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BH3-only protein Noxa is a mediator of hypoxic cell death induced by hypoxia-inducible factor 1alpha.

Kim JY, Ahn HJ, Ryu JH, Suk K, Park JH - J. Exp. Med. (2003)

Bottom Line: Noxa is a candidate molecule mediating p53-induced apoptosis.We show that Noxa promoter responds directly to hypoxia via hypoxia-inducible factor (HIF)-1alpha.Suppression of Noxa expression by antisense oligonucleotides rescued cells from hypoxia-induced cell death and decreased infarction volumes in an animal model of ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, College of Medicine, Kyung Hee University, #1 Hoegi-dong, Dongdaemoon-Koo, Seoul 130-701, Korea.

ABSTRACT
Hypoxia is a common cause of cell death and is implicated in many disease processes including stroke and chronic degenerative disorders. In response to hypoxia, cells express a variety of genes, which allow adaptation to altered metabolic demands, decreased oxygen demands, and the removal of irreversibly damaged cells. Using polymerase chain reaction-based suppression subtractive hybridization to find genes that are differentially expressed in hypoxia, we identified the BH3-only Bcl-2 family protein Noxa. Noxa is a candidate molecule mediating p53-induced apoptosis. We show that Noxa promoter responds directly to hypoxia via hypoxia-inducible factor (HIF)-1alpha. Suppression of Noxa expression by antisense oligonucleotides rescued cells from hypoxia-induced cell death and decreased infarction volumes in an animal model of ischemia. Further, we show that reactive oxygen species and resultant cytochrome c release participate in Noxa-mediated hypoxic cell death. Altogether, our results show that Noxa is induced by HIF-1alpha and mediates hypoxic cell death.

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ROS mediates Noxa-induced hypoxic cell death. (A) Saos-2 cells were transiently transfected with 1 μg pcDNA-Noxa or mock vector. After 16 h of transfection, cells were stained with DCF-DA and subjected to flow cytometric analysis. (B) The cells were untreated or treated with 20 μmol/liter SE or AS oligonucleotides before 4 h of hypoxic exposure, subjected to hypoxia for 3 h, and followed by reoxygenation for 1 h. The cells were double stained with PI and DCF-DA and analyzed by flow cytometry using CELLQuest™ software. (C) Saos-2 cells were transiently transfected with 1 μg pcDNA-Noxa or mock vector. After 16 h of transfection, cells were treated with or without NAC for an additional 12 h and cell death assay was performed using the trypan blue exclusion test.
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fig6: ROS mediates Noxa-induced hypoxic cell death. (A) Saos-2 cells were transiently transfected with 1 μg pcDNA-Noxa or mock vector. After 16 h of transfection, cells were stained with DCF-DA and subjected to flow cytometric analysis. (B) The cells were untreated or treated with 20 μmol/liter SE or AS oligonucleotides before 4 h of hypoxic exposure, subjected to hypoxia for 3 h, and followed by reoxygenation for 1 h. The cells were double stained with PI and DCF-DA and analyzed by flow cytometry using CELLQuest™ software. (C) Saos-2 cells were transiently transfected with 1 μg pcDNA-Noxa or mock vector. After 16 h of transfection, cells were treated with or without NAC for an additional 12 h and cell death assay was performed using the trypan blue exclusion test.

Mentions: ROS plays a central role in the arena of cell injuries in response to hypoxia by perturbation of mitochondria, e.g., permeability transition or membrane peroxidation. It has been known that Noxa, targeting to mitochondria, induces loss of mitochondrial membrane potential (Δψm; reference 17). In addition, mitochondria are the major organelle responsible for intracellular ROS generation. Therefore, we hypothesized that Noxa might mediate hypoxic cell death by production of ROS. To examine the possibility that Noxa may perturb mitochondria by ROS generation, we transiently transfected Saos-2 cells with pcDNA-Noxa and determined the changes of ROS level using ROS-sensitive fluorescent dye, 2′, 7′-dichlorofluorescein diacetate (DCF-DA). As presented in Fig. 6 A, an increase in the ROS level was observed in Noxa-transfected cells in comparison with mock-transfected cells. Next, we analyzed the suppression effects of endogenous Noxa on ROS production in hypoxia. Cells whose endogenous Noxa expression was blocked with AS were cultured in hypoxic condition for 3 h followed by 1 h of reoxygenation. Cells were then sequentially stained with DCF-DA and propidium iodide (PI) followed by FACS® analysis. The populations of living cells, as indicated by their ability to exclude PI, were analyzed to discard the possibility that ROS were byproducts of cell death. As shown in Fig. 6 B, AS suppressed ROS generation induced by hypoxia, whereas SE failed to reduce ROS levels. Further, to establish that ROS contributed directly to Noxa-mediated hypoxic cell death, cells were transiently transfected with 1 μg pcDNA-Noxa in the presence or absence of antioxidant N-acetylcysteine (NAC). NAC reduced the ROS level and protected cell death in pcDNA-Noxa–transfected cells in a concentration-dependent manner (Fig. 6 C). Our results showed that ROS generation was crucial in Noxa-triggered cell death.


