inducible factor-1alpha in two kinds of rats asphyxiation death models].

PubMed

Zhang, Bei-lei; Yang, Zhi-hui; Ran, Peng; Liang, Wei-bo; Zhou, Bin; Zhang, Geng-qian; Lu, Mei-li; Zhang, Lin

2007-02-15

To investigate the expression of hypoxia-inducible factor 1-alpha (HIF1-alpha) in the heart, lung, liver and kidney in rats died of two typical models of asphyxia. Two asphyxia models were made and tissue samples of the dead rats were collected from different groups at various postmortem duration. The expression and the changes of HIF1-alpha in various tissues were examined by immunohistochemistry and image analysis techniques. Results Significant expression of HIF1-alpha was observed in the myocardial fibers, kidney cells, liver cells and lung cells in both asphyxia models, but not in the control group. The expression of HIF1-alpha in various tissues in the rat died of nitrogen gas breathing was found in the nuclei at 0 hour and the expression level decreased gradually thereafter. The HIF1-alpha expression level and duration in various tissues of the rat died of hanging were higher and longer than that of the former group, with a peak of the expression level observed 6 hours after death, and then started to decline in all tissues except the heart where the expression still showed an increase 24 hours after death. The control groups showed a steady expression in the cytoplasm but not in the nuclei. HIF1-alpha appears to be a valuable biomarker in the diagnosis of asphyxia within 24 hours after death.

Systemic pretreatment with dimethyloxalylglycine increases myocardial HIF-1α and VEGF production and improves functional recovery after acute ischemia/reperfusion.

PubMed

Poynter, Jeffrey A; Manukyan, Mariuxi C; Wang, Yue; Brewster, Benjamin D; Herrmann, Jeremy L; Weil, Brent R; Abarbanell, Aaron M; Meldrum, Daniel R

2011-08-01

Stem cells protect the heart from ischemic damage in part by the release of cytoprotective growth factors, particularly vascular endothelial growth factor (VEGF). Production of VEGF is regulated in part by levels of the transcription factor hypoxia inducible factor 1-α (HIF-1α). Dimethyloxalylglycine (DMOG) prevents the deactivation of HIF-1α and increases VEGF production. However, the effects of systemic DMOG treatment on myocardial tolerance for ischemia are unknown. We hypothesized that systemic pretreatment with DMOG would improve myocardial ischemic tolerance. To study this hypothesis, adult male rats were randomly given an intraperitoneal injection of DMOG (40 mg/kg in 1 mL saline, n = 5) or saline (1 mL, n = 6) 24 h before cardiectomy and isolated heart perfusion. All hearts were subjected to 15 min equilibration, 25 min ischemia and 40 min reperfusion. Myocardial function was continuously monitored. Following reperfusion, myocardial homogenates were analyzed for HIF-1α and VEGF production. We observed that hearts in the DMOG group exhibited greater recovery of left ventricular developed pressure LVDP, +dP/dt and -dP/dt. Myocardial HIF-1α and VEGF levels were increased by DMOG therapy. In conclusion, systemic pretreatment with DMOG augments post-ischemic myocardial functional recovery through increased HIF-1α levels and greater VEGF production. Copyright © 2011 Mosby, Inc. All rights reserved.

DNA Methylation and BMI: Investigating Identified Methylation Sites at HIF3A in a Causal Framework

PubMed Central

Richmond, Rebecca C.; Ward, Mary E.; Fraser, Abigail; Lyttleton, Oliver; McArdle, Wendy L.; Ring, Susan M.; Gaunt, Tom R.; Lawlor, Debbie A.; Davey Smith, George; Relton, Caroline L.

2016-01-01

Multiple differentially methylated sites and regions associated with adiposity have now been identified in large-scale cross-sectional studies. We tested for replication of associations between previously identified CpG sites at HIF3A and adiposity in ∼1,000 mother-offspring pairs from the Avon Longitudinal Study of Parents and Children (ALSPAC). Availability of methylation and adiposity measures at multiple time points, as well as genetic data, allowed us to assess the temporal associations between adiposity and methylation and to make inferences regarding causality and directionality. Overall, our results were discordant with those expected if HIF3A methylation has a causal effect on BMI and provided more evidence for causality in the reverse direction (i.e., an effect of BMI on HIF3A methylation). These results are based on robust evidence from longitudinal analyses and were also partially supported by Mendelian randomization analysis, although this latter analysis was underpowered to detect a causal effect of BMI on HIF3A methylation. Our results also highlight an apparent long-lasting intergenerational influence of maternal BMI on offspring methylation at this locus, which may confound associations between own adiposity and HIF3A methylation. Further work is required to replicate and uncover the mechanisms underlying the direct and intergenerational effect of adiposity on DNA methylation. PMID:26861784

Pachymic Acid Sensitizes Gastric Cancer Cells to Radiation Therapy by Upregulating Bax through Hypoxia.

PubMed

Lu, Chunwei; Cai, Dingfang; Ma, Jun

2018-05-08

We have previously shown that pachymic acid (PA) inhibited tumorigenesis of gastric cancer (GC) cells. However, the exact mechanism underlying the radiation response of GC was still elusive. To evaluate the effects of PA treatment on radiation response of GC cell lines both in vitro and in vivo, a colony formation assay and xenograft mouse model were employed. Changes in Bax and HIF1[Formula: see text] expressions were assessed in GC cells following PA treatment. Luciferase reporter and chromatin immune-precipitation assays were carried out to investigate the regulation of Bax through HIF1[Formula: see text]. Stable HIF1[Formula: see text] knockdown was introduced into GC cells to further study the mechanism underlying PA-enhanced response to radiation both in vitro and in vivo. PA greatly enhanced the sensitivity of GC cells to radiation in vitro and in vivo, upregulated Bax expression and inhibited hypoxia. Bax expression was under hypoxia inhibition, and PA increased Bax expression through repressing HIF1[Formula: see text]. Stable HIF1[Formula: see text] overexpression in GC cells abolished the sensitizing effect of PA on GC cells to radiation both in vitro and in vivo. PA functions as a radiation sensitizing compound in GC. PA treatment induces the expression of pro-apoptotic factor Bax by inhibiting hypoxia/HIF1[Formula: see text], supporting the therapeutic potential of PA in radiation therapy against GC.

Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hyung Gyun; Han, Eun Hee; Im, Ji Hye

2015-09-25

Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Takenmore » together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.« less

Curcumin inhibits hepatic stellate cell activation via suppression of succinate-associated HIF-1α induction.

