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1.
PLoS One ; 18(3): e0282223, 2023.
Article in English | MEDLINE | ID: mdl-36862715

ABSTRACT

The microenvironment of solid tumors is characterized by oxygen and glucose deprivation. Acss2/HIF-2 signaling coordinates essential genetic regulators including acetate-dependent acetyl CoA synthetase 2 (Acss2), Creb binding protein (Cbp), Sirtuin 1 (Sirt1), and Hypoxia Inducible Factor 2α (HIF-2α). We previously shown in mice that exogenous acetate augments growth and metastasis of flank tumors derived from fibrosarcoma-derived HT1080 cells in an Acss2/HIF-2 dependent manner. Colonic epithelial cells are exposed to the highest acetate levels in the body. We reasoned that colon cancer cells, like fibrosarcoma cells, may respond to acetate in a pro-growth manner. In this study, we examine the role of Acss2/HIF-2 signaling in colon cancer. We find that Acss2/HIF-2 signaling is activated by oxygen or glucose deprivation in two human colon cancer-derived cell lines, HCT116 and HT29, and is crucial for colony formation, migration, and invasion in cell culture studies. Flank tumors derived from HCT116 and HT29 cells exhibit augmented growth in mice when supplemented with exogenous acetate in an Acss2/HIF-2 dependent manner. Finally, Acss2 in human colon cancer samples is most frequently localized in the nucleus, consistent with it having a signaling role. Targeted inhibition of Acss2/HIF-2 signaling may have synergistic effects for some colon cancer patients.


Subject(s)
Colonic Neoplasms , Fibrosarcoma , Humans , Animals , Mice , Acetate-CoA Ligase , Signal Transduction , Basic Helix-Loop-Helix Transcription Factors/genetics , Tumor Microenvironment
2.
Am J Respir Crit Care Med ; 206(1): 56-69, 2022 07 01.
Article in English | MEDLINE | ID: mdl-35417304

ABSTRACT

Rationale: Genetic studies of idiopathic pulmonary fibrosis (IPF) have improved our understanding of this disease, but not all causal loci have been identified. Objectives: To identify genes enriched with rare deleterious variants in IPF and familial pulmonary fibrosis. Methods: We performed gene burden analysis of whole-exome data, tested single variants for disease association, conducted KIF15 (kinesin family member 15) functional studies, and examined human lung single-cell RNA sequencing data. Measurements and Main Results: Gene burden analysis of 1,725 cases and 23,509 control subjects identified heterozygous rare deleterious variants in KIF15, a kinesin involved in spindle separation during mitosis, and three telomere-related genes (TERT [telomerase reverse transcriptase], RTEL1 [regulator of telomere elongation helicase 1], and PARN [poly(A)-specific ribonuclease]). KIF15 was implicated in autosomal-dominant models of rare deleterious variants (odds ratio [OR], 4.9; 95% confidence interval [CI], 2.7-8.8; P = 2.55 × 10-7) and rare protein-truncating variants (OR, 7.6; 95% CI, 3.3-17.1; P = 8.12 × 10-7). Meta-analyses of the discovery and replication cohorts, including 2,966 cases and 29,817 control subjects, confirm the involvement of KIF15 plus the three telomere-related genes. A common variant within a KIF15 intron (rs74341405; OR, 1.6; 95% CI, 1.4-1.9; P = 5.63 × 10-10) is associated with IPF risk, confirming a prior report. Lymphoblastoid cells from individuals heterozygous for the common variant have decreased KIF15 and reduced rates of cell growth. Cell proliferation is dependent on KIF15 in the presence of an inhibitor of Eg5/KIF11, which has partially redundant function. KIF15 is expressed specifically in replicating human lung cells and shows diminished expression in replicating epithelial cells of patients with IPF. Conclusions: Both rare deleterious variants and common variants in KIF15 link a nontelomerase pathway of cell proliferation with IPF susceptibility.


