NF-kappaB plays a key role in hypoxia-inducible factor-1-regulated erythropoietin gene expression.
OBJECTIVE
The aim of this study was to further define the signal transduction pathways leading to hypoxia-inducible factor-1 (HIF-1) erythropoietin (EPO) gene expression.


METHODS
Human hepatocellular carcinoma cells (Hep3B) were exposed to hypoxia (1% oxygen) and examined for mRNA expression, as well as gene transactivation with RT-PCR and luciferase reporter gene assays, respectively.


RESULTS
Treatment with LY294002 (a selective pharmacological inhibitor of phosphatidylinositol 3-kinase) significantly inhibited EPO protein and mRNA expression in Hep3B cells exposed to hypoxia for 24 hours, while treatment with PD098059 or SB203580 (selective pharmacological inhibitors of the MEK and p38 mitogen-activated protein kinase pathways, respectively) had no significant effects. The activity of AKT, a downstream target of PI3K, was increased by hypoxia and was also inhibited by LY294002. Genetic inhibition of AKT resulted in significant inhibition of NF-kappaB and HIF-1-mediated transactivation, as well as EPO gene expression, in response to hypoxia. Overexpression of constitutively active AKT resulted in increased NF-kappaB and HIF-1 transactivation. The selective inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), significantly blocked HIF-1 protein expression. Inhibition of NF-kappaB with a superrepressor dominant negative IkappaBalpha genetic construct also significantly blocked NF-kappaB and HIF-1 transactivation, as well as EPO gene expression.


CONCLUSIONS
We propose a key role for NF-kappaB in EPO gene regulation in response to hypoxia.
Figueroa
Yanira G
YG
Department of Pharmacology, Cancer Center, Tulane Health Sciences Center, 1430 Tulane Avenue, New Orleans, LA 70112, USA.
Chan
Anna K
AK
Ibrahim
Rania
R
Tang
Yan
Y
Burow
Matthew E
ME
Alam
Jawed
J
Scandurro
Aline B
AB
Beckman
Barbara S
BS
D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D004921:Erythropoietin / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology*; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014157:Transcription Factors; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D004921:Erythropoietin; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger; D014157:Transcription Factors; C497442:HIF1A protein, human; C494918:AKT1 protein, human