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

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.

Figure 1

A) Hif-1 α protein expression in whole cell lysates of H9c2 cells transfected with either pEGFP or pcDNA3-HA-HIF-1α and subjected to normoxia or hypoxia for 12 h. Immunoblotting with antibodies against HIF-1β, the nuclear dimerization partner of HIF-1α was used to monitor equal protein loading. B) HIF-1α mediates hypoxia-induced apoptosis in H9c2 cells transfected with pEGFP or pcDNA3-HA-HIF-1α expressing the wild type HIF-1α or pcDNA3-Flag-DN-HIF-1α expressing the dominant negative version as described in methods. Cells were exposed to normoxia or hypoxia for 12 h in glucose-deficient medium. The number of cells displaying condensed and fragmented nuclei was counted only in cells positive for each respective epitope tag or EGFP. The results are representative of five independent experiments. *P < 0.01 vs. hypoxia in pEGFP and pcDNA3-HA-Hif-1α.

Figure 2

Effect of shRNA treatment on HIF-1α protein expression in H9c2 cells. H9c2 cells were transfected with shRNAi_HIF-1α (200 nM) or an shRNAi targeted to a scrambled mRNA sequence (Scr siRNA, nonspecific control), and 24 h later were subjected to hypoxia for an additional 24 h and subsequently whole cell lysates were isolated for Western blot analysis for HIF-1α protein expression. The data shown is from two independent experiments. As a loading control the lysates were also probed with an antibody against Hif-1β, the dimerization partner of Hif-1α.

Figure 3

Downregulation of HIF-1α by shRNAi treatment reduces hypoxia-induced apoptosis in H9c2 cells. After co-transfection with shRNA_HIF-1α or an shRNAi targeted to an unrelated mRNA sequence (Scr shRNA, nonspecific control) or with the U6RNAi empty vector and pEGFP the H9c2 cells were cultured under normoxic or hypoxic conditions for an additional 24 h. (A) After transfection with respective shRNAi constructs and exposure to normoxic or hypoxic conditions the cells were analysed for caspase 3 activity. Results are mean ± SEM of 4 separate experiments. (B) Cells were stained with Hoechst 33258 and visualized under ultraviolet light. The data represent the mean ± SEM of triplicate wells of three independent experiments. *P < 0.05 vs. hypoxia in U6 RNAi vector and Scr shRNAi.

Figure 4

Flow cytometry analysis followed by Bax protein expression. H9c2 cells were transfected with pEGFP, pEGFP and pcDNA3-HA- HIF-1α or pcDNA3-Flag-DN-HIF-1α and were sorted in a flow cytometer. The EGFP positive sorted cells were subjected to hypoxia for 6 h. Total Bax expression in the same lysates was used to monitor protein loading. N = Normoxia; H = Hypoxia. The data are representative of 3 separate experiments and only 2 experiments using dominant negative Hif-1α (right panel).

Figure 5

Effect of HIF-1α overexpression on apoptosis in HK-2 subjected to 16 h of hypoxia. (A) HIF-1α protein expression in HK-2 cells transfected with either the empty vector or pcDNA3-HA-Hif1α and subjected to hypoxia or normoxia. Immunoblotting of the same lysates with Hif-1β to monitor protein loading is also shown. (B) Morphological analysis of apoptosis with Hoechst 33258. *P < 0.05 vs hypoxia in pcDNA3. pEGFP was co-transfected alongwith the vector and HIF-1 alpha construct to aid in the identification of apoptotic nuclei in EGFP transfected cells. (C) Apoptotic cells as determined by TUNEL staining. *P < 0.001 vs hypoxia after transfection with pcDNA3. Data shown is mean ± SEM of six separate experiments and (D) Caspase 3 activity (O.D. units 405 nm).*P < 0.001 vs. hypoxia with the empty vector pcDNA3. Data in panels B, C and D are means ± SEM of triplicate wells of six separate experiments.

Figure 6

HIF-1α promotes Bax activation in HK-2 cells. Cells were subjected to normoxia or hypoxia of 16 h after sorting in a flow cytometer. Active and Total Bax protein expression of the whole cell lysates of EGFP positive cells is shown of cells transfected with either pEGFP or pEGFP and pcDNA3-HA-Hif-1α or pcDNA3-DN-Hif-1α. N = Normoxia; H = Hypoxia. Data shown is representative of two separate experiments.