PubMed

She, Linlin; Xu, Dan; Wang, Zixia; Zhang, Yirui; Wei, Qingli; Aa, Jiye; Wang, Guangji; Liu, Baolin; Xie, Yuan

2018-05-07

Aberrant succinate accumulation emerges as a unifying mechanism for inflammation and oxidative stress. This study aims to investigate whether curcumin ameliorates hepatic fibrosis via blocking succinate signaling. We investigated the effects of curcumin on hepatic succinate accumulation and liver fibrosis in mice fed a high-fat diet (HFD). Meanwhile, we stimulated mouse primary hepatic stellate cells (HSCs) with succinate and observed the inhibitory effects of curcumin on succinate signaling. Oral administration of curcumin and metformin combated mitochondrial fatty acid oxidation and reduced hepatic succinate accumulation due to the inhibition of succinate dehydrogenase (SDH) activity and demonstrated inhibitory effect on hepatic fibrosis. In mouse primary HSCs, curcumin prevented succinate- and CoCl 2 -induced hypoxia-inducible transcription factor-1α (HIF-1α) induction via suppression of ROS production and effectively reduced gene expressions of Col1α, Col3α, fibronectin and TGF-β1 with inflammation inhibition. Knockdown of HIF-1α with small interfering RNA blocked the action of succinate to induce HSCs activation, indicative of the essential role of HIF-1α in succinate signaling. Hepatic succinate accumulation served as a metabolic signal to promote liver fibrosis through HIF-1α induction. Curcumin reduced succinate accumulation by combating fatty acid oxidation and prevented HSCs activation by blocking succinate/HIF-1α signaling pathway. Copyright © 2018. Published by Elsevier B.V.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tariq, Mohammad; Graduate School of Science and Engineering, Saitama University, 645 Shimo-Okubo, Sakura-ku, Saitama 338-8570; Ito, Akihiro, E-mail: akihiro-i@riken.jp

The eukaryotic initiation factor 5A (eIF5A) is an essential protein involved in translation elongation and cell proliferation. eIF5A undergoes several post-translational modifications including hypusination and acetylation. Hypusination is indispensable for the function of eIF5A. On the other hand, the precise function of acetylation remains unknown, but it may render the protein inactive since hypusination blocks acetylation. Here, we report that acetylation of eIF5A increases under hypoxia. During extended hypoxic periods an increase in the level of eIF5A acetylation correlated with a decrease in HIF-1α, suggesting involvement of eIF5A activity in HIF-1α expression under hypoxia. Indeed, suppression of eIF5A by siRNAmore » oligo-mediated knockdown or treatment with GC7, a deoxyhypusine synthase inhibitor, led to significant reduction of HIF-1α activity. Furthermore, knockdown of eIF5A or GC7 treatment reduced tumor spheroid formation with a concomitant decrease in HIF-1α expression. Our results suggest that functional, hypusinated eIF5A is necessary for HIF-1α expression during hypoxia and that eIF5A is an attractive target for cancer therapy. - Highlights: • Hypoxia induces acetylation of eIF5A. • Active eIF5A is necessary for HIF-1α activation in hypoxia. • Active eIF5A is important for tumor spheroid growth.« less

Separate necdin domains bind ARNT2 and HIF1{alpha} and repress transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedman, Eitan R.; Fan Chenming

2007-11-09

PWS is caused by the loss of expression of a set of maternally imprinted genes including NECDIN (NDN). NDN is expressed in post-mitotic neurons and plays an essential role in PWS as mouse models lacking only the Ndn gene mimic aspects of this disease. Patients haploid for SIM1 develop a PW-like syndrome. Here, we report that NDN directly interacts with ARNT2, a bHLH-PAS protein and dimer partner for SIM1. We also found that NDN can interact with HIF1{alpha}. We showed that NDN can repress transcriptional activation mediated by ARNT2:SIM1 as well as ARNT2:HIF1{alpha}. The N-terminal 115 residues of NDN aremore » sufficient for interaction with the bHLH domains of ARNT2 or HIF1{alpha} but not for transcriptional repression. Using GAL4-NDN fusion proteins, we determined that NDN possesses multiple repression domains. We thus propose that NDN regulates neuronal function and hypoxic response by regulating the activities of the ARNT2:SIM1 and ARNT2:HIF1{alpha} dimers, respectively.« less

Copper activates HIF-1α/GPER/VEGF signalling in cancer cells

PubMed Central

Rigiracciolo, Damiano Cosimo; Scarpelli, Andrea; Lappano, Rosamaria; Pisano, Assunta; Santolla, Maria Francesca; De Marco, Paola; Cirillo, Francesca; Cappello, Anna Rita; Dolce, Vincenza; Belfiore, Antonino; Maggiolini, Marcello; De Francesco, Ernestina Marianna

2015-01-01

Copper promotes tumor angiogenesis, nevertheless the mechanisms involved remain to be fully understood. We have recently demonstrated that the G-protein estrogen receptor (GPER) cooperates with hypoxia inducible factor-1α (HIF-1α) toward the regulation of the pro-angiogenic factor VEGF. Here, we show that copper sulfate (CuSO4) induces the expression of HIF-1α as well as GPER and VEGF in breast and hepatic cancer cells through the activation of the EGFR/ERK/c-fos transduction pathway. Worthy, the copper chelating agent TEPA and the ROS scavenger NAC prevented the aforementioned stimulatory effects. We also ascertained that HIF-1α and GPER are required for the transcriptional activation of VEGF induced by CuSO4. In addition, in human endothelial cells, the conditioned medium from breast cancer cells treated with CuSO4 promoted cell migration and tube formation through HIF-1α and GPER. The present results provide novel insights into the molecular mechanisms involved by copper in triggering angiogenesis and tumor progression. Our data broaden the therapeutic potential of copper chelating agents against tumor angiogenesis and progression. PMID:26415222

Ferrous glycinate regulates cell energy metabolism by restrictinghypoxia-induced factor-1α expression in human A549 cells.

PubMed

Kuo, Yung-Ting; Jheng, Jhong-Huei; Lo, Mei-Chen; Chen, Wei-Lu; Wang, Shyang-Guang; Lee, Horng-Mo

2018-06-04

Iron or oxygen regulates the stability of hypoxia inducible factor-1α (HIF-1α). We investigated whether ferrous glycinate would affect HIF-1α accumulation, aerobic glycolysis and mitochondrial energy metabolism in human A549 lung cancer cells. Incubation of A549 cells with ferrous glycinate decreased the protein levels of HIF-1α, which was abrogated by proteosome inhibitor, or prolyl hydroxylase inhibitor. The addition of ferrous glycinate decreased protein levels of glucose transporter-1, hexokinase-2, and lactate dehydrogenase A, and decreased pyruvate dehydrogenase kinase-1 (PDK-1) and pyruvate dehydrogenase (PDH) phosphorylation in A549 cells. Ferrous glycinate also increased the expression of the mitochondrial transcription factor A (TFAM), and the mitochondrial protein, cytochrome c oxidase (COX-IV). Silencing of HIF-1α expression mimicked the effects of ferrous glycinate on PDK-1, PDH, TFAM and COX-IV in A549 cells. Ferrous glycinate increased mitochondrial membrane potential and ATP production in A549 cells. These results suggest that ferrous glycinate may reverse Warburg effect through down regulating HIF-1α in A549 cells.

Increasing AR by HIF-2α inhibitor (PT-2385) overcomes the side-effects of sorafenib by suppressing hepatocellular carcinoma invasion via alteration of pSTAT3, pAKT and pERK signals

PubMed Central

Xu, Junjie; Zheng, Longbo; Chen, Jiang; Sun, Yin; Lin, Hui; Jin, Ren-an; Tang, Minyue; Liang, Xiao; Cai, Xiujun

2017-01-01

Although sorafenib is currently used as a standard treatment for advanced hepatocellular carcinoma, low response rate, transient and limited efficacy, primary and acquired resistance and negative side-effects gain increasing attentions, suggesting the need for better efficacious combination therapy. Here, we demonstrated that the sorafenib-induced or hypoxia-induced hypoxia inducible factor (HIF)-2α could bind to an hypoxia responsive element within 500 bp region of androgen receptor (AR) promoter and thus transcriptionally suppress AR. Importantly, In vitro and In vivo studies suggested a specific and potent HIF-2α inhibitor, PT-2385, could significantly enhance sorafenib efficacy by suppressing HIF-2α, increasing AR and suppressing downstream pSTAT3/pAKT/pERK pathways. Clinical samples further confirmed the role of HIF-2α and AR. It is promising that PT-2385 could alleviate the undesirable side-effects of sorafenib treatment by sorafenib-PT-2385 combination therapy, which may shed light for late-stage HCC patients. PMID:29022906

Increasing AR by HIF-2α inhibitor (PT-2385) overcomes the side-effects of sorafenib by suppressing hepatocellular carcinoma invasion via alteration of pSTAT3, pAKT and pERK signals.