Subject(s)
Idiopathic Pulmonary Fibrosis , Kinesins , Telomerase , Exome , Humans , Idiopathic Pulmonary Fibrosis/genetics , Kinesins/genetics , Telomerase/genetics , Telomere
3.
J Biol Chem ; 297(3): 101037, 2021 09.
Article in English | MEDLINE | ID: mdl-34343565

ABSTRACT

Besides contributing to anabolism, cellular metabolites serve as substrates or cofactors for enzymes and may also have signaling functions. Given these roles, multiple control mechanisms likely ensure fidelity of metabolite-generating enzymes. Acetate-dependent acetyl CoA synthetases (ACS) are de novo sources of acetyl CoA, a building block for fatty acids and a substrate for acetyltransferases. Eukaryotic acetate-dependent acetyl CoA synthetase 2 (Acss2) is predominantly cytosolic, but is also found in the nucleus following oxygen or glucose deprivation, or upon acetate exposure. Acss2-generated acetyl CoA is used in acetylation of Hypoxia-Inducible Factor 2 (HIF-2), a stress-responsive transcription factor. Mutation of a putative nuclear localization signal in endogenous Acss2 abrogates HIF-2 acetylation and signaling, but surprisingly also results in reduced Acss2 protein levels due to unmasking of two protein destabilization elements (PDE) in the Acss2 hinge region. In the current study, we identify up to four additional PDE in the Acss2 hinge region and determine that a previously identified PDE, the ABC domain, consists of two functional PDE. We show that the ABC domain and other PDE are likely masked by intramolecular interactions with other domains in the Acss2 hinge region. We also characterize mice with a prematurely truncated Acss2 that exposes a putative ABC domain PDE, which exhibits reduced Acss2 protein stability and impaired HIF-2 signaling. Finally, using primary mouse embryonic fibroblasts, we demonstrate that the reduced stability of select Acss2 mutant proteins is due to a shortened half-life, which is a result of enhanced degradation via a nonproteasome, nonautophagy pathway.


Subject(s)
Acetate-CoA Ligase/chemistry , Acetate-CoA Ligase/metabolism , Acetates/metabolism , Acetate-CoA Ligase/genetics , Amino Acid Sequence , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Fibroblasts/chemistry , Fibroblasts/enzymology , Humans , Mice , Protein Binding , Protein Domains , Protein Stability , Sequence Alignment
4.
J Appl Physiol (1985) ; 130(4): 1122-1132, 2021 04 01.
Article in English | MEDLINE | ID: mdl-33539264

ABSTRACT

Olfactory receptor (Olfr) 78 is expressed in the carotid bodies (CB) and participates in CB responses to acute hypoxia. Olfr78 is also expressed in the kidney, which is a major site of erythropoietin (Epo) production by hypoxia. The present study examined the role of Olfr78 in cardiorespiratory and renal Epo gene responses to hypobaric hypoxia (HH), simulating low O2 condition experienced at high altitude. Studies were performed on adult, male wild-type (WT) and Olfr78 null mice treated with 18 h of HH (0.4 atmospheres). HH-treated WT mice exhibited increased baseline breathing, augmented hypoxic ventilatory response, elevated blood pressure, and plasma norepinephrine (NE) levels. These effects were associated with increased baseline CB sensory nerve activity and augmented CB sensory nerve response to subsequent acute hypoxia. In contrast, HH-treated Olfr78 null mice showed an absence of cardiorespiratory and CB sensory nerve responses, suggesting impaired CB-dependent cardiorespiratory adaptations. WT mice responded to HH with activation of the renal Epo gene expression and elevated plasma Epo levels, and these effects were attenuated or absent in Olfr78 null mice. The attenuated Epo activation by HH was accompanied with markedly reduced hypoxia-inducible factor (HIF)-2α protein and reduced activation of HIF-2 target gene Sod-1 in Olfr78 null mice, suggesting impaired transcriptional activation of HIF-2 contributes to attenuated Epo responses to HH. These results demonstrate a hitherto uncharacterized role for Olfr78 in cardiorespiratory adaptations and renal Epo gene activation by HH such as that experienced at high altitude.NEW & NOTEWORTHY In this study, we delineated a previously uncharacterized role for olfactory receptor 78 (Olfr78), a G-protein-coupled receptor in regulation of erythropoietin and cardiorespiratory responses to hypobaric hypoxia. Our results demonstrate a striking loss of cardiorespiratory adaptations accompanied by an equally striking absence of carotid body sensory nerve responses to hypobaric hypoxia in Olfr78 null mice. We further demonstrate a hitherto uncharacterized role for Olfr78 in erythropoietin activation by hypobaric hypoxia.


