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. 2007 Aug;27(16):5737-45.
doi: 10.1128/MCB.02265-06. Epub 2007 Jun 11.

Mitochondrial reactive oxygen species trigger hypoxia-inducible factor-dependent extension of the replicative life span during hypoxia

Eric L Bell  1 , Tatyana A Klimova, James Eisenbart, Paul T Schumacker, Navdeep S Chandel
Affiliations

Mitochondrial reactive oxygen species trigger hypoxia-inducible factor-dependent extension of the replicative life span during hypoxia

Eric L Bell et al. Mol Cell Biol. 2007 Aug.

Abstract

Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span.

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Figures

FIG. 1.
FIG. 1.
Hypoxia increases replicative life span of human lung fibroblasts and mitochondrion-generated ROS. (a) PHLFs were cultured in either 21% O2 or 1.5% O2 and their PDs were monitored. (b) Senescence was detected by β-galactosidase activity in normoxia (PD29 and PD55) or hypoxia (PD27, PD55, and PD66). Scale bar, 132 μm. (c) Relative intracellular H2O2 levels of PHLFs, as determined by Amplex Red in 21% O2, 1.5% O2, or antimycin A (1 μg/ml) for 4 h in the presence of the mitochondrion-targeted antioxidant MitoQ (1 μM) or control compound TPMP (1 μM). *, P < 0.05 for TPMP at 21% O2 compared to TPMP at 1.5% O2 or antimycin A (n = 3). (d) Cytosolic ROS levels were detected by roGFP in PHLFs exposed to 21% O2 and 1.5% O2 for 24 and 48 h. Intracellular oxidant levels are displayed as a percentage of roGFP maximally oxidized by treating cells with 1 mM H2O2 for 5 min (n = 3).
FIG. 2.
FIG. 2.
Hypoxic increase in cytosolic ROS does not induce DNA double-stranded break response. (a) Relative intracellular H2O2 levels of PHLFs, as determined by Amplex Red in 21% O2, 3% O2, or 1.5% O2. *, P < 0.05 for 3% O2 or 1.5% O2 compared to 21% O2 (n = 3). (b) Phosphorylation status of H2AX in acid-extracted proteins from PHLF exposed to 21% O2, 3% O2, 1.5% O2, or staurosporine.
FIG. 3.
FIG. 3.
Hypoxia-induced generation of mitochondrial ROS triggers HIF activation in PHLFs. (a) PHLFs transiently transfected with the firefly luciferase reporter construct driven by 3×HRE and the Renilla luciferase construct driven by the thymidine kinase promoter and exposed to 21% O2, 3% O2, or 1.5% O2. *, P < 0.05 for 3% O2 or 1.5% O2 compared to 21% O2 (n = 3). (b) HIF-1α and RNA Pol II protein levels in nuclear extracts from PHLFs exposed to 21% O2 and 1.5% O2 for 4 h in the presence of either 5 μM MitoQ or TPMP. (c) PHLFs transiently transfected with the firefly luciferase reporter construct driven by 3×HRE and the Renilla luciferase construct driven by the thymidine kinase promoter. Cells were exposed to 2 μM MitoQ or TPMP under 21% O2 and 1.5% O2 for 16 h. Relative luciferase values were determined by normalizing firefly to Renilla luciferase. *, P < 0.05 for TPMP at 21% O2 compared to TPMP at 1.5% O2 (n = 3).
FIG. 4.
FIG. 4.
Stabilization of HIF under normal oxygen conditions is sufficient to increase hTERT transcription and telomerase activity. HIF-1α protein levels (a), relative hTERT mRNA levels normalized to the ribosomal protein L19 (b), and telomerase activity (c) in PHLF exposed to 21% O2, PHLF infected with adenovirus encoding the ODDDwt or ODDD(P564A) of HIF-1α (amino acids 531 to 575) in 21% O2, or PHLF in 1.5% O2 *, P < 0.05 for ODDDwt compared to ODDD(P564A) (n = 3).
FIG. 5.
FIG. 5.
HIF is necessary for the hypoxic increase in replicative life span. (a) Transcriptional activity of PHLFs stably expressing HIF-DN or the vector control pLXIN was assayed with a firefly luciferase reporter construct driven by 3×HRE and with a control Renilla luciferase construct driven by the thymidine kinase promoter. Relative luciferase values were determined by normalizing values of firefly to Renilla luciferase (n = 3). (b) Telomerase activity of PHLFs stably expressing HIF-DN or pLXIN exposed to 21% O2 or 1.5% O2 (n = 3). PHLFs stably expressing HIF-DN or pLXIN were grown in 21% O2 (c) or 1.5% O2 (d), and their PDs were monitored postinfection.
FIG. 6.
FIG. 6.
HIF is sufficient for the increase in replicative life span. PHLF cells with activated endogenous HIF were generated by stably expressing the ODDDwt construct, with ODDD(P564A) as a control. Transcriptional activity of HIF (a) and PDs (b) were monitored under 21% O2 at the indicated days postinfection. * P < 0.05 for ODDDwt compared to ODDD(P564A) (n = 3).
FIG. 7.
FIG. 7.
Loss of the pVHL tumor suppressor protein that regulates HIF increases replicative life span in PHLF. (a) pVHL protein levels from whole-cell lysates of PHLF stably expressing an shRNA against pVHL. (b) HIF-1α protein levels in cells stably expressing shRNA for dHIF and pVHL that were exposed to 21% O2 or 1.5% O2. N, normoxia; H, hypoxia. (c) PHLFs expressing an shRNA against pVHL or dHIF (control) were grown in 21% O2, and their PDs were monitored postinfection.
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References

    1. Abe, J., M. Takahashi, M. Ishida, J. D. Lee, and B. C. Berk. 1997. c-Src is required for oxidative stress-mediated activation of big mitogen-activated protein kinase 1. J. Biol. Chem. 272:20389-20394. - PubMed
    1. Beckman, K. B., and B. N. Ames. 1998. The free radical theory of aging matures. Physiol. Rev. 78:547-581. - PubMed
    1. Bell, E. L., T. A. Klimova, J. Eisenbart, C. T. Moraes, M. Murphy, G. R. S. Budinger, and N. S. Chandel. 11 June 2007, posting date. The Qo site of the mitochondrial complex III is required for the transduction of hypoxic signaling via reactive oxygen species production. J. Cell Biol. doi: 10.1083/jcb.200609074v1. - DOI - PMC - PubMed
    1. Bodnar, A. G., M. Ouellette, M. Frolkis, S. E. Holt, C. P. Chiu, G. B. Morin, C. B. Harley, J. W. Shay, S. Lichtsteiner, and W. E. Wright 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279:349-352. - PubMed
    1. Brunelle, J. K., E. L. Bell, N. M. Quesada, K. Vercauteren, V. Tiranti, M. Zeviani, R. C. Scarpulla, and N. S. Chandel. 2005. Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation. Cell Metab. 1:409-414. - PubMed

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