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

Ricky Malhotra,Frank C Brosius,David W Tyson,Henry M Rosevear

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.

Highlights

Hypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia
Whenever Hif-1α is induced in a cell in response to low oxygen conditions it primarily triggers a number of pro-survival events such as increased expression of vascular endothelial growth factor (VEGF) and angiogenesis, increased expression of genes for various glycolytic enzymes as well as for the glucose transporter 1 (GLUT1) thereby enhancing glycolysis, and activation of the erythropoietin gene and erythropoiesis [4]
These findings were consistent with proteasomal degradation of Hif-1α in the presence of O2, as the oxygen degradation domain is intact in both the wild type and dominant negative constructs. (Fig. 1A)

Summary

Introduction

Hypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia. Over the past decade it has become clear that hypoxia induces programmed cell death, or apoptosis, through a mitochondrial dependent process [5,6] involving release of cytochrome c, activation of caspase 9 and subsequent cleavage and activation of downstream, effector caspases [7,8]. Multiple factors are involved in this programmed cell death pathway but because it largely determines the cellular response to hypoxia, Hif-1 has been a candidate to regulate hypoxia-induced apoptosis. Despite its clear role in cellular protection, there have been a number of reports demonstrating that Hif-1 contributes to programmed cell death during hypoxia [e.g., [9,10,11,12,13]]. The role of Hif-1 in apoptosis remains somewhat controversial and appears to be cell type and context specific

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Conclusion