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. 2012 Jul 3;5(231):ra47.
doi: 10.1126/scisignal.2002712.

Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription

Abu-Bakr Al-Mehdi  1 , Viktor M Pastukh, Brad M Swiger, Darla J Reed, Mita R Patel, Gina C Bardwell, Viktoriya V Pastukh, Mikhail F Alexeyev, Mark N Gillespie
Affiliations

Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription

Abu-Bakr Al-Mehdi et al. Sci Signal. .

Abstract

Mitochondria can govern local concentrations of second messengers, such as reactive oxygen species (ROS), and mitochondrial translocation to discrete subcellular regions may contribute to this signaling function. Here, we report that exposure of pulmonary artery endothelial cells to hypoxia triggered a retrograde mitochondrial movement that required microtubules and the microtubule motor protein dynein and resulted in the perinuclear clustering of mitochondria. This subcellular redistribution of mitochondria was accompanied by the accumulation of ROS in the nucleus, which was attenuated by suppressing perinuclear clustering of mitochondria with nocodazole to destabilize microtubules or with small interfering RNA-mediated knockdown of dynein. Although suppression of perinuclear mitochondrial clustering did not affect the hypoxia-induced increase in the nuclear abundance of hypoxia-inducible factor 1α (HIF-1α) or the binding of HIF-1α to an oligonucleotide corresponding to a hypoxia response element (HRE), it eliminated oxidative modifications of the VEGF (vascular endothelial growth factor) promoter. Furthermore, suppression of perinuclear mitochondrial clustering reduced HIF-1α binding to the VEGF promoter and decreased VEGF mRNA accumulation. These findings support a model for hypoxia-induced transcriptional regulation in which perinuclear mitochondrial clustering results in ROS accumulation in the nucleus and causes oxidative base modifications in the VEGF HRE that are important for transcriptional complex assembly and VEGF mRNA expression.

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Conflict of interest statement

