Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells.
BACKGROUND
Hypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia. There is evidence that HIF-1 can also trigger apoptosis, possibly when cellular responses are inadequate to meet energy demands under hypoxic conditions.


METHODS
Cardiac derived H9c2 and renal tubular epithelial HK-2 cells expressing either the wild type oxygen regulated subunit of HIF-1 (pcDNA3-Hif-1alpha) or a dominant negative version that lacked both DNA binding and transactivation domains (pcDNA3-DN-Hif-1alpha), were maintained in culture and exposed to hypoxia. An RNA interference approach was also employed to selectively knockdown expression of Hif-1alpha. Apoptosis was analyzed in both H9c2 and HK-2 cells by Hoechst and TUNEL staining, caspase 3 activity assays and activation of pro-apoptotic Bcl2 family member Bax.


RESULTS
Overexpression of pcDNA3-DN-Hif-1alpha led to a significant reduction in hypoxia -induced apoptosis (17 +/- 2%, P < 0.01) in H9c2 cells compared to both control-transfected and wild type Hif-1alpha transfected cells. Moreover, selective ablation of HIF-1alpha protein expression by RNA interference in H9c2 cells led to 55% reduction of caspase 3 activity and 46% reduction in the number of apoptotic cells as determined by Hoechst 33258 staining, after hypoxia. Finally, upregulation of the pro-apoptotic protein, Bax, was found in H9c2 cells overexpressing full-length pcDNA3-HA-HIF-1alpha exposed to hypoxia. In HK-2 cells overexpression of wild-type Hif-1alpha led to a two-fold increase in Hif-1alpha levels during hypoxia. This resulted in a 3.4-fold increase in apoptotic cells and a concomitant increase in caspase 3 activity during hypoxia when compared to vector transfected control cells. HIF-1alpha also induced upregulation of Bax in HK-2 cells. In addition, introduction of dominant negative Hif-1alpha constructs in both H9c2 and HK-2 -cells led to decreased active Bax expression.


CONCLUSIONS
These data demonstrate that HIF-1alpha is an important component of the apoptotic signaling machinery in the two cell types.
Malhotra
Ricky
R
Department of Internal Medicine/Nephrology, University of Michigan Medical Center, Ann Arbor, MI-48109-0676, USA. m.r3078@gmail.com
Tyson
David W
DW
Rosevear
Henry M
HM
Brosius
Frank C
FC
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D020287:In Situ Nick-End Labeling; D007668:Kidney / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D014162:Transfection; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494826:BAX protein, human; C494828:Bax protein, rat; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3