Analysis of ARD1 function in hypoxia response using retroviral RNA interference.
Cellular hypoxia response is regulated at the level of hypoxia-inducible factor (HIF) activity. A number of recently identified oxygen sensors are HIF-modifying enzymes that respond to low oxygen by altering HIF modification and thus lead to its activation. In addition to the HIF proline hydroxylases and asparagine hydroxylases, ARD1 is recently described as a HIF-1alpha acetylase that regulates its stability. We found that ARD1 is down-regulated in a number of cell lines in response to hypoxia and hypoxia mimic compounds. After surveying these lines for erythropoietin production and retroviral transfection efficiency, we chose to use HepG2 cells to study the function of ARD1. ARD1 short hairpin RNA delivered by a retroviral vector caused >80% reduction in ARD1 message. We observed decreases in erythropoietin and vascular endothelial growth factor protein production, whereas there was no change in the HIF-1alpha protein level. A gene chip analysis of HepG2 cells transduced with virus expressing ARD1 short hairpin RNA under normoxia and hypoxia conditions or with virus overexpressing recombinant ARD1 confirmed that inhibition of ARD1 does not cause activation of HIF and downstream target genes. However, this analysis revealed that ARD1 is involved in cell proliferation and in regulating a series of cellular metabolic pathways that are regulated during hypoxia response. The role of ARD1 in cell proliferation is confirmed using fluorescence labeling analysis of cell division. From these studies we conclude that ARD1 is not required to suppress HIF but is required to maintain cell proliferation in mammalian cells.
Fisher
Tim S
TS
Department of Antibacterials, Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340, USA.
Etages
Shelley Des
SD
Hayes
Lisa
L
Crimin
Kim
K
Li
Baiyong
B
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases; D034622:RNA Interference; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D012190:Retroviridae / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D000123:Acetyltransferases; D004722:Endoribonucleases; D010749:Phosphoprotein Phosphatases; C097081:PPP1R8 protein, human