PubMed

Xu, Junjie; Zheng, Longbo; Chen, Jiang; Sun, Yin; Lin, Hui; Jin, Ren-An; Tang, Minyue; Liang, Xiao; Cai, Xiujun

2017-10-12

Although sorafenib is currently used as a standard treatment for advanced hepatocellular carcinoma, low response rate, transient and limited efficacy, primary and acquired resistance and negative side-effects gain increasing attentions, suggesting the need for better efficacious combination therapy. Here, we demonstrated that the sorafenib-induced or hypoxia-induced hypoxia inducible factor (HIF)-2α could bind to an hypoxia responsive element within 500 bp region of androgen receptor (AR) promoter and thus transcriptionally suppress AR. Importantly, In vitro and In vivo studies suggested a specific and potent HIF-2α inhibitor, PT-2385, could significantly enhance sorafenib efficacy by suppressing HIF-2α, increasing AR and suppressing downstream pSTAT3/pAKT/pERK pathways. Clinical samples further confirmed the role of HIF-2α and AR. It is promising that PT-2385 could alleviate the undesirable side-effects of sorafenib treatment by sorafenib-PT-2385 combination therapy, which may shed light for late-stage HCC patients.

Hypoxia inducible factor 1 (HIF-1) and cardioprotection

PubMed Central

Tekin, Demet; Dursun, Ali D; Xi, Lei

2010-01-01

Since its discovery in early 1990s, hypoxia inducible factor 1 (HIF-1) has been increasingly recognized for its key role in transcriptional control of more than a hundred genes that regulate a wide-spectrum of cellular functional events, including angiogenesis, vasomotor control, glucose and energy metabolism, erythropoiesis, iron homeostasis, pH regulation, cell proliferation and viability. Evidence accumulated during the past 7 years suggests a critical role for HIF-1α in mediating cardioprotection. The purpose of our present article is to provide an updated overview on this important regulator of gene expression in the cellular stress-responsive and adaptive process. We have particularly emphasized the involvement of HIF-1 in the induction of cardioprotective molecules, such as inducible nitric oxide synthase (iNOS), hemeoxygenase 1 (HO-1), and erythropoietin (EPO), which in turn alleviate myocardial damages caused by harmful events such as ischemia-reperfusion injury. Despite these advances, further in-depth studies are needed to elucidate the possible coordination or interaction between HIF-1α and other key transcription factors in regulating protein expression that leads to cardioprotection. PMID:20711226

Interrelated Roles for the Aryl Hydrocarbon Receptor and Hypoxia Inducible Factor-1α in the Immune Response to Infection

PubMed Central

Wagage, Sagie; Hunter, Christopher A.

2015-01-01

Cells of the immune system utilize multiple mechanisms to respond to environmental signals and recent studies have demonstrated roles for two closely related proteins, the aryl hydrocarbon receptor (AHR) and hypoxia inducible factor-1α (HIF1α), in these processes. The AHR is a transcription factor that is activated by diverse ligands found in the diet and environmental pollution as well as by microbial and host-derived products. In contrast, HIF1α is a transcription factor that is active under low oxygen conditions and mediates cellular responses to hypoxia. These evolutionarily conserved proteins have roles in the interrelated processes of metabolism, tumorigenesis, and vascular development. Additionally, the AHR and HIF1α have multiple effects on innate and adaptive immunity. This article provides an overview of the biology of these transcription factors and reviews the effects of AHR and HIF1α signaling on immunity to infection. There are many parallels between these two pathways and their functions highlight the importance of AHR and HIF1α activity particularly at barrier surfaces in coordinating responses to pathogens.

Click Chemistry-based Discovery of [3-Hydroxy-5-(1H-1,2,3-triazol-4-yl)picolinoyl]glycines as Orally Active Hypoxia Inducing Factor Prolyl Hydroxylase Inhibitors with Favorable Safety Profiles for the Treatment of Anemia.

PubMed

Wu, Yue; Jiang, Zhensheng; Li, Zhihong; Gu, Jing; You, Qi-Dong; Zhang, Xiaojin

2018-06-01

As a gene associated with anemia, the erythropoiesis gene is physiologically expressed under hypoxia regulated by hypoxia-inducing factor-α (HIF-α). Thus, stabilizing HIF-α is a potent strategy to stimulate the expression and secretion of erythropoiesis. In this study we applied click chemistry to the discovery of HIF prolyl hydroxylase 2 (HIF-PHD2) inhibitors for the first time and a series of triazole compounds showed preferable inhibitory activity in fluorescence polarization assay. Of particular note was the orally active HIF-PHD inhibitor 15i (IC50 = 62.23 nM), which was almost ten times more active than the phase III drug FG-4592 (IC50 = 591.4 nM). Furthermore, it can upregulate the hemoglobin of cisplatin induced anemia mice (120 g/L) to normal levels (160 g/L) with no apparent toxicity observed in vivo. These results confirm that triazole compound 15i is a promising candidate for the treatment of renal anemia.

Hypoxia-inducible factor-1α promotes cell survival during ammonia stress response in ovarian cancer stem-like cells

PubMed Central

Kitajima, Shojiro; Lee, Kian Leong; Hikasa, Hiroki; Sun, Wendi; Huang, Ruby Yun-Ju; Yang, Henry; Matsunaga, Shinji; Yamaguchi, Takehiro; Araki, Marito; Kato, Hiroyuki

2017-01-01

Ammonia is a toxic by-product of metabolism that causes cellular stresses. Although a number of proteins are involved in adaptive stress response, specific factors that counteract ammonia-induced cellular stress and regulate cell metabolism to survive against its toxicity have yet to be identified. We demonstrated that the hypoxia-inducible factor-1α (HIF-1α) is stabilized and activated by ammonia stress. HIF-1α activated by ammonium chloride compromises ammonia-induced apoptosis. Furthermore, we identified glutamine synthetase (GS) as a key driver of cancer cell proliferation under ammonia stress and glutamine-dependent metabolism in ovarian cancer stem-like cells expressing CD90. Interestingly, activated HIF-1α counteracts glutamine synthetase function in glutamine metabolism by facilitating glycolysis and elevating glucose dependency. Our studies reveal the hitherto unknown functions of HIF-1α in a biphasic ammonia stress management in the cancer stem-like cells where GS facilitates cell proliferation and HIF-1α contributes to the metabolic remodeling in energy fuel usage resulting in attenuated proliferation but conversely promoting cell survival. PMID:29383096

A new pharmacological agent (AKB-4924) stabilizes hypoxia inducible factor-1 (HIF-1) and increases skin innate defenses against bacterial infection.

PubMed

Okumura, Cheryl Y M; Hollands, Andrew; Tran, Dan N; Olson, Joshua; Dahesh, Samira; von Köckritz-Blickwede, Maren; Thienphrapa, Wdee; Corle, Courtney; Jeung, Seung Nam; Kotsakis, Anna; Shalwitz, Robert A; Johnson, Randall S; Nizet, Victor

2012-09-01

Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 increases HIF-1 levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinetobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections.