Competing interests: The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Perinuclear mitochondrial clustering in hypoxia: role of microtubules and the dynein motor system. (A) Left image: The arrow points to the nucleus of a capillary endothelial cell in a perfused rat lung. Mitochondrial labeling is green. Right image: Arrows point to clustered mitochondria after 3 hours of hypoxia. Scale bar, 20 μm. (B) Rat PAECs stained with MitoTracker Red, Oregon Green paclitaxel (microtubules), and Hoechst 33342 (nuclei, blue) were cultured under normoxia or hypoxia for 3 hours. Yellow circle denotes perinuclear region. Scale bar, 30 μm. (C) Quantification of perinuclear mitochondrial distribution in normoxic (NORM) and hypoxic PAECs. (D) Distribution of mitochondria in concentric rings radiating from the nucleus outward in normoxic and hypoxic PAECs. (E) Mitochondrial distribution in normoxic and hypoxic PASMCs. (F) Impact of the microtubule-destabilizing agent nocodazole (Noc) on mitochondrial distribution in normoxic and hypoxic PAECs. (G) Impact of siRNA knockdown of the dynein heavy chain (siDYN) on mitochondrial distribution in normoxic and hypoxic PAECs. n = 6 different culture dishes with three to six cells analyzed per dish. *P < 0.05, different from normoxia.
Fig. 2
Fig. 2
Impact of perinuclear mitochondrial clustering on hypoxia-induced pan-cellular ROS generation. (A) Pseudo-colored intensity plots of roGFP signal in normoxic PAECs and PAECs cultured in hypoxia (H) or cultured in hypoxia in the presence of nocodazole or after siRNA-mediated dynein knockdown (siDYN). roGFP signal intensity (indicated by the color bar) correlates with ROS concentrations. Scale bar, 15 μm. (B) Quantitative assessment of nuclear and cytoplasmic roGFP signals in the absence and presence of nocodazole (Noc) in normoxic (N) and hypoxic (H) PAECs. (C) Effect of dynein heavy chain–specific siRNA (siDYN) or scrambled siRNA (Scram) on hypoxia-induced changes in nuclear and cytoplasmic roGFP signals. n = 3 to 5 different culture dishes with three to six cells analyzed per dish for all panels. *P < 0.05, increased from normoxia.
Fig. 3
Fig. 3
Hypoxia induces a redistribution of nuclear ROS that requires microtubule- and dynein-dependent perinuclear mitochondrial clustering. (A) Time-dependent effects of hypoxia on nuclear-targeted roGFP signals as depicted by pseudo-colored, ratiometric images of a PAEC nucleus. roGFP signal intensity (indicated by the color bar) correlates with ROS concentrations. Scale bar, 15 μm. (B) Time-dependent effects of hypoxia on nuclear roGFP fluorescence ratio in PAECs. (C) Effect of nocodazole on hypoxia-induced changes in roGFP fluorescence ratio in normoxic (NORM, N) PAECs and in PAECs cultured in hypoxia (HYP, H) for 60 min. (D) Effect of dynein-specific siRNA (siDYN) or scrambled siRNA (Scram) on roGFP fluorescence ratio in normoxic and hypoxic PAECs. n = 3 to 5 different culture dishes with 3 to 6 cells analyzed per dish for all panels. *P < 0.05, increased from normoxia.
Fig. 4
Fig. 4
Hypoxia causes oxidative base modifications in the HRE of the VEGF promoter that require perinuclear mitochondrial clustering. (A) Effect of nocodazole on Fpg-detectable oxidative base damage in the VEGF HRE in PAECS cultured in normoxia (NORM, N) or hypoxia (HYP, H). (B) Effect of dynein-specific siRNA (siDYN) or scrambled siRNA (Scram) on Fpg-detectable oxidative base damage in the VEGF HRE in normoxic and hypoxic PAECS. (C) ChIP analysis of 8-oxoguanine–containing VEGF HRE sequences in PAECs treated with nocodazole. (D) ChIP analysis of 8-oxoguanine–containing HRE sequences in PAECs transfected with dynein-specific siRNA or scrambled siRNA. n = 4 to 6 different culture dishes for all panels. *P < 0.05, increased from normoxia.
Fig. 5
Fig. 5
Base modifications in the VEGF HRE that occur after perinuclear clustering mitochondria are required for HIF-1α binding and VEGF mRNA expression. (A) Top: Western analyses of HIF-1α and the nuclear marker lamin A/C in PAECs cultured for 3 hours under normoxia (NORM) or hypoxia (HYP) in the presence of nocodazole (Noc) or after transfection with dynein-specific siRNA (siDYN). Representative of four experiments. Bottom: Quantification of HIF-1α abundance normalized to lamin A/C calculated as a percentage of the normoxic control. n = 4 separate culture dishes per experimental group. *P < 0.05, increased from normoxia. (B) Western blot analysis of HIF-1α associating with a 65-mer oligonucleotide model of the VEGF HRE (DNA affinity precipitation analysis). Oligonucleotide-associated HIF-1α was derived from nuclear extracts isolated from normoxic and hypoxic control PAECs or PAECs treated with nocodazole or transfected with dynein-specific siRNA. Data are representative of three separate experiments. (C) ChIP analysis of VEGF HRE sequences immunoprecipitating with HIF-1α recovered from PAECs incubated under normoxia or hypoxia in the presence of nocodazole. (D) ChIP assays for VEGF HRE sequences immunoprecipitating with HIF-1α from PAECs transfected with dynein-specific (siDYN) or scrambled siRNA (Scram). (E) Quantitative RT-PCR analysis of VEGF mRNA expression by PAECs in the presence of nocodazole. (F) Quantitative RT-PCR analysis of VEGF mRNA expression in PAECs transfected with dynein-specific or scrambled siRNA. n = 4 to 6 separate culture dishes per experimental group. *P < 0.05, increased from normoxia. **P < 0.05, different from normoxia and hypoxia alone.
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