scholarly journals Up-regulation of the manganese transporter SLC30A10 by hypoxia-inducible factors defines a homeostatic response to manganese toxicity

2021 ◽  
Vol 118 (35) ◽  
pp. e2107673118
Author(s):  
Chunyi Liu ◽  
Thomas Jursa ◽  
Michael Aschner ◽  
Donald R. Smith ◽  
Somshuvra Mukhopadhyay
Keyword(s):  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Hypoxia Response Element ◽  
Mn Toxicity ◽  
Cellular Protection ◽  
Regulated Expression ◽  
Therapeutic Development ◽  
Prolyl Hydroxylation ◽  
Homeostatic Response

Manganese (Mn) is an essential metal that induces incurable parkinsonism at elevated levels. However, unlike other essential metals, mechanisms that regulate mammalian Mn homeostasis are poorly understood, which has limited therapeutic development. Here, we discovered that the exposure of mice to a translationally relevant oral Mn regimen up-regulated expression of SLC30A10, a critical Mn efflux transporter, in the liver and intestines. Mechanistic studies in cell culture, including primary human hepatocytes, revealed that 1) elevated Mn transcriptionally up-regulated SLC30A10, 2) a hypoxia response element in the SLC30A10 promoter was necessary, 3) the transcriptional activities of hypoxia-inducible factor (HIF) 1 or HIF2 were required and sufficient for the SLC30A10 response, 4) elevated Mn activated HIF1/HIF2 by blocking the prolyl hydroxylation of HIF proteins necessary for their degradation, and 5) blocking the Mn-induced up-regulation of SLC30A10 increased intracellular Mn levels and enhanced Mn toxicity. Finally, prolyl hydroxylase inhibitors that stabilize HIF proteins and are in advanced clinical trials for other diseases reduced intracellular Mn levels and afforded cellular protection against Mn toxicity and also ameliorated the in vivo Mn-induced neuromotor deficits in mice. These findings define a fundamental homeostatic protective response to Mn toxicity—elevated Mn levels activate HIF1 and HIF2 to up-regulate SLC30A10, which in turn reduces cellular and organismal Mn levels, and further indicate that it may be possible to repurpose prolyl hydroxylase inhibitors for the management of Mn neurotoxicity.

scholarly journals The pro-oncogenic adaptor CIN85 inhibits hypoxia-inducible factor prolyl hydroxylase-2

2018 ◽  
Author(s):  
Nina Kozlova ◽  
Daniela Mennerich ◽  
Anatoly Samoylenko ◽  
Elitsa Y. Dimova ◽  
Peppi Koivunen ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Survival Function ◽  
Hypoxia Inducible Factor ◽  
Adaptor Protein ◽  
Prolyl Hydroxylase ◽  
Binding Partner ◽  
And Migration ◽  
Hif Prolyl Hydroxylase ◽  
Promote Breast Cancer

SummaryThe EGFR-adaptor protein CIN85 has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill-defined. Here, we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SH3 domains of CIN85 interact with the proline-arginine rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF-degradation. This activity is essential in vivo, as specific loss of the CIN85-PHD2 interaction in CRISPR/Cas9 edited cells affected growth and migration properties as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2, and uncovered an essential survival function in tumor cells linking growth factor adaptors with hypoxia signaling.

scholarly journals Regulation of adult erythropoiesis by prolyl hydroxylase domain proteins

Blood ◽  
2008 ◽  
Vol 111 (6) ◽  
pp. 3229-3235 ◽  
Author(s):  
Kotaro Takeda ◽  
Hector L. Aguila ◽  
Nehal S. Parikh ◽  
Xiping Li ◽  
Katie Lamothe ◽  
...  
Keyword(s):  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Heterozygous Mutation ◽  
Adult Liver ◽  
Double Knockout ◽  
Adult Mice ◽  
Liver And Kidney ◽  
Prolyl Hydroxylation ◽  
Α Subunits ◽  
Prolyl Hydroxylase Domain

Abstract Polycythemia is often associated with erythropoietin (EPO) overexpression and defective oxygen sensing. In normal cells, intracellular oxygen concentrations are directly sensed by prolyl hydroxylase domain (PHD)–containing proteins, which tag hypoxia-inducible factor (HIF) α subunits for polyubiquitination and proteasomal degradation by oxygen-dependent prolyl hydroxylation. Here we show that different PHD isoforms differentially regulate HIF-α stability in the adult liver and kidney and suppress Epo expression and erythropoiesis through distinct mechanisms. Although Phd1−/− or Phd3−/− mice had no apparent defects, double knockout of Phd1 and Phd3 led to moderate erythrocytosis. HIF-2α, which is known to activate Epo expression, accumulated in the liver. In adult mice deficient for PHD2, the prototypic Epo transcriptional activator HIF-1α accumulated in both the kidney and liver. Elevated HIF-1α levels were associated with dramatically increased concentrations of both Epo mRNA in the kidney and Epo protein in the serum, which led to severe erythrocytosis. In contrast, heterozygous mutation of Phd2 had no detectable effects on blood homeostasis. These findings suggest that PHD1/3 double deficiency leads to erythrocytosis partly by activating the hepatic HIF-2α/Epo pathway, whereas PHD2 deficiency leads to erythrocytosis by activating the renal Epo pathway.