A New Pharmacological Agent (AKB-4924) Stabilizes Hypoxia Inducible Factor (HIF) and Increases Skin Innate Defenses Against Bacterial Infection

PubMed Central

Okumura, Cheryl Y.M.; Hollands, Andrew; Tran, Dan N.; Olson, Joshua; Dahesh, Samira; von Köckritz-Blickwede, Maren; Thienphrapa, Wdee; Corle, Courtney; Jeung, Seung Nam; Kotsakis, Anna; Shalwitz, Robert A.; Johnson, Randall S.; Nizet, Victor

2013-01-01

Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 (Akebia Therapeutics) increases HIF-1α levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and -resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinitobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections. PMID:22371073

Copper activates HIF-1α/GPER/VEGF signalling in cancer cells.

PubMed

Rigiracciolo, Damiano Cosimo; Scarpelli, Andrea; Lappano, Rosamaria; Pisano, Assunta; Santolla, Maria Francesca; De Marco, Paola; Cirillo, Francesca; Cappello, Anna Rita; Dolce, Vincenza; Belfiore, Antonino; Maggiolini, Marcello; De Francesco, Ernestina Marianna

2015-10-27

Copper promotes tumor angiogenesis, nevertheless the mechanisms involved remain to be fully understood. We have recently demonstrated that the G-protein estrogen receptor (GPER) cooperates with hypoxia inducible factor-1α (HIF-1α) toward the regulation of the pro-angiogenic factor VEGF. Here, we show that copper sulfate (CuSO4) induces the expression of HIF-1α as well as GPER and VEGF in breast and hepatic cancer cells through the activation of the EGFR/ERK/c-fos transduction pathway. Worthy, the copper chelating agent TEPA and the ROS scavenger NAC prevented the aforementioned stimulatory effects. We also ascertained that HIF-1α and GPER are required for the transcriptional activation of VEGF induced by CuSO4. In addition, in human endothelial cells, the conditioned medium from breast cancer cells treated with CuSO4 promoted cell migration and tube formation through HIF-1α and GPER. The present results provide novel insights into the molecular mechanisms involved by copper in triggering angiogenesis and tumor progression. Our data broaden the therapeutic potential of copper chelating agents against tumor angiogenesis and progression.

Tissue mechanics promote IDH1-dependent HIF1α–tenascin C feedback to regulate glioblastoma aggression

PubMed Central

Miroshnikova, Yekaterina A.; Mouw, Janna K.; Barnes, J. Matthew; Pickup, Michael W.; Lakins, Johnathan N.; Kim, Youngmi; Lobo, Khadjia; Persson, Anders I.; Reis, Gerald F.; McKnight, Tracy R.; Holland, Eric C.; Phillips, Joanna J.; Weaver, Valerie M.

2017-01-01

Increased overall survival for patients with glioma brain tumours is associated with mutations in the metabolic regulator isocitrate dehydrogenase 1 (IDH1). Gliomas develop within a mechanically challenged microenvironment that is characterized by a dense extracellular matrix (ECM) that compromises vascular integrity to induce hypoxia and activate HIF1α. We found that glioma aggression and patient prognosis correlate with HIF1α levels and the stiffness of a tenascin C (TNC)-enriched ECM. Gain- and loss-of-function xenograft manipulations demonstrated that a mutant IDH1 restricts glioma aggression by reducing HIF1α-dependent TNC expression to decrease ECM stiffness and mechanosignalling. Recurrent IDH1-mutant patient gliomas had a stiffer TNC-enriched ECM that our studies attributed to reduced miR-203 suppression of HIF1α and TNC mediated via a tension-dependent positive feedback loop. Thus, our work suggests that elevated ECM stiffness can independently foster glioblastoma aggression and contribute to glioblastoma recurrence via bypassing the protective activity of IDH1 mutational status. PMID:27820599

Hypoxia-Inducible Factors Link Iron Homeostasis and Erythropoiesis

PubMed Central

Shah, Yatrik M.; Xie, Liwei

2014-01-01

Iron is required for efficient oxygen transport, and hypoxia signaling links erythropoiesis with iron homeostasis. Hypoxia induces a highly conserved signaling pathway in cells under conditions of low O2. One component of this pathway, hypoxia-inducible factor (HIF), is a transcription factor that is highly active in hypoxic cells. The first HIF target gene characterized was EPO, which encodes erythropoietin—a glycoprotein hormone that controls erythropoiesis. The past decade has led to fundamental advances in our understanding of how hypoxia regulates iron levels to support erythropoiesis and maintain systemic iron homeostasis. We review the cell-type specific effects of hypoxia and HIFs in adaptive response to changes in oxygen and iron availability, as well as potential uses of HIF modulators for patients with iron-related disorders. PMID:24389303

VHL and Hypoxia Signaling: Beyond HIF in Cancer

PubMed Central

Zhang, Jing

2018-01-01

Von Hippel-Lindau (VHL) is an important tumor suppressor that is lost in the majority of clear cell carcinoma of renal cancer (ccRCC). Its regulatory pathway involves the activity of E3 ligase, which targets hypoxia inducible factor α (including HIF1α and HIF2α) for proteasome degradation. In recent years, emerging literature suggests that VHL also possesses other HIF-independent functions. This review will focus on VHL-mediated signaling pathways involving the latest identified substrates/binding partners, including N-Myc downstream-regulated gene 3 (NDRG3), AKT, and G9a, etc., and their physiological roles in hypoxia signaling and cancer. We will also discuss the crosstalk between VHL and NF-κB signaling. Lastly, we will review the latest findings on targeting VHL signaling in cancer. PMID:29562667

Early Diagnosis of Clear Cell Kidney Cancer via VHL/HIF Pathway Regulated-Circulating microRNA

DTIC Science & Technology

2016-05-01

Award Number: W81XWH-11-1-0715 TITLE: Early Diagnosis of Clear Cell Kidney Cancer via VHL/HIF Pathway-Regulated Circulating microRNA PRINCIPAL...TITLE AND SUBTITLE Sa. CONTRACT NUMBER Early Diagnosis of Clear Cell Kidney Cancer via VHL/HIF Pathway- Regulated Circulating microRNA Sb. GRANT NUMBER...panel of diagnostic miRNAs that are measurable in serum and will be able to identify kidney cancer in its earliest stages. We hypothesized that serum

Development of a Combination Therapy for Prostate Cancer by Targeting Stat3 and HIF-1alpha

DTIC Science & Technology

2013-07-01

inflammation-induced cancer, making it an attractive target (25-27). A3. Innovation 1. TEL03 is a novel anti-cancer agent from Chinese herbal medicine ...agents from Chinese herbal medicine (CHM) that targets HIF-1α /2α for prostate cancer therapy. Hypoxia orchestrated by HIF-1αis crucial for tumor...Stat3 for treatment of prostate and other cancers. TEL03, which is a novel anti-cancer agent derived from Chinese herbal medicine (CHM: Hypocrella

Cobalt chloride compromises transepithelial barrier properties of CaCo-2 BBe human gastrointestinal epithelial cell layers.