Subject(s)
Carotid Body , Erythropoietin , Receptors, Odorant , Animals , Hypoxia , Male , Mice , Respiration
5.
Bioconjug Chem ; 31(5): 1449-1462, 2020 05 20.
Article in English | MEDLINE | ID: mdl-32302483

ABSTRACT

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analogue of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.


Subject(s)
Amines/chemistry , Imides/chemistry , Proteins/chemistry , Hydrogen-Ion Concentration , Kinetics , Lysine/chemistry , Solvents/chemistry , Sulfhydryl Compounds/chemistry , Thermodynamics
6.
PLoS One ; 14(11): e0225105, 2019.
Article in English | MEDLINE | ID: mdl-31725783

ABSTRACT

The response to environmental stresses by eukaryotic organisms includes activation of protective biological mechanisms, orchestrated in part by transcriptional regulators. The tri-member Hypoxia Inducible Factor (HIF) family of DNA-binding transcription factors include HIF-2, which is activated under conditions of oxygen or glucose deprivation. Although oxygen-dependent protein degradation is a key mechanism by which HIF-1 and HIF-2 activity is regulated, HIF-2 is also influenced substantially by the coupled action of acetylation and deacetylation. The acetylation/deacetylation process that HIF-2 undergoes employs a specific acetyltransferase and deacetylase. Likewise, the supply of the acetyl donor, acetyl CoA, used for HIF-2 acetylation originates from a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (Acss2). Although Acss2 is predominantly cytosolic, a subset of the Acss2 cellular pool is enriched in the nucleus following oxygen or glucose deprivation. Prevention of nuclear localization by a directed mutation in a putative nuclear localization signal in Acss2 abrogates HIF-2 acetylation and blunts HIF-2 dependent signaling as well as flank tumor growth for knockdown/rescue cancer cells expressing ectopic Acss2. In this study, we report generation of a novel mouse strain using CRISPR/Cas9 mutagenesis that express this mutant Acss2 allele in the mouse germline. The homozygous mutant mice have impaired induction of the canonical HIF-2 target gene erythropoietin and blunted recovery from acute anemia. Surprisingly, Acss2 protein levels are dramatically reduced in these mutant mice. Functional studies investigating the basis for this phenotype reveal multiple protein instability domains in the Acss2 carboxy terminus. The findings described herein may be of relevance in the regulation of native Acss2 protein as well as for humans carrying missense mutations in these domains.


Subject(s)
Acetate-CoA Ligase/chemistry , Acetate-CoA Ligase/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Conserved Sequence , Mutation , Protein Interaction Domains and Motifs , Signal Transduction , Amino Acid Sequence , Animals , Genes, Reporter , Genotype , Humans , Mice , Protein Stability
7.
PLoS One ; 12(12): e0190241, 2017.
Article in English | MEDLINE | ID: mdl-29281714

ABSTRACT

Survival of cancer cells in the harsh tumor microenvironment, characterized by oxygen and glucose deprivation, requires rapid initiation of cytoprotective measures. Metabolites whose levels change during stress are ideal signaling cues, particularly if used in post-translational modifications of stress-responsive signal transducers. In cancer cells exposed to oxygen or glucose deprivation, there is an increase in cellular levels of acetate, a substrate for acetate-dependent acetyl CoA synthetase 2 (Acss2) that also stimulates translocation of Acss2 from the cytosol to the nucleus. Nuclear, but not cytosolic, Acss2 promotes acetylation of the stress-responsive Hypoxia Inducible Factor 2α (HIF-2α) subunit by the acetyltransferase/coactivator Creb binding protein (Cbp), a process that facilitates stable Cbp/HIF-2α complex formation. In addition to promoting de novo transcription, Cbp and HIF-2α act in concert to regulate local histone 3 epigenetic marks. Exogenous acetate augments Acss2/HIF-2 dependent cancer growth and metastasis in cell culture and mouse models. Thus, an acetate switch in mammals links nutrient intake and stress signaling with tumor growth and metastasis.