BH3-only protein Noxa is a mediator of hypoxic cell death induced by hypoxia-inducible factor 1alpha.

Kim JY, Ahn HJ, Ryu JH, Suk K, Park JH - J. Exp. Med. (2003)

ROS mediates Noxa-induced hypoxic cell death. (A) Saos-2 cells were transiently transfected with 1 μg pcDNA-Noxa or mock vector. After 16 h of transfection, cells were stained with DCF-DA and subjected to flow cytometric analysis. (B) The cells were untreated or treated with 20 μmol/liter SE or AS oligonucleotides before 4 h of hypoxic exposure, subjected to hypoxia for 3 h, and followed by reoxygenation for 1 h. The cells were double stained with PI and DCF-DA and analyzed by flow cytometry using CELLQuest™ software. (C) Saos-2 cells were transiently transfected with 1 μg pcDNA-Noxa or mock vector. After 16 h of transfection, cells were treated with or without NAC for an additional 12 h and cell death assay was performed using the trypan blue exclusion test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1887730&req=5

fig6: ROS mediates Noxa-induced hypoxic cell death. (A) Saos-2 cells were transiently transfected with 1 μg pcDNA-Noxa or mock vector. After 16 h of transfection, cells were stained with DCF-DA and subjected to flow cytometric analysis. (B) The cells were untreated or treated with 20 μmol/liter SE or AS oligonucleotides before 4 h of hypoxic exposure, subjected to hypoxia for 3 h, and followed by reoxygenation for 1 h. The cells were double stained with PI and DCF-DA and analyzed by flow cytometry using CELLQuest™ software. (C) Saos-2 cells were transiently transfected with 1 μg pcDNA-Noxa or mock vector. After 16 h of transfection, cells were treated with or without NAC for an additional 12 h and cell death assay was performed using the trypan blue exclusion test.
Mentions: ROS plays a central role in the arena of cell injuries in response to hypoxia by perturbation of mitochondria, e.g., permeability transition or membrane peroxidation. It has been known that Noxa, targeting to mitochondria, induces loss of mitochondrial membrane potential (Δψm; reference 17). In addition, mitochondria are the major organelle responsible for intracellular ROS generation. Therefore, we hypothesized that Noxa might mediate hypoxic cell death by production of ROS. To examine the possibility that Noxa may perturb mitochondria by ROS generation, we transiently transfected Saos-2 cells with pcDNA-Noxa and determined the changes of ROS level using ROS-sensitive fluorescent dye, 2′, 7′-dichlorofluorescein diacetate (DCF-DA). As presented in Fig. 6 A, an increase in the ROS level was observed in Noxa-transfected cells in comparison with mock-transfected cells. Next, we analyzed the suppression effects of endogenous Noxa on ROS production in hypoxia. Cells whose endogenous Noxa expression was blocked with AS were cultured in hypoxic condition for 3 h followed by 1 h of reoxygenation. Cells were then sequentially stained with DCF-DA and propidium iodide (PI) followed by FACS® analysis. The populations of living cells, as indicated by their ability to exclude PI, were analyzed to discard the possibility that ROS were byproducts of cell death. As shown in Fig. 6 B, AS suppressed ROS generation induced by hypoxia, whereas SE failed to reduce ROS levels. Further, to establish that ROS contributed directly to Noxa-mediated hypoxic cell death, cells were transiently transfected with 1 μg pcDNA-Noxa in the presence or absence of antioxidant N-acetylcysteine (NAC). NAC reduced the ROS level and protected cell death in pcDNA-Noxa–transfected cells in a concentration-dependent manner (Fig. 6 C). Our results showed that ROS generation was crucial in Noxa-triggered cell death.

Bottom Line: Noxa is a candidate molecule mediating p53-induced apoptosis.We show that Noxa promoter responds directly to hypoxia via hypoxia-inducible factor (HIF)-1alpha.Suppression of Noxa expression by antisense oligonucleotides rescued cells from hypoxia-induced cell death and decreased infarction volumes in an animal model of ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, College of Medicine, Kyung Hee University, #1 Hoegi-dong, Dongdaemoon-Koo, Seoul 130-701, Korea.

ABSTRACT
Hypoxia is a common cause of cell death and is implicated in many disease processes including stroke and chronic degenerative disorders. In response to hypoxia, cells express a variety of genes, which allow adaptation to altered metabolic demands, decreased oxygen demands, and the removal of irreversibly damaged cells. Using polymerase chain reaction-based suppression subtractive hybridization to find genes that are differentially expressed in hypoxia, we identified the BH3-only Bcl-2 family protein Noxa. Noxa is a candidate molecule mediating p53-induced apoptosis. We show that Noxa promoter responds directly to hypoxia via hypoxia-inducible factor (HIF)-1alpha. Suppression of Noxa expression by antisense oligonucleotides rescued cells from hypoxia-induced cell death and decreased infarction volumes in an animal model of ischemia. Further, we show that reactive oxygen species and resultant cytochrome c release participate in Noxa-mediated hypoxic cell death. Altogether, our results show that Noxa is induced by HIF-1alpha and mediates hypoxic cell death.

Show MeSH
Related in: MedlinePlus