PubMed

DiGuilio, K M; Valenzano, M C; Rybakovsky, E; Mullin, J M

2018-01-05

Elevation of the transcription factor HIF-1 is a prominent mediator of not only processes that accompany hypoxia, but also the tumor microenvironment and tissue regeneration. This study uses mediators of "chemical hypoxia" to ask the question whether HIF-1α elevation in a healthy epithelial cell layer leads to leakiness in its tight junctional seals. Transepithelial electrical resistance and transepithelial diffusion of 14 C-D-mannitol and other radiolabeled probes are used as indicators of transepithelial barrier function of CaCo-2 BBe human gastrointestinal epithelial cell layers cultured on permeable supports. Western immunoblot analyses of integral tight junctional proteins (occludin and claudins) are used as further indicators of barrier function change. Cobalt, an inhibitor of the prolyl hydroxylase enzymes governing HIF-1α breakdown in the cell, induces transepithelial leakiness in CaCo-2 BBe cell layers in a time and concentration-dependent manner. This increased leakiness is accompanied by significant changes in certain specific integral tight junctional (TJ) proteins such as a decreased level of occludin and increased level of claudin-5. Similar results regarding barrier function compromise also occur with other chemical inhibitors of HIF-1α breakdown, namely ciclopiroxolamine (CPX) and dimethyloxalylglycine (DMOG). The increased leak is manifested by both decreased transepithelial electrical resistance (R t ) and increased paracellular diffusion of D-mannitol (J m ). The induced transepithelial leak shows significant size selectivity, consistent with induced effects on TJ permeability. Less-differentiated cell layers were significantly more affected than well-differentiated cell layers regarding induced transepithelial leak. A genetically modified CaCo-2 variant with reduced levels of HIF-1β, showed reduced transepithelial leak in response to cobalt exposure, further indicating that elevation of HIF-1α levels induced by agents of "chemical hypoxia" is responsible for the compromised barrier function of the CaCo-2 BBe cell layers. Exposure to inducers of chemical hypoxia elevated HIF-1α levels and increased transepithelial leak. The degree of epithelial differentiation has significant effects on this action, possibly explaining the varying effects of HIF-1 modulation in epithelial and endothelial barrier function in different physiological and pathophysiological conditions.

HIF-1α and HIF-2α: siblings in promoting angiogenesis of residual hepatocellular carcinoma after high-intensity focused ultrasound ablation.

PubMed

Wu, Lun; Fu, Zhihao; Zhou, Shiji; Gong, Jianping; Liu, Chang An; Qiao, Zhengrong; Li, Shengwei

2014-01-01

High-intensity focused ultrasound (HIFU) is a widely applied to treatment for unresectable hepatocellular carcinoma. However, insufficient HIFU can result in rapid progression of the residual tumor. The mechanism of such rapid growth of the residual tumor after HIFU ablation is poorly understood. The aim of this study was to investigate the dynamic angiogenesis of residual tumor, and the temporal effect and mechanism of the HIF-1, 2α in the residual tumor angiogenesis. Xenograft tumors of HepG2 cells were created by subcutaneously inoculating nude mice (athymic BALB/c nu/nu mice) with hepatoma cells. About thirty days after inoculation, all mice (except control group) were treated by HIFU and assigned randomly to 7 groups according to various time intervals (1st, 3rd, 5th day (d) and 1st, 2nd, 3rd, 4th week (w)). The residual tumor tissues were obtained from the experimental groups at various time points. Protein levels of HIF-1α, HIF-2α, VEGF-A, and EphA2 were quantified by immunohistochemistry analysis and Western Blot assays, and mRNA levels measured by Q-PCR. Microvascular density was calculated with counting of CD31 positive vascular endothelial cells by immunohistochemical staining. Compared with the control group, protein and mRNA levels of HIF-1α reached their highest levels on the 3rd day (P<0.01), then decreased (P<0.05). HIF-2α expression reached its highest level on the 2nd week compared with control group (P<0.01), then decreased (2 w-4 w) (P<0.05). The protein and mRNA levels of VEGF-A and EphA2 in the residual tumor tissues group that received HIFU were significantly decreased until 1 week compared with the control group (P<0.01). However, the levels increased compared to controls in 2-4 weeks (P<0.05). Similar results were obtained for MVD expression (P<0.05). Insufficient HIFU ablation promotes the angiogenesis in residual carcinoma tissue over time. The data indicate that the HIF-1, 2α/VEGFA/EphA2 pathway is involved.

The c-Abl signaling network in the radioadaptive response

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chi-Min, Yuan

2014-01-28

The radioadaptive response, or radiation hormesis, i.e. a low dose of radiation can protect cells and organisms from the effects of a subsequent higher dose, is a widely recognized phenomenon. Mechanisms underlying such radiation hormesis, however, remain largely unclear. Preliminary studies indicate an important role of c-Abl signaling in mediating the radioadaptive response. We propose to investigate how c-Abl regulates the crosstalk between p53 and NFκB in response to low doses irradiation. We found in our recent study that low dose IR induces a reciprocal p53 suppression and NFκB activation, which induces HIF-a and subsequently a metabolic reprogramming resulting inmore » a transition from oxidative phosphorylation to glycolysis. Of importance is that this glycolytic switch is essential for the radioadaptive response. This low-dose radiationinduced HIF1α activation was in sharp contrast with the high-dose IR-induced p53 activation and HIF1α inhibition. HIF1α and p53 seem to play distinct roles in mediating the radiation dose-dependent metabolic response. The induction of HIF1α-mediated glycolysis is restricted to a low dose range of radiation, which may have important implications in assessing the level of radiation exposure and its potential health risk. Our results support a dose-dependent metabolic response to IR. When IR doses are below the threshold of causing detectable DNA damage (<0.2Gy) and thus little p53 activation, HIF1α is induced resulting in induction of glycolysis and increased radiation resistance. When the radiation dose reaches levels eliciting DNA damage, p53 is activated and diminishes the activity of HIF1α and glycolysis, leading to the induction of cell death. Our work challenges the LNT model of radiation exposure risk and provides a metabolic mechanism of radioadaptive response. The study supports a need for determining the p53 and HIF1α activity as a potential reliable biological readout of radiation exposure in humans. The exquisite sensitivity of cellular metabolism to low doses of radiation could also serve as a valuable biomarker for estimating the health effects of low-level radiation exposure.« less

Mechanism of salutary effects of melatonin-mediated liver protection after trauma-hemorrhage: p38 MAPK-dependent iNOS/HIF-1α pathway.

PubMed

Hsu, Jun-Te; Le, Puo-Hsien; Lin, Chun-Jung; Chen, Tsung-Hsing; Kuo, Chia-Jung; Chiang, Kun-Chun; Yeh, Ta-Sen

2017-05-01

Although melatonin attenuates the increases in inflammatory mediators and reduces organ injury during trauma-hemorrhage, the mechanisms remain unclear. This study explored whether melatonin prevents liver injury after trauma-hemorrhage through the p38 mitogen-activated protein kinase (MAPK)-dependent, inducible nitrite oxide (iNOS)/hypoxia-inducible factor (HIF)-1α pathway. After a 5-cm midline laparotomy, male rats underwent hemorrhagic shock (mean blood pressure ~40 mmHg for 90 min) followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, melatonin (2 mg/kg), melatonin plus p38 MAPK inhibitor SB203580 (2 mg/kg), or melatonin plus the melatonin receptor antagonist luzindole (2.5 mg/kg). At 2 h after trauma-hemorrhage, histopathology score of liver injury, liver tissue myeloperoxidase activity, malondialdehyde, adenosine triphosphate, serum alanine aminotransferase, and asparate aminotransferase levels were significantly increased compared with sham-operated control. Trauma-hemorrhage resulted in a significant decrease in the p38 MAPK activation compared with that in the sham-treated animals. Administration of melatonin after trauma-hemorrhage normalized liver p38 MAPK phosphorylation and iNOS and HIF-1α expression and attenuated cleaved caspase 3 and receptor interacting protein kinase-1 levels. Coadministration of SB203580 or luzindole abolished the melatonin-mediated attenuation of the trauma-hemorrhage-induced increase of iNOS/HIF-1α protein expression and liver injury markers. Taken together, our results suggest that melatonin prevents trauma-hemorrhage-induced liver injury in rats, at least in part, through melatonin receptor-related, p38 MAPK-dependent iNOS/HIF-1α pathway. NEW & NOTEWORTHY Trauma-hemorrhage resulted in a significant decrease in liver p38 MAPK activation and increase in nitrite oxide synthase (iNOS) and hypoxia-inducible factor (HIF)-1α expression. Administration of melatonin after trauma-hemorrhage normalized liver p38 MAPK phosphorylation and iNOS and HIF-1α expression, which was abolished by coadministration of SB203580 or luzindole. Melatonin prevents trauma-hemorrhage-induced liver injury in rats via the melatonin receptor-related, p38 MAPK-dependent iNOS/HIF-1α pathway. Copyright © 2017 the American Physiological Society.