Subject(s)
Acetate-CoA Ligase/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Epigenesis, Genetic , Neoplasms/metabolism , Oxidative Stress , Signal Transduction , Acetyl Coenzyme A/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/chemistry , Cell Line, Tumor , HEK293 Cells , Humans , Lysine/metabolism , Mice , Mice, Nude , Neoplasms/genetics , Neoplasms/pathology , Real-Time Polymerase Chain Reaction
9.
Nature ; 523(7559): 226-30, 2015 Jul 09.
Article in English | MEDLINE | ID: mdl-26098368

ABSTRACT

Although the adult mammalian heart is incapable of meaningful functional recovery following substantial cardiomyocyte loss, it is now clear that modest cardiomyocyte turnover occurs in adult mouse and human hearts, mediated primarily by proliferation of pre-existing cardiomyocytes. However, fate mapping of these cycling cardiomyocytes has not been possible thus far owing to the lack of identifiable genetic markers. In several organs, stem or progenitor cells reside in relatively hypoxic microenvironments where the stabilization of the hypoxia-inducible factor 1 alpha (Hif-1α) subunit is critical for their maintenance and function. Here we report fate mapping of hypoxic cells and their progenies by generating a transgenic mouse expressing a chimaeric protein in which the oxygen-dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen-inducible CreERT2 recombinase. In mice bearing the creERT2-ODD transgene driven by either the ubiquitous CAG promoter or the cardiomyocyte-specific α myosin heavy chain promoter, we identify a rare population of hypoxic cardiomyocytes that display characteristics of proliferative neonatal cardiomyocytes, such as smaller size, mononucleation and lower oxidative DNA damage. Notably, these hypoxic cardiomyocytes contributed widely to new cardiomyocyte formation in the adult heart. These results indicate that hypoxia signalling is an important hallmark of cycling cardiomyocytes, and suggest that hypoxia fate mapping can be a powerful tool for identifying cycling cells in adult mammals.


Subject(s)
Myocardium/cytology , Myocytes, Cardiac/cytology , Recombinant Fusion Proteins/metabolism , Animals , Cell Hypoxia , Cell Proliferation/genetics , Female , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Mice, Transgenic , Myocytes, Cardiac/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinases/genetics , Recombinases/metabolism , Signal Transduction , Stem Cells/cytology , Stem Cells/metabolism
10.
PLoS One ; 10(2): e0116515, 2015.
Article in English | MEDLINE | ID: mdl-25689462

ABSTRACT

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.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Signal Transduction , Tumor Microenvironment , Acetate-CoA Ligase/metabolism , Acetates/metabolism , Animals , CREB-Binding Protein/metabolism , Cell Line, Tumor , Disease Models, Animal , Glucose/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Oxygen/metabolism , Sirtuin 1/metabolism
11.
Nat Med ; 20(9): 1018-26, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25108527

ABSTRACT

The hormone erythropoietin (EPO), which is synthesized in the kidney or liver of adult mammals, controls erythrocyte production and is regulated by the stress-responsive transcription factor hypoxia-inducible factor-2 (HIF-2). We previously reported that the lysine acetyltransferase CREB-binding protein (CBP) is required for HIF-2α acetylation and efficient HIF-2-dependent EPO induction during hypoxia. We now show that these processes require acetate-dependent acetyl CoA synthetase 2 (ACSS2). In human Hep3B hepatoma cells and in EPO-generating organs of hypoxic or acutely anemic mice, acetate levels rise and ACSS2 is required for HIF-2α acetylation, CBP-HIF-2α complex formation, CBP-HIF-2α recruitment to the EPO enhancer and efficient induction of EPO gene expression. In acutely anemic mice, acetate supplementation augments stress erythropoiesis in an ACSS2-dependent manner. Moreover, in acquired and inherited chronic anemia mouse models, acetate supplementation increases EPO expression and the resting hematocrit. Thus, a mammalian stress-responsive acetate switch controls HIF-2 signaling and EPO induction during pathophysiological states marked by tissue hypoxia.


Subject(s)
Acetates/metabolism , Erythropoiesis , Stress, Physiological , Acetylation , Animals , Cell Hypoxia , Erythropoietin/genetics , Mice , Signal Transduction , Transcription Factors/metabolism
12.
Cell ; 157(3): 565-79, 2014 Apr 24.
Article in English | MEDLINE | ID: mdl-24766806

ABSTRACT

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.