scholarly journals Evidence for the Capability of Roxadustat (FG-4592), an Oral HIF Prolyl-Hydroxylase Inhibitor, to Perturb Membrane Ionic Currents: An Unidentified yet Important Action

2019 ◽  
Vol 20 (23) ◽  
pp. 6027 ◽  
Author(s):  
Wei-Ting Chang ◽  
Yi-Ching Lo ◽  
Zi-Han Gao ◽  
Sheng-Nan Wu
Keyword(s):  
Time Course ◽  
Hypoxia Inducible Factor ◽  
Ionic Currents ◽  
Membrane Depolarization ◽  
Prolyl Hydroxylase ◽  
Gh3 Cells ◽  
Heart Cells ◽  
Patch Clamp Technique ◽  
Hif Prolyl Hydroxylase

Roxadustat (FG-4592), an analog of 2-oxoglutarate, is an orally-administered, heterocyclic small molecule known to be an inhibitor of hypoxia inducible factor (HIF) prolyl hydroxylase. However, none of the studies have thus far thoroughly investigated its possible perturbations on membrane ion currents in endocrine or heart cells. In our studies, the whole-cell current recordings of the patch-clamp technique showed that the presence of roxadustat effectively and differentially suppressed the peak and late components of IK(DR) amplitude in response to membrane depolarization in pituitary tumor (GH3) cells with an IC50 value of 5.71 and 1.32 μM, respectively. The current inactivation of IK(DR) elicited by 10-sec membrane depolarization became raised in the presence of roxadustatt. When cells were exposed to either CoCl2 or deferoxamine (DFO), the IK(DR) elicited by membrane depolarization was not modified; however, nonactin, a K+-selective ionophore, in continued presence of roxadustat, attenuated roxadustat-mediated inhibition of the amplitude. The steady-state inactivation of IK(DR) could be constructed in the presence of roxadustat. Recovery of IK(DR) block by roxadustat (3 and 10 μM) could be fitted by a single exponential with 382 and 523 msec, respectively. The roxadustat addition slightly suppressed erg-mediated K+ or hyperpolarization-activated cation currents. This drug also decreased the peak amplitude of voltage-gated Na+ current with a slowing in inactivation rate of the current. Likewise, in H9c2 heart-derived cells, the addition of roxadustat suppressed IK(DR) amplitude in combination with the shortening in inactivation time course of the current. In high glucose-treated H9c2 cells, roxadustat-mediated inhibition of IK(DR) remained unchanged. Collectively, despite its suppression of HIF prolyl hydroxylase, inhibitory actions of roxadustat on different types of ionic currents possibly in a non-genomic fashion might provide another yet unidentified mechanism through which cellular functions are seriously perturbed, if similar findings occur in vivo.

scholarly journals Cystathione β-synthase regulates HIF-1α stability through persulfidation of PHD2

2020 ◽  
Vol 6 (27) ◽  
pp. eaaz8534
Author(s):  
Anindya Dey ◽  
Shubhangi Prabhudesai ◽  
Yushan Zhang ◽  
Geeta Rao ◽  
Karthikeyan Thirugnanam ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Hypoxia Inducible Factor ◽  
Point Mutations ◽  
Prolyl Hydroxylase ◽  
Zinc Finger Motif ◽  
Hydroxylase Activity ◽  
Zebrafish Model ◽  
Enzymatic Action

The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine β-synthase (CBS) produces H2S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys21 and Cys33 (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H2S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H2S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H2S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.

scholarly journals Multiple Factors Affecting Cellular Redox Status and Energy Metabolism Modulate Hypoxia-Inducible Factor Prolyl Hydroxylase Activity In Vivo and In Vitro

2006 ◽  
Vol 27 (3) ◽  
pp. 912-925 ◽  
Author(s):  
Yi Pan ◽  
Kyle D. Mansfield ◽  
Cara C. Bertozzi ◽  
Viktoriya Rudenko ◽  
Denise A. Chan ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Hypoxia Inducible Factor ◽  
Simultaneous Detection ◽  
Tca Cycle ◽  
Cellular Responses ◽  
Prolyl Hydroxylase ◽  
In Vitro Assays ◽  
Tca Cycle Intermediates

