Hypoxia-inducible factor-1alpha Is a Critical Mediator of Hypoxia Induced Apoptosis in Cardiac H9c2 and Kidney Epithelial HK-2 Cells

BMC Cardiovasc Disord. 2008 Apr 30;8:9. doi: 10.1186/1471-2261-8-9.

Abstract

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.

Conclusion: These data demonstrate that HIF-1alpha is an important component of the apoptotic signaling machinery in the two cell types.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis*
  • Caspase 3 / metabolism
  • Cell Hypoxia
  • Cell Line
  • Epithelial Cells / enzymology
  • Epithelial Cells / metabolism*
  • Epithelial Cells / pathology
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism*
  • In Situ Nick-End Labeling
  • Kidney / enzymology
  • Kidney / metabolism*
  • Kidney / pathology
  • Myocytes, Cardiac / enzymology
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / pathology
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Rats
  • Signal Transduction*
  • Transfection
  • bcl-2-Associated X Protein / metabolism

Substances

  • BAX protein, human
  • Bax protein, rat
  • HIF1A protein, human
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • RNA, Small Interfering
  • bcl-2-Associated X Protein
  • CASP3 protein, human
  • Casp3 protein, rat
  • Caspase 3