Subject(s)
Cell Cycle Checkpoints , Myocytes, Cardiac/cytology , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Animals , Cell Proliferation/drug effects , DNA Damage , Free Radical Scavengers/pharmacology , Mice , Mitochondria/metabolism , Myocytes, Cardiac/metabolism , Zebrafish
13.
Proc Natl Acad Sci U S A ; 110(19): E1788-96, 2013 May 07.
Article in English | MEDLINE | ID: mdl-23610397

ABSTRACT

Breathing and blood pressure are under constant homeostatic regulation to maintain optimal oxygen delivery to the tissues. Chemosensory reflexes initiated by the carotid body and catecholamine secretion from the adrenal medulla are the principal mechanisms for maintaining respiratory and cardiovascular homeostasis; however, the underlying molecular mechanisms are not known. Here, we report that balanced activity of hypoxia-inducible factor-1 (HIF-1) and HIF-2 is critical for oxygen sensing by the carotid body and adrenal medulla, and for their control of cardio-respiratory function. In Hif2α(+/-) mice, partial HIF-2α deficiency increased levels of HIF-1α and NADPH oxidase 2, leading to an oxidized intracellular redox state, exaggerated hypoxic sensitivity, and cardio-respiratory abnormalities, which were reversed by treatment with a HIF-1α inhibitor or a superoxide anion scavenger. Conversely, in Hif1α(+/-) mice, partial HIF-1α deficiency increased levels of HIF-2α and superoxide dismutase 2, leading to a reduced intracellular redox state, blunted oxygen sensing, and impaired carotid body and ventilatory responses to chronic hypoxia, which were corrected by treatment with a HIF-2α inhibitor. None of the abnormalities observed in Hif1α(+/-) mice or Hif2α(+/-) mice were observed in Hif1α(+/-);Hif2α(+/-) mice. These observations demonstrate that redox balance, which is determined by mutual antagonism between HIF-α isoforms, establishes the set point for hypoxic sensing by the carotid body and adrenal medulla, and is required for maintenance of cardio-respiratory homeostasis.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Carotid Body/physiology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Oxygen/metabolism , Adrenal Medulla/physiology , Animals , Blood Pressure , Cardiovascular System , Carotid Body/metabolism , Catecholamines/metabolism , Heterozygote , Homeostasis , Hypoxia , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Transgenic , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Oxidation-Reduction , PC12 Cells , Rats , Superoxide Dismutase/metabolism
14.
Nat Chem Biol ; 9(4): 271-6, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23434853

ABSTRACT

Hypoxia inducible factors (HIFs) are heterodimeric transcription factors induced in many cancers where they frequently promote the expression of protumorigenic pathways. Though transcription factors are typically considered 'undruggable', the PAS-B domain of the HIF-2α subunit contains a large cavity within its hydrophobic core that offers a unique foothold for small-molecule regulation. Here we identify artificial ligands that bind within this pocket and characterize the resulting structural and functional changes caused by binding. Notably, these ligands antagonize HIF-2 heterodimerization and DNA-binding activity in vitro and in cultured cells, reducing HIF-2 target gene expression. Despite the high sequence identity between HIF-2α and HIF-1α, these ligands are highly selective and do not affect HIF-1 function. These chemical tools establish the molecular basis for selective regulation of HIF-2, providing potential therapeutic opportunities to intervene in HIF-2-driven tumors, such as renal cell carcinomas.


Subject(s)
Antineoplastic Agents/pharmacology , Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Allosteric Regulation , Antineoplastic Agents/chemistry , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , High-Throughput Screening Assays , Humans , Kinetics , Ligands , Molecular Docking Simulation , Neoplasm Proteins/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Tertiary , Small Molecule Libraries/chemistry
15.
Atherosclerosis ; 225(1): 91-8, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22959701

