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.

Reactive oxygen species (ROS) generated in hypoxic pulmonary artery endothelial cells cause transient oxidative base modifications in the hypoxic response element (HRE) of the VEGF gene that bear a conspicuous relationship to induction of VEGF mRNA expression (FASEB J 19: 387-394, 2005). If such base modifications are indeed linked to transcriptional regulation, then they should be detected in HRE sequences associated with transcriptionally active nucleosomes. Southern blot analysis of the VEGF HRE associated with nucleosome fractions prepared by micrococcal nuclease digestion indicated that hypoxia redistributed some HRE sequences from multi-nucleosomes to transcriptionally-active mono-and di-nucleosome fractions. A simple PCR method revealed that VEGF HRE sequences harboring oxidative base modifications were found exclusively in mononucleosomes. Inhibition of hypoxia-induced ROS generation with myxathiozol prevented formation of oxidative base modifications but not the redistribution of HRE sequences into mono-and dinucleosome fractions. The histone deacetylase inhibitor, trichostatin A, caused retention of HRE sequences in compacted nucleosome fractions and prevented formation of oxidative base modifications. These findings suggest that the hypoxia-induced oxidant stress directed at the VEGF HRE requires the sequence to be repositioned into mono-nucleosomes, and support the prospect that oxidative modifications in this sequence are an important step in transcriptional activation.

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Changes in oxygen tension and shear stress in vascular endothelial cells (ECs) activate specific signaling pathways utilizing second messengers produced by mitochondria. Since mitochondria are motile signaling organelles, mitochondrial redistribution may be an essential response to the changing requirements under hypoxia and flow. We hypothesize that hypoxia and shear stress results in cytoplasmic mitochondrial redistribution in ECs. To study this, rat pulmonary artery and microvascular ECs were subjected to either 3 h hypoxia or 6 h flow at 14 dynes/cm2, respectively. MitoTracker Red (MTR) was utilized to image and track mitochondrial movement. MTR signal was quantified in individual cells at different time points. Our results show increased perinuclear MTR signal at 3 and 6 h of flow and with 3 h hypoxia. Nocodozole abolished the mitochondrial translocation response in both hypoxia and flow. The probe DCF and a reactive‐oxygen species (ROS) sensitive GFP construct with a nuclear localizing sequence were used to study nuclear ROS. DCF studies revealed sustained nuclear ROS production with flow, and increased nuclear ROS production with hypoxia. Preliminary roGFP flow studies indicate nuclear oxidant stress at 1 h and 6 h. We conclude that hypoxia and flow induce microtubule dependent mitochondrial translocation, which may serve to create a nuclear ROS‐signaling mechanism.

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