Assessing the association between hypoxia during craniofacial development and oral clefts.

PubMed

Küchler, Erika Calvano; Silva, Lea Assed da; Nelson-Filho, Paulo; Sabóia, Ticiana M; Rentschler, Angela M; Granjeiro, José Mauro; Oliveira, Driely; Tannure, Patricia N; Silva, Raquel Assed da; Antunes, Leonardo Santos; Tsang, Michael; Vieira, Alexandre R

2018-01-01

Objectives To evaluate the association between hypoxia during embryo development and oral clefts in an animal model, and to evaluate the association between polymorphisms in the HIF-1A gene with oral clefts in human families. Material and Methods The study with the animal model used zebrafish embryos at 8 hours post-fertilization submitted to 30% and 50% hypoxia for 24 hours. At 5 days post-fertilization, the larvae were fixed. The cartilage structures were stained to evaluate craniofacial phenotypes. The family-based association study included 148 Brazilian nuclear families with oral clefts. The association between the genetic polymorphisms rs2301113 and rs2057482 in HIF-1A with oral clefts was tested. We used real time PCR genotyping approach. ANOVA with Tukey's post-test was used to compare means. The transmission/disequilibrium test was used to analyze the distortion of the inheritance of alleles from parents to their affected offspring. Results For the hypoxic animal model, the anterior portion of the ethmoid plate presented a gap in the anterior edge, forming a cleft. The hypoxia level was associated with the severity of the phenotype (p<0.0001). For the families, there was no under-transmitted allele among the affected progeny (p>0.05). Conclusion Hypoxia is involved in the oral cleft etiology, however, polymorphisms in HIF-1A are not associated with oral clefts in humans.

Assessing the association between hypoxia during craniofacial development and oral clefts

PubMed Central

Rentschler, Angela M.; Oliveira, Driely; da Silva, Raquel Assed; Antunes, Leonardo Santos

2018-01-01

Abstract Objectives To evaluate the association between hypoxia during embryo development and oral clefts in an animal model, and to evaluate the association between polymorphisms in the HIF-1A gene with oral clefts in human families. Material and Methods The study with the animal model used zebrafish embryos at 8 hours post-fertilization submitted to 30% and 50% hypoxia for 24 hours. At 5 days post-fertilization, the larvae were fixed. The cartilage structures were stained to evaluate craniofacial phenotypes. The family-based association study included 148 Brazilian nuclear families with oral clefts. The association between the genetic polymorphisms rs2301113 and rs2057482 in HIF-1A with oral clefts was tested. We used real time PCR genotyping approach. ANOVA with Tukey's post-test was used to compare means. The transmission/disequilibrium test was used to analyze the distortion of the inheritance of alleles from parents to their affected offspring. Results For the hypoxic animal model, the anterior portion of the ethmoid plate presented a gap in the anterior edge, forming a cleft. The hypoxia level was associated with the severity of the phenotype (p<0.0001). For the families, there was no under-transmitted allele among the affected progeny (p>0.05). Conclusion Hypoxia is involved in the oral cleft etiology, however, polymorphisms in HIF-1A are not associated with oral clefts in humans. PMID:29791568

SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization

PubMed Central

Finley, Lydia W.S.; Carracedo, Arkaitz; Lee, Jaewon; Souza, Amanda; Egia, Ainara; Zhang, Jiangwen; Teruya-Feldstein, Julie; Moreira, Paula I.; Cardoso, Sandra M.; Clish, Clary B.; Pandolfi, Pier Paolo; Haigis, Marcia C.

2011-01-01

Summary Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression. PMID:21397863

Inhibitory effects of epigallocatechin-3-gallate on cell proliferation and the expression of HIF-1α and P-gp in the human pancreatic carcinoma cell line PANC-1.

PubMed

Zhu, Zhenni; Wang, Yu; Liu, Zhiqing; Wang, Fan; Zhao, Qiu

2012-05-01

The aim of this study was to verify the inhibitory effects of epigallocatechin-3-gallate (EGCG) on cell proliferation and the expression of hypoxia-inducible factor 1 (HIF-1α) and multidrug resistance protein 1 (MDR1/P-gp) in the human pancreatic carcinoma cell line PANC-1, thereby, reversing drug resistance of pancreatic carcinoma and improving its sensitivity to cancer chemotherapy. The human pancreatic carcinoma cell line PANC-1 was incubated under hypoxic conditions with different concentrations of epigallocatechin-3-gallate (EGCG) for indicated hours. The effects of EGCG on the mRNA or protein expression of HIF-1α and MDR1 were determined by RT-PCR or western blotting. Cellular proliferation and viability assays were measured using Cell Counting Kit-8. Western blotting revealed that EGCG inhibits the expression of the HIF-1α protein in a dose-dependent manner, while RT-PCR showed that it does not have any effects on HIF-1α mRNA. In addition, EGCG attenuated the mRNA and protein levels of P-gp in a dose-dependent manner, reaching a peak at the highest concentration. Furthermore, EGCG inhibited the proliferation of PANC-1 cells in a concentration- and time-dependent manner. The attenuation of HIF-1α and the consequently reduced P-gp could contribute to the inhibitory effects of EGCG on the proliferation of PANC-1 cells.

Shikonin suppresses proliferation and induces cell cycle arrest through the inhibition of hypoxia-inducible factor-1α signaling.

PubMed

Li, Ming Yue; Mi, Chunliu; Wang, Ke Si; Wang, Zhe; Zuo, Hong Xiang; Piao, Lian Xun; Xu, Guang Hua; Li, Xuezheng; Ma, Juan; Jin, Xuejun

2017-08-25

Hypoxia enhances the development of solid tumors. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that is dominantly expressed under hypoxia in solid tumor cells and is a key factor of tumor regulation. HIF-1α regulates several target genes involved in many aspects of cancer progression, including angiogenesis, metastasis, and cell proliferation, as well as imparting resistance to cancer treatment. In this study, we assessed shikonin, which derives from the traditional medical herb Lithospermum erythrorhizon, for its anti-cancer effects in hypoxia-induced human colon cancer cell lines. Shikonin showed potent inhibitory activity against hypoxia-induced HIF-1α activation in various human cancer cell lines and efficient scavenging activity of hypoxia-induced reactive oxygen species in tumor cells. Further analysis revealed that shikonin inhibited HIF-1α protein synthesis without affecting the expression of HIF-1α mRNA or degrading HIF-1α protein. It was subsequently shown to attenuate the activation of downstream mTOR/p70S6K/4E-BP1/eIF4E kinase. Shikonin also dose-dependently caused the cell cycle arrest of activated HCT116 cells and inhibited the proliferation of HCT116 and SW620 cells. Moreover, it significantly inhibited tumor growth in a xenograft modal. These findings suggest that shikonin could be considered for use as a potential drug in human colon cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

PubMed

Schörg, Alexandra; Santambrogio, Sara; Platt, James L; Schödel, Johannes; Lindenmeyer, Maja T; Cohen, Clemens D; Schrödter, Katrin; Mole, David R; Wenger, Roland H; Hoogewijs, David

2015-07-13

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the -82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the -82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the -82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Valproic acid inhibits the angiogenic potential of cervical cancer cells via HIF-1α/VEGF signals.

