Abstract
The nuclear factor-κB (NF-κB) family of transcription factors can directly or indirectly regulate many important areas of biology, including immunity, inflammation and cell survival. One intriguing aspect of NF-κB crosstalk with other cell signalling pathways is its regulation of mitochondrial biology, including biogenesis, metabolism and apoptosis. In addition to regulating the expression of mitochondrial genes encoded in the nucleus, NF-κB signalling components are also found within mitochondria themselves and associated with mitochondrial DNA. However, complete biochemical analysis of mitochondrial and sub-mitochondrial localisation of all NF-κB subunits has not been undertaken. Here, we show that only the RelA NF-κB subunit and its inhibitor IκBα reside within mitochondria, whilst p50 is found in the endoplasmic reticulum (ER). Fractionation of mitochondria revealed that only RelA was found in the mitoplast, the location of the mtDNA. We demonstrate that hypoxia leads to a very rapid but transient accumulation of RelA and IκBα in mitochondria. This effect required reactive oxygen species (ROS) but was not dependent on the hypoxia sensing transcription factor subunit HIF1α or intracellular Ca2+ release. We also observed rapid mitochondrial localisation of transcription factor STAT3 following hypoxia. Inhibition of STAT3 blocked RelA and IκBα mitochondrial localisation revealing a previously unknown aspect of crosstalk between these key cellular regulators.
Highlights
The nuclear factor-κB (NF-κB) family of transcription factors regulate the expression of a vast array of genes in response to extra- and intracellular stimuli by binding to κB DNA responsive elements within their promoters [1]
Several studies have demonstrated that members of the NF-κB signalling pathway can be present within mitochondrial protein extracts, including RelA, p50, IκBα, IKKα, IKKβ and IKKγ, where they are thought to control apoptosis and mitochondrial metabolism [20,21,23,26,27]
Mitochondria are closely associated with the endoplasmic reticulum (ER) [30] and some protocols are not able to distinguish between localisation in different compartments of the mitochondria or potential ER contamination
Summary
The nuclear factor-κB (NF-κB) family of transcription factors regulate the expression of a vast array of genes in response to extra- and intracellular stimuli by binding to κB DNA responsive elements within their promoters [1]. The C-termini of RelA, RelB and c-Rel contain transactivation domains, essential for controlling the expression of target genes [6,7]. RelA and c-Rel are inactivated by the ankyrin repeat containing Inhibitor of NF-κB (IκB) proteins such as IκBα [8]. IκBs are phosphorylated and targeted for proteasomal degradation via the IκB kinase (IKK) complex, consisting of IKKα, IKKβ and NEMO [13]. This allows the nuclear localisation of the active NF-κB complexes, followed by target gene promoter binding and transcriptional regulation.
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