Differentiating the functional role of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha (EPAS-1) by the use of RNA interference: erythropoietin is a HIF-2alpha target gene in Hep3B and Kelly cells.
Activation of the hypoxia-inducible factor alpha-subunits, HIF-1alpha and HIF-2alpha, seems to be subject to similar regulatory mechanisms, and transgene approaches suggested partial functional redundancy. Here, we used RNA interference to determine the contribution of HIF-1alpha vs. HIF-2alpha to the hypoxic gene induction. Surprisingly, most genes tested were responsive only to the HIF-1alpha siRNA, showing no effect by HIF-2alpha knock-down. The same was found for the activation of reporter genes driven by hypoxia-responsive elements (HREs) from the erythropoietin (EPO), vascular endothelial growth factor, or phosphoglycerate kinase gene. Interestingly, EPO was the only gene investigated that showed responsiveness only to HIF-2alpha knock-down, as observed in Hep3B and Kelly cells. In contrast to the EPO-HRE reporter, the complete EPO enhancer displayed dependency on HIF-2alpha regulation, indicating that additional cis-acting elements confer HIF-2alpha specificity within this region. In 786-0 cells lacking HIF-1alpha protein, the identified HIF-1alpha target genes were regulated by HIF-2alpha. Overexpression of the HIFalpha subunits in different cell lines also led to a loss of target gene specificity. In conclusion, we found a remarkably restricted target gene specificity of the HIFalpha subunits, which can be overcome in cells with perturbations in the pVHL/HIF system and under forced expression.
Warnecke
Christina
C
Department of Nephrology and Medical Intensive Care, Virchow Clinic, Charité University Medicine, Berlin, Germany. christina.warnecke@med4.imed.uni-erlangen.de
Zaborowska
Zaneta
Z
Kurreck
Jens
J
Erdmann
Volker A
VA
Frei
Ulrich
U
Wiesener
Michael
M
Eckardt
Kai-Uwe
KU
D051792:Basic Helix-Loop-Helix Proteins; D002460:Cell Line; D045744:Cell Line, Tumor; D004742:Enhancer Elements, Genetic / Q000235:genetics; D004921:Erythropoietin / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D000099162:Endothelial PAS Domain-Containing Protein 1
D051792:Basic Helix-Loop-Helix Proteins; D004921:Erythropoietin; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D021122:Protein Subunits; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human