ABSTRACT

BACKGROUND: Vascular stent coverage by endothelial cells, derived from endothelial progenitor cells (EPC) is considered a surrogate for healing. However, the effects of antiproliferative drugs used in current drug-eluting stents (DES) on EPC proliferative and antithrombotic function remains poorly defined. METHOD AND RESULTS: Herein, we studied and compared the in vitro and in vivo effects of four antiproliferative drugs - paclitaxel, sirolimus, everolimus, and zotarolimus on several EPC properties including colony forming units (CFU), cell proliferation, apoptosis, antithrombotic and prothrombotic gene expression and nitric oxide (NO) as well as prostacyclin (PGI(2)) release. We also examined EPC migration and adhesion under flow conditions. We find that whereas all antiproliferative agents inhibited EPC proliferation and caused cell apoptosis, only paclitaxel and sirolimus reduced CFU formation. Paclitaxel treatment also resulted in the greatest down-regulation of antithrombotic gene expression and up-regulation of prothrombotic gene expression. NO release, migration, and adhesion of EPC under shear stress were inhibited by all antiproliferative drugs, most notably by paclitaxel and sirolimus. CONCLUSIONS: These results indicate that antiproliferative drugs on DES, particularly paclitaxel, impair the proliferative and antithrombotic functions of EPC, and thereby could contribute to incomplete vascular healing and increase the risk of stent thrombosis.


Subject(s)
Endothelium, Vascular/cytology , Paclitaxel/pharmacology , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Stem Cells/drug effects , Animals , Apoptosis/drug effects , Cell Adhesion/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Coronary Restenosis/etiology , Drug-Eluting Stents , Endothelial Cells/drug effects , Epoprostenol/metabolism , Everolimus , Humans , Male , Nitric Oxide/metabolism , Rats , Stents/adverse effects
16.
J Biol Chem ; 287(36): 30800-11, 2012 Aug 31.
Article in English | MEDLINE | ID: mdl-22807441

ABSTRACT

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.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , CREB-Binding Protein/metabolism , Peptide Fragments/metabolism , Sialoglycoproteins/metabolism , Signal Transduction/physiology , Sirtuin 1/metabolism , p300-CBP Transcription Factors/metabolism , Acetylation , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , CREB-Binding Protein/genetics , Cell Line , Erythropoietin/biosynthesis , Erythropoietin/genetics , Humans , Mice , Peptide Fragments/genetics , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Protein Structure, Tertiary , Sialoglycoproteins/genetics , Sirtuin 1/genetics , p300-CBP Transcription Factors/genetics
17.
J Cardiovasc Transl Res ; 5(5): 654-65, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22566269

ABSTRACT

Recent reports indicate that the adult mammalian heart is capable of limited, but measurable, cardiomyocyte turnover. While the lineage origin of the newly formed cardiomyocytes is not entirely understood, mounting evidence suggest that the epicardium and subepicardium may represent an important source of cardiac stem or progenitor cells. Stem cell niches are characterized by low oxygen tension, where stem cells preferentially utilize cytoplasmic glycolysis to meet their energy demands. However, it is unclear if the heart harbors similar hypoxic regions, or whether these regions house metabolically distinct cardiac progenitor populations. Here we identify the epicardium and subepicardium as the cardiac hypoxic niche based on [corrected] capillary density quantification, and localization of Hif-1α in the uninjured heart. We further demonstrate that this hypoxic microenvironment houses a metabolically distinct population of glycolytic progenitor cells. Finally, we show that Hif-1α regulates the glycolytic phenotype and progenitor properties of these cells. These findings highlight important anatomical and functional properties of the epicardial and subepicardial microenvironment, and the potential role of hypoxia signaling in regulation of cardiac progenitors.


Subject(s)
Cellular Microenvironment , Energy Metabolism , Myocytes, Cardiac/metabolism , Oxygen/metabolism , Pericardium/metabolism , Stem Cells/metabolism , Animals , Cell Differentiation , Cell Hypoxia , Cell Lineage , Cell Proliferation , Cells, Cultured , Coculture Techniques , Gene Expression Regulation , Glycolysis , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Oxidative Stress , Perfusion , Phenotype , RNA Interference , Signal Transduction , Transfection
18.
Proc Natl Acad Sci U S A ; 108(7): 3065-70, 2011 Feb 15.
Article in English | MEDLINE | ID: mdl-21288809