PubMed

Zhao, Y; You, W; Zheng, J; Chi, Y; Tang, W; Du, R

2016-11-01

Cervical cancer is one of the most prevalent malignancies in women worldwide. Therefore, the investigation about the molecular pathogenesis and related therapy targets of cervical cancer is an emergency. The objective of the present study is to investigate the effects of valproic acid (VPA), a histone deacetylase inhibitor, on the angiogenesis of cervical cancer. The effects and mechanisms of VPA on in vitro angiogenesis and vascular endothelial growth factor (VEGF) expression of human cervical cancer HeLa and SiHa cells were investigated. Our present study reveals that 1 mM VPA can significantly inhibit the in vitro angiogenic potential and VEGF expression of human cervical cancer HeLa and SiHa cells. Further, the transcription and protein levels of hypoxia inducible factor-1α (HIF-1α), and not HIF-1β, are significantly inhibited in VPA-treated cervical cancer cells. Over expression of HIF-1α can obviously reverse VPA-induced VEGF down regulation. VPA-treatment decreases the activation of Akt and ERK1/2 in both HeLa and SiHa cells in a time-dependent manner. The inhibitor of Akt (LY 294002) or ERK1/2 (PD98059) can inhibit VEGF alone and cooperatively reinforce the suppression effects of VPA on HIF-1α and VEGF expression. Collectively, our data reveal that the inhibition of PI3K/Akt and ERK1/2 signals are involved in VPA-induced HIF-1α and VEGF suppression of cervical cancer cells.

Targeting Hypoxia-Inducible Factor 1α in a New Orthotopic Model of Glioblastoma Recapitulating the Hypoxic Tumor Microenvironment.

PubMed

Nigim, Fares; Cavanaugh, Jill; Patel, Anoop P; Curry, William T; Esaki, Shin-ichi; Kasper, Ekkehard M; Chi, Andrew S; Louis, David N; Martuza, Robert L; Rabkin, Samuel D; Wakimoto, Hiroaki

2015-07-01

Tissue hypoxia and necrosis represent pathophysiologic and histologic hallmarks of glioblastoma (GBM). Although hypoxia inducible factor 1α (HIF-1α) plays crucial roles in the malignant phenotypes of GBM, developing HIF-1α-targeted agents has been hampered by the lack of a suitable preclinical model that recapitulates the complex biology of clinical GBM. We present a new GBM model, MGG123, which was established from a recurrent human GBM. Orthotopic xenografting of stem-like MGG123 cells reproducibly generated lethal tumors that were characterized by foci of palisading necrosis, hypervascularity, and robust stem cell marker expression. Perinecrotic neoplastic cells distinctively express HIF-1α and are proliferative in both xenografts and the patient tissue. The xenografts contain scattered hypoxic foci that were consistently greater than 50 μm distant from blood vessels, indicating intratumoral heterogeneity of oxygenation. Hypoxia enhanced HIF-1α expression in cultured MGG123 cells, which was abrogated by the HIF-1α inhibitors digoxin or ouabain. In vivo, treatment of orthotopic MGG123 xenografts with digoxin decreased HIF-1α expression, vascular endothelial growth factor mRNA levels, and CD34-positive vasculature within the tumors, and extended survival of mice bearing the aggressive MGG123 GBM. This preclinical tumor model faithfully recapitulates the GBM-relevant hypoxic microenvironment and stemness and is a suitable platform for studying disease biology and developing hypoxia-targeted agents.

Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling.

PubMed

Senavirathna, Lakmini Kumari; Huang, Chaoqun; Yang, Xiaoyun; Munteanu, Maria Cristina; Sathiaseelan, Roshini; Xu, Dao; Henke, Craig A; Liu, Lin

2018-02-09

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3-6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.

HIF-1α signaling activation by post-ischemia treatment with astragaloside IV attenuates myocardial ischemia-reperfusion injury.

PubMed

Si, Jingwen; Wang, Ning; Wang, Huan; Xie, Juan; Yang, Jian; Yi, Hui; Shi, Zixuan; Ma, Jing; Wang, Wen; Yang, Lifang; Yu, Shiqiang; Li, Junchang

2014-01-01

In this study, we evaluated the effect of astragaloside IV (Ast IV) post-ischemia treatment on myocardial ischemia-reperfusion (IR) injury (IRI). We also examined whether hypoxia inducible factor-1α (HIF-1α) and its downstream gene-inducible nitric oxide (NO) synthase (iNOS) play roles in the cardioprotective effect of Ast IV. Cultured cardiomyocytes and perfused isolated rat hearts were exposed to Ast IV during reperfusion in the presence or absence of the HIF-1α inhibitor 2-methoxyestradiol (2-MeOE2). The post-ischemia treatment with Ast IV protected cardiomyocytes from the apoptosis and death induced by simulated IRI (SIRI). Additionally, in cardiomyocytes, 2-MeOE2 and HIF-1α siRNA treatment each not only abolished the anti-apoptotic effect of post-ischemia treatment with Ast IV but also reversed the upregulation of HIF-1α and iNOS expression. Furthermore, after treatment with Ast IV, post-ischemic cardiac functional recovery and lactate dehydrogenase (LDH) release in the coronary flow (CF) were improved, and the myocardial infarct size was decreased. Moreover, the number of apoptotic cells was reduced, and the upregulation of the anti-apoptotic protein Bcl2 and downregulation of the pro-apoptotic protein Caspase3 were reversed. 2-MeOE2 reversed these effects of Ast IV on IR-injured hearts. These results suggest that post-ischemia treatment with Ast IV can attenuate IRI by upregulating HIF-1α expression, which transmits a survival signal to the myocardium.

Elevated Endothelial Hypoxia-Inducible Factor-1α Contributes to Glomerular Injury and Promotes Hypertensive Chronic Kidney Disease.

PubMed

Luo, Renna; Zhang, Weiru; Zhao, Cheng; Zhang, Yujin; Wu, Hongyu; Jin, Jianping; Zhang, Wenzheng; Grenz, Almut; Eltzschig, Holger K; Tao, Lijian; Kellems, Rodney E; Xia, Yang

2015-07-01

Hypertensive chronic kidney disease is one of the most prevalent medical conditions with high morbidity and mortality in the United States and worldwide. However, early events initiating the progression to hypertensive chronic kidney disease are poorly understood. We hypothesized that elevated endothelial hypoxia-inducible factor-1α (HIF-1α) is a common early insult triggering initial glomerular injury leading to hypertensive chronic kidney disease. To test our hypothesis, we used an angiotensin II infusion model of hypertensive chronic kidney disease to determine the specific cell type and mechanisms responsible for elevation of HIF-1α and its role in the progression of hypertensive chronic kidney disease. Genetic studies coupled with reverse transcription polymerase chain reaction profiling revealed that elevated endothelial HIF-1α is essential to initiate glomerular injury and progression to renal fibrosis by the transcriptional activation of genes encoding multiple vasoactive proteins. Mechanistically, we found that endothelial HIF-1α gene expression was induced by angiotensin II in a nuclear factor-κB-dependent manner. Finally, we discovered reciprocal positive transcriptional regulation of endothelial Hif-1α and Nf-κb genes is a key driving force for their persistent activation and disease progression. Overall, our findings revealed that the stimulation of HIF-1α gene expression in endothelial cells is detrimental to induce kidney injury, hypertension, and disease progression. Our findings highlight early diagnostic opportunities and therapeutic approaches for hypertensive chronic kidney disease. © 2015 American Heart Association, Inc.