ABSTRACT Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-α) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O2)-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O2 deprivation (hypoxia). Using a fusion protein containing the human HIF-1α O2-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell-free systems. This novel assay allows the simultaneous detection of both hydroxylated and nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1α during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1α accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O2 depletion.

scholarly journals Control of the Hypoxic Response in Drosophila melanogaster by the Basic Helix-Loop-Helix PAS Protein Similar

2002 ◽  
Vol 22 (19) ◽  
pp. 6842-6853 ◽  
Author(s):  
Sofía Lavista-Llanos ◽  
Lázaro Centanin ◽  
Maximiliano Irisarri ◽  
Daniela M. Russo ◽  
Jonathan M. Gleadle ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Time Course ◽  
Hypoxia Inducible Factor ◽  
Ectopic Expression ◽  
Prolyl Hydroxylase ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix ◽  
Oxygen Homeostasis ◽  
Multicellular Organisms

ABSTRACT In mammalian systems, the heterodimeric basic helix-loop-helix (bHLH)-PAS transcription hypoxia-inducible factor (HIF) has emerged as the key regulator of responses to hypoxia. Here we define a homologous system in Drosophila melanogaster, and we characterize its activity in vivo during development. By using transcriptional reporters in developing transgenic flies, we show that hypoxia-inducible activity rises to a peak in late embryogenesis and is most pronounced in tracheal cells. We show that the bHLH-PAS proteins Similar (Sima) and Tango (Tgo) function as HIF-α and HIF-β homologues, respectively, and demonstrate a conserved mode of regulation for Sima by oxygen. Sima protein, but not its mRNA, was upregulated in hypoxia. Time course experiments following pulsed ectopic expression demonstrated that Sima is stabilized in hypoxia and that degradation relies on a central domain encompassing amino acids 692 to 863. Continuous ectopic expression overrode Sima degradation, which remained cytoplasmic in normoxia, and translocated to the nucleus only in hypoxia, revealing a second oxygen-regulated activation step. Abrogation of the Drosophila Egl-9 prolyl hydroxylase homologue, CG1114, caused both stabilization and nuclear localization of Sima, indicating a central involvement in both processes. Tight conservation of the HIF/prolyl hydroxylase system in Drosophila provides a new focus for understanding oxygen homeostasis in intact multicellular organisms.

scholarly journals The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α

2016 ◽  
Vol 36 (18) ◽  
pp. 2328-2343 ◽  
Author(s):  
Patrick R. Arsenault ◽  
Daisheng Song ◽  
Yu Jin Chung ◽  
Tejvir S. Khurana ◽  
Frank S. Lee
Keyword(s):  
High Altitude ◽  
Zinc Finger ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Loss Of Function ◽  
Domain Protein ◽  
Factor Α ◽  
Prolyl Hydroxylase Domain

Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical bothin vitroandin vivo. We show thatin vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α.In vivo, ablation of zinc finger function by a C36S/C42SEgln1knock-in mutation results in upregulation of theerythropoietingene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks ofEgln1loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-functionEGLN1allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude.

scholarly journals A Feedback Loop Involving the Phd3 Prolyl Hydroxylase Tunes the Mammalian Hypoxic Response In Vivo

2009 ◽  
Vol 29 (21) ◽  
pp. 5729-5741 ◽  
Author(s):  
Yoji Andrew Minamishima ◽  
Javid Moslehi ◽  
Robert F. Padera ◽  
Roderick T. Bronson ◽  
Ronglih Liao ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Target Genes ◽  
Hypoxia Inducible Factor ◽  
Premature Mortality ◽  
Prolyl Hydroxylase ◽  
Α Subunit ◽  
Von Hippel Lindau ◽  
Regulatory Feedback Loop

ABSTRACT Hypoxia-inducible factor (HIF), consisting of a labile α subunit and a stable β subunit, is a master regulator of hypoxia-responsive mRNAs. HIFα undergoes oxygen-dependent prolyl hydroxylation, which marks it for polyubiquitination by a complex containing the von Hippel-Lindau protein (pVHL). Among the three Phd family members, Phd2 appears to be the primary HIF prolyl hydroxylase. Phd3 is induced by HIF and, based on findings from in vitro studies, may participate in a HIF-regulatory feedback loop. Here, we report that Phd3 loss exacerbates the HIF activation, hepatic steatosis, dilated cardiomyopathy, and premature mortality observed in mice lacking Phd2 alone and produces a closer phenocopy of the changes seen in mice lacking pVHL than the loss of Phd2 alone. Importantly, the degree to which Phd3 can compensate for Phd2 loss and the degree to which the combined loss of Phd2 and Phd3 resembles pVHL loss appear to differ for different HIF-responsive genes and in different tissues. These findings highlight that the responses of different HIF target genes to changes in prolyl hydroxylase activity differ, quantitatively and qualitatively, in vivo and have implications for the development of paralog-specific prolyl hydroxylase inhibitors as therapeutic agents.