ABSTRACT

Cardiorespiratory functions in mammals are exquisitely sensitive to changes in arterial O(2) levels. Hypoxia-inducible factors (e.g., HIF-1 and HIF-2) mediate transcriptional responses to reduced oxygen availability. We demonstrate that haploinsufficiency for the O(2)-regulated HIF-2α subunit results in augmented carotid body sensitivity to hypoxia, irregular breathing, apneas, hypertension, and elevated plasma norepinephrine levels in adult Hif-2α(+/-) mice. These dysregulated autonomic responses were associated with increased oxidative stress and decreased mitochondrial electron transport chain complex I activity in adrenal medullae as a result of decreased expression of major cytosolic and mitochondrial antioxidant enzymes. Systemic administration of a membrane-permeable antioxidant prevented oxidative stress, normalized hypoxic sensitivity of the carotid body, and restored autonomic functions in Hif-2α(+/-) mice. Thus, HIF-2α-dependent redox regulation is required for maintenance of carotid body function and cardiorespiratory homeostasis.


Subject(s)
Adrenal Medulla/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Carotid Body/physiology , Hypertension/physiopathology , Hypoxia/physiopathology , Oxidative Stress/physiology , Respiratory Mechanics/physiology , Analysis of Variance , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Blood Pressure , Blotting, Western , Electron Transport Complex I/metabolism , Gene Expression Profiling , Immunohistochemistry , Mice , Mice, Knockout , Norepinephrine/metabolism , Oxidation-Reduction , Oxygen Consumption , Plethysmography, Whole Body , Reverse Transcriptase Polymerase Chain Reaction
19.
J Biol Chem ; 286(16): 13869-78, 2011 Apr 22.
Article in English | MEDLINE | ID: mdl-21345792

ABSTRACT

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.


Subject(s)
Gene Expression Regulation , Hypoxia-Inducible Factor 1/metabolism , Hypoxia , Sirtuin 1/biosynthesis , Acetylation , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line, Tumor , Chromatin Immunoprecipitation , Humans , Mice , Mice, Inbred C57BL , Protein Processing, Post-Translational , Signal Transduction , Transcriptional Activation
20.
Biol Reprod ; 82(6): 1227-36, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20181618

ABSTRACT

Spermatogenesis, a process involving the differentiation of spermatogonial stem cells into mature spermatozoa, takes place throughout masculine life. A complex system in the testis, including endocrine signaling, physical interactions between germ and somatic cells, spermatocyte meiosis, and timely release of spermatozoa, controls this cycle. We demonstrate herein that decreased O(2) levels and Epas1 activation are critical components of spermatogenesis. Postnatal Epas1 ablation leads to male infertility, with reduced testis size and weight. While immature spermatogonia and spermatocytes are present in Epas1(Delta/Delta) testes, spermatid and spermatozoan numbers are dramatically reduced. This is not due to germ cell-intrinsic defects. Rather, Epas(Delta/Delta) Sertoli cells exhibit decreased ability to form tight junctions, thereby disrupting the blood-testis barrier necessary for proper spermatogenesis. Reduced numbers of tight junction complexes are due to decreased expression of multiple genes encoding tight junction proteins, including TJP1 (ZO1), TJP2 (ZO2), and occludin. Furthermore, Epas1(Delta/Delta) testes exhibit disrupted basement membranes surrounding the seminiferous tubules, causing the premature release of incompletely differentiated germ cells. We conclude that low O(2) levels in the male gonad regulate germ cell homeostasis in this organ via EPAS1.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Blood-Testis Barrier/metabolism , Spermatogenesis , Spermatogonia/metabolism , Testis/metabolism , Animals , Basement Membrane/chemistry , Basement Membrane/pathology , Basic Helix-Loop-Helix Transcription Factors/deficiency , Blood-Testis Barrier/pathology , Male , Membrane Proteins/analysis , Mice , Occludin , Organ Size , Phosphoproteins/analysis , Seminiferous Tubules/chemistry , Seminiferous Tubules/pathology , Sertoli Cells/chemistry , Sertoli Cells/pathology , Spermatids/metabolism , Spermatids/pathology , Spermatogonia/pathology , Testis/pathology , Tight Junctions/chemistry , Tight Junctions/pathology , Zonula Occludens-1 Protein , Zonula Occludens-2 Protein
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