Dysregulation of RyR Calcium Channel Causes the Onset of Mitochondrial Retrograde Signaling

This study shows that multiple modes of mitochondrial stress generated by partial mtDNA depletion or cytochrome c oxidase disruption cause ryanodine receptor channel (RyR) dysregulation, which instigates the release of Ca2+ in the cytoplasm of C2C12 myoblasts and HCT116 carcinoma cells. We also observed a reciprocal downregulation of IP3R channel activity and reduced mitochondrial uptake of Ca2+. Ryanodine, an RyR antagonist, abrogated the mitochondrial stress-mediated increase in [Ca2+]c and the entire downstream signaling cascades of mitochondrial retrograde signaling. Interestingly, ryanodine also inhibited mitochondrial stress-induced invasive behavior in mtDNA-depleted C2C12 cells and HCT116 carcinoma cells. In addition, co-immunoprecipitation shows reduced FKBP12 protein binding to RyR channel proteins, suggesting the altered function of the Ca2+ channel. These results document how the endoplasmic reticulum-associated RyR channels, in combination with inhibition of the mitochondrial uniporter system, modulate cellular Ca2+ homeostasis and signaling under mitochondrial stress conditions

HnRNPA2 is a Novel Histone Acetyltransferase That Mediates Mitochondrial Stress-Induced Nuclear Gene Expression

Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies

Impaired Mitochondrial Respiratory Functions and Oxidative Stress in Streptozotocin-Induced Diabetic Rats

We have previously shown a tissue-specific increase in oxidative stress in the early stages of streptozotocin (STZ)-induced diabetic rats. In this study, we investigated oxidative stress-related long-term complications and mitochondrial dysfunctions in the different tissues of STZ-induced diabetic rats (>15 mM blood glucose for 8 weeks). These animals showed a persistent increase in reactive oxygen and nitrogen species (ROS and RNS, respectively) production. Oxidative protein carbonylation was also increased with the maximum effect observed in the pancreas of diabetic rats. The activities of mitochondrial respiratory enzymes ubiquinol: cytochrome c oxidoreductase (Complex III) and cytochrome c oxidase (Complex IV) were significantly decreased while that of NADH:ubiquinone oxidoreductase (Complex I) and succinate:ubiquinone oxidoreductase (Complex II) were moderately increased in diabetic rats, which was confirmed by the increased expression of the 70 kDa Complex II sub-unit. Mitochondrial matrix aconitase, a ROS sensitive enzyme, was markedly inhibited in the diabetic rat tissues. Increased expression of oxidative stress marker proteins Hsp-70 and HO-1 was also observed along with increased expression of nitric oxide synthase. These results suggest that mitochondrial respiratory complexes may play a critical role in ROS/RNS homeostasis and oxidative stress related changes in type 1 diabetes and may have implications in the etiology of diabetes and its complications

Activation of Akt Is Essential for the Propagation of Mitochondrial Respiratory