scholarly journals Pseudomonas expression of an oxygen sensing prolyl hydroxylase homologue regulates neutrophil host responses in vitro and in vivo

2017 ◽  
Vol 2 ◽  
pp. 104 ◽  
Author(s):  
Rebecca S. Dickinson ◽  
Fiona Murphy ◽  
Catherine Doherty ◽  
Sam Williams ◽  
Ananda Mirchandani ◽  
...  
Keyword(s):  
Active Sites ◽  
Hypoxia Inducible Factor ◽  
Elongation Factor ◽  
Prolyl Hydroxylase ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
Wild Type ◽  
Prolyl Hydroxylases

Background: Pseudomonas species are adapted to evade innate immune responses and can persist at sites of relative tissue hypoxia, including the mucus-plugged airways of patients with cystic fibrosis and bronchiectasis.  The ability of these bacteria to directly sense and respond to changes in local oxygen availability is in part consequent upon expression of the 2-oxoglutarate oxygenase, Pseudomonas prolyl hydroxylase (PPHD), which acts on elongation factor Tu (EF-Tu), and is homologous with the human hypoxia inducible factor (HIF) prolyl hydroxylases. We report that PPHD expression regulates the neutrophil response to acute pseudomonal infection. Methods: In vitro co-culture experiments were performed with human neutrophils and PPHD-deficient and wild-type bacteria and supernatants, with viable neutrophil counts determined by flow cytometry. In vivo consequences of infection with PPHD deficient P. aeruginosa were determined in an acute pneumonia mouse model following intra-tracheal challenge. Results: Supernatants of PPHD-deficient bacterial cultures contained higher concentrations of the phenazine exotoxin pyocyanin and induced greater acceleration of neutrophil apoptosis than wild-type PAO1 supernatants in vitro.  In vivo infection with PPHD mutants compared to wild-type PAO1 controls resulted in increased levels of neutrophil apoptosis and impaired control of infection, with higher numbers of P. aeruginosa recovered from the lungs of mice infected with the PPHD-deficient strain.  This resulted in an overall increase in mortality in mice infected with the PPHD-deficient strain. Conclusions: Our data show that Pseudomonas expression of its prolyl hydroxylase influences the outcome of host-pathogen interactions in vitro and in vivo, demonstrating the importance of considering how both host and pathogen adaptations to hypoxia together define outcomes of infection. Given that inhibitors for the HIF prolyl hydroxylases are in late stage trials for the treatment of anaemia and that the active sites of PPHD and human HIF prolyl hydroxylases are closely related, the results are of current clinical interest.

Metallic Elements (Cu, Zn, Ni and Mn) Toxicity Effects Determination on a Fresh Water Fish Cyprinus Carpio (Common Carp) Laboratory Acclimatized

2017 ◽  
Vol 68 (8) ◽  
pp. 1711-1715
Author(s):  
Stefania Gheorghe ◽  
Gabriela Geanina Vasile ◽  
Cristina Gligor ◽  
Irina Eugenia Lucaciu ◽  
Mihai Nita Lazar
Keyword(s):  
Cyprinus Carpio ◽  
Aquatic Organisms ◽  
Control Fish ◽  
Toxicity Tests ◽  
Fresh Water Fish ◽  
Metallic Elements ◽  
Mn Toxicity ◽  
Vitro Effect

Metallic elements copper (Cu), zinc (Zn), nickel (Ni) and manganese (Mn) are some of the most commonly found in water and sediment samples collected from the Danube - Danube Delta. These elements are important as essential micronutrients, being normally present at low concentrations in biological organisms, but in high concentrations they become toxic with immediate and delayed effects. The role of this metals is still controversial, that�s why bioconcentration potential is so important. In this non-clinical study, we tested in vitro effect of heavy metals on carp, Cyprinus carpio, reproducing in vivo presence of Cu, Zn, Ni and Mn in the Romanian�s surface water. The toxicity tests were performed according to OECD 203 by detecting the average (50%) lethal concentration - LC50 on aquatic organisms (freshwater fish) at 96h. The results pointed out that, copper value for LC 50 at 96h was estimated as 3.4 mg/L (concentrations tested in the range of 0.1 - 4.75 mg/L). Zinc value for LC 50 at 96h was estimated as 20.8 mg/L (concentrations tested in the range of 0.028 � 29.6 mg/L). Nickel value for LC 50 at 96h was estimated as 40.1 mg/L (concentrations tested in the range of 0.008 - 84.5 mg/L). For manganese the mortality effects has recorded at LC 50 at 96h at estimated value higher than 53 mg/L (concentrations tested in the range of 0.04 - 53.9 mg/L). The accuracy of the testing metals concentration was insured by the screening of the dilution water, as well as food and control fish, acclimated in laboratory conditions.

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