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.
Wolf
Gunter
G
Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, DE-20246 Hamburg, Germany. wolf@uke.uni-hamburg.de
Schroeder
Regine
R
Stahl
Rolf A K
RA
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