Angiotensin II induces hypoxia-inducible factor-1 alpha in PC 12 cells through a posttranscriptional mechanism: role of AT2 receptors.
BACKGROUND
Angiotensin II (ANG II) inhibits proliferation and induces differentiation in PC 12 cells via AT(2) receptor activation. Using differential display analysis, we previously isolated SM-20/PHD3 as a key factor, which is downregulated by ANG II treatment. Subsequently, it turned out that SM-20/PHD3 is a rat homologue of PHD3, a key prolyl hydroxylase involved in the initial steps fostering the degradation of hypoxia-inducible factor (HIF). The present study was undertaken to investigate whether the ANG-II-mediated suppression of SM-20/PHD3/PHD3 may be associated with an increase in HIF-1 alpha.


METHODS
HIF-1 alpha protein expression was assessed by Western blots. mRNA levels for HIF-1 alpha were measured by real-time PCR and for SM-20/PHD3 by Northern blots. Binding of HIF-1 alpha to consensus oligonucleotides in vitro was determined with gel shift analysis. SM-20/PHD3 was transiently overexpressed in PC 12 cells using an inducible expression system.


RESULTS
ANG II stimulated HIF-1alpha protein expression. This effect was already detected after 30 min and peaked at 6 h, but was not detectable anymore after 24- 48 h of stimulation. PD 123177, but not losartan, antagonized this effect, indicating transduction through AT(2) receptors. Real-time PCR failed to show a significant increase in HIF-1 alpha transcripts after ANG II challenge at any time point. Gel shift analysis revealed that ANG-II-induced nuclear HIF-1 alpha protein binds to consensus sites. A reduction in SM-20/PHD3 mRNA expression paralleled the increase in HIF-1 alpha. Overexpression of SM-20/PHD3 transiently resulted in a decrease in HIF-1 alpha protein concentrations under basal conditions as well as after stimulation with ANG II.


CONCLUSIONS
ANG II stimulates HIF-1 alpha expression by a posttranscriptional mechanism via AT(2) receptors. This increase is likely caused by a downregulation of SM-20/PHD3. The ANG-II-mediated increase in HIF-1 alpha expression could be potentially involved in physiological as well as pathophysiological processes such as differentiation, growth inhibition, and remodeling.
Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, DE-20246 Hamburg, Germany. wolf@uke.uni-hamburg.de
Gunter
G
Wolf
Regine
R
Schroeder
Rolf A K
RA
Stahl
D000804:Angiotensin II / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology; D057912:Angiotensin II Type 2 Receptor Blockers; D000818:Animals; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007093:Imidazoles / Q000494:pharmacology; D019808:Losartan / Q000494:pharmacology; D016716:PC12 Cells; D011725:Pyridines / Q000494:pharmacology; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D044139:Receptor, Angiotensin, Type 2 / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics*; D015854:Up-Regulation / Q000187:drug effects; D014662:Vasoconstrictor Agents / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D047228:Angiotensin II Type 1 Receptor Blockers; D057912:Angiotensin II Type 2 Receptor Blockers; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007093:Imidazoles; D011725:Pyridines; D012333:RNA, Messenger; D044139:Receptor, Angiotensin, Type 2; D014157:Transcription Factors; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; C066253:PD 123177; D019808:Losartan