Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cellsReport as inadecuate




Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells - Download this document for free, or read online. Document in PDF available to download.

BMC Cardiovascular Disorders

, 8:9

First Online: 30 April 2008Received: 06 August 2007Accepted: 30 April 2008DOI: 10.1186-1471-2261-8-9

Cite this article as: Malhotra, R., Tyson, D.W., Rosevear, H.M. et al. BMC Cardiovasc Disord 2008 8: 9. doi:10.1186-1471-2261-8-9

Abstract

BackgroundHypoxia 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.

MethodsCardiac derived H9c2 and renal tubular epithelial HK-2 cells expressing either the wild type oxygen regulated subunit of HIF-1 pcDNA3-Hif-1α or a dominant negative version that lacked both DNA binding and transactivation domains pcDNA3-DN-Hif-1α, were maintained in culture and exposed to hypoxia. An RNA interference approach was also employed to selectively knockdown expression of Hif-1α. 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.

ResultsOverexpression of pcDNA3-DN-Hif-1α 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-1α transfected cells. Moreover, selective ablation of HIF-1α 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-1α exposed to hypoxia. In HK-2 cells overexpression of wild-type Hif-1α led to a two-fold increase in Hif-1α 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-1α also induced upregulation of Bax in HK-2 cells. In addition, introduction of dominant negative Hif-1α constructs in both H9c2 and HK-2 -cells led to decreased active Bax expression.

ConclusionThese data demonstrate that HIF-1α is an important component of the apoptotic signaling machinery in the two cell types.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2261-8-9 contains supplementary material, which is available to authorized users.

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Author: Ricky Malhotra - David W Tyson - Henry M Rosevear - Frank C BrosiusIII

Source: https://link.springer.com/



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