A Myeloid Hypoxia-inducible Factor 1α-Krüppel-like Factor 2 Pathway Regulates Gram-positive Endotoxin-mediated Sepsis*

PubMed Central

Mahabeleshwar, Ganapati H.; Qureshi, Muhammad Awais; Takami, Yoichi; Sharma, Nikunj; Lingrel, Jerry B.; Jain, Mukesh K.

2012-01-01

Although Gram-positive infections account for the majority of cases of sepsis, the molecular mechanisms underlying their effects remains poorly understood. We investigated how cell wall components of Gram-positive bacteria contribute to the development of sepsis. Experimental observations derived from cultured primary macrophages and the cell line indicate that Gram-positive bacterial endotoxins induce hypoxia-inducible factor 1α (HIF-1α) mRNA and protein expression. Inoculation of live or heat-inactivated Gram-positive bacteria with macrophages induced HIF-1 transcriptional activity in macrophages. Concordant with these results, myeloid deficiency of HIF-1α attenuated Gram-positive bacterial endotoxin-induced cellular motility and proinflammatory gene expression in macrophages. Conversely, Gram-positive bacteria and their endotoxins reduced expression of the myeloid anti-inflammatory transcription factor Krüppel-like transcription factor 2 (KLF2). Sustained expression of KLF2 reduced and deficiency of KLF2 enhanced Gram-positive endotoxins induced HIF-1α mRNA and protein expression in macrophages. More importantly, KLF2 attenuated Gram-positive endotoxins induced cellular motility and proinflammatory gene expression in myeloid cells. Consistent with these results, mice deficient in myeloid HIF-1α were protected from Gram-positive endotoxin-induced sepsis mortality and clinical symptomatology. By contrast, myeloid KLF2-deficient mice were susceptible to Gram-positive sepsis induced mortality and clinical symptoms. Collectively, these observations identify HIF-1α and KLF2 as critical regulators of Gram-positive endotoxin-mediated sepsis. PMID:22110137

Inhibitory effect of a redox-silent analogue of tocotrienol on hypoxia adaptation in prostate cancer cells.

PubMed

Shiozawa, Nobuya; Sugahara, Ryosuke; Namiki, Kozue; Sato, Chiaki; Ando, Akira; Sato, Ayami; Virgona, Nantiga; Yano, Tomohiro

2017-03-01

Prostate cancer (PCa) is one of the most common cancers in Western countries and acquires a malignant phenotype, androgen-independent growth. PCa under hypoxia often has resistance to chemotherapy and radiotherapy. However, an effective therapy against PCa under hypoxia has not yet been established. In this report, we investigated the inhibitory effect of a redox-silent analogue of tocotrienol on the survival of a human androgen-independent PCa cell line (PC3) under hypoxia. We found that the redox-silent analogue exerted a cytotoxic effect on PC3 cells in a dose-dependent manner irrespective of either hypoxia or normoxia. Moreover, under hypoxia, the analogue dose dependently reduced the protein levels of hypoxia-inducible factor (HIF)-1α and HIF-2α. In addition, a specific inhibitor toward HIF-1α induced cytotoxicity on PC3 cells, whereas selective inhibition of HIF-2α exerted no effect. Furthermore, suppression of HIFs levels by the analogue in hypoxic PC3 cells was closely associated with the inactivation of Fyn, a member of the nonreceptor tyrosine kinase family, as confirmed by the action of a specific inhibitor toward the kinase (PP2). Taken together, these results suggest that the tocotrienol analogue could inhibit the survival of PC3 cells under hypoxia, mainly by the inhibition of Fyn/HIF-1α signaling, and this may lead to the establishment of a new effective therapy for androgen-independent PCa.

Impairment of hypoxia-induced HIF-1α signaling in keratinocytes and fibroblasts by sulfur mustard is counteracted by a selective PHD-2 inhibitor.

PubMed

Deppe, Janina; Popp, Tanja; Egea, Virginia; Steinritz, Dirk; Schmidt, Annette; Thiermann, Horst; Weber, Christian; Ries, Christian

2016-05-01

Skin exposure to sulfur mustard (SM) provokes long-term complications in wound healing. Similar to chronic wounds, SM-induced skin lesions are associated with low levels of oxygen in the wound tissue. Normally, skin cells respond to hypoxia by stabilization of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α). HIF-1α modulates expression of genes including VEGFA, BNIP3, and MMP2 that control processes such as angiogenesis, growth, and extracellular proteolysis essential for proper wound healing. The results of our studies revealed that exposure of primary normal human epidermal keratinocytes (NHEK) and primary normal human dermal fibroblasts (NHDF) to SM significantly impaired hypoxia-induced HIF-1α stabilization and target gene expression in these cells. Addition of a selective inhibitor of the oxygen-sensitive prolyl hydroxylase domain-containing protein 2 (PHD-2), IOX2, fully recovered HIF-1α stability, nuclear translocation, and target gene expression in NHEK and NHDF. Moreover, functional studies using a scratch wound assay demonstrated that the application of IOX2 efficiently counteracted SM-mediated deficiencies in monolayer regeneration under hypoxic conditions in NHEK and NHDF. Our findings describe a pathomechanism by which SM negatively affects hypoxia-stimulated HIF-1α signaling in keratinocytes and fibroblasts and thus possibly contributes to delayed wound healing in SM-injured patients that could be treated with PHD-2 inhibitors.

IL-1β upregulates Muc5ac expression via NF-κB-induced HIF-1α in asthma.

PubMed

Wu, Shouzhen; Li, Hailong; Yu, Lijuan; Wang, Ning; Li, Xu; Chen, Wei

2017-12-01

The manifest and important feature in respiratory diseases, including asthma and COPD (chronic obstructive pulmonary disease), is the increased numbers and hypersecretion of goblet cells and overexpression of mucins, especially Muc5ac. Many proinflammatory cytokines play important roles in goblet cell metaplasia and overproduction of Muc5ac. However, the effect of IL-1β on Muc5ac expression in asthma remains unknown. Here, we detected the correlation between IL-1β and Muc5ac in asthma patients and further explored the mechanism of IL-1β-induced Muc5ac overexpression. Our results showed that Muc5ac and IL-1β were up-regulated in 41 patients with asthma and that Muc5ac overexpression was related with IL-1β in asthma (R 2 =0.668, p≪0.001). Furthermore, the correlation between IL-1β and Muc5ac is higher in severe group than that in moderate group. In vitro experiments with normal human bronchial epithelial cells (NHBECs) showed that IL-1β up-regulated Muc5ac expression in NHBEC in a time- and dosage-dependent manner. Hypoxia-induced HIF-1α was responsible for Muc5ac expression mediated by IL-1β. Knocking down HIF-1α by siRNA decreased Muc5ac expression under hypoxia even in IL-1β-treated NHBEC cells. Luciferase reporter assay showed that HIF-1α enhanced Muc5ac promoter activity in HEK293T cells. HIF-1α could specifically bind to the promoter of Muc5ac by EMSA. The correlation among IL-1β, HIF-1α and Muc5ac was observed in patients with asthma. Mechanically, NF-κB activation was essential to IL-1β-induced HIF-1α upregulation via the canonical pathway of NF-κB. The level of nuclear p65, a subunit of NF-κB, was obviously increased in NHBEC cells under IL-1β treatment. IL-1β did not change either HIF-1α or Muc5ac expression when inhibiting NF-κB signaling with Bay11-7082, an inhibitor of NF-κB. Collectively, we concluded that IL-1β up-regulated Muc5ac expression via NF-κB-induced HIF-1α in asthma and provided a potential therapeutic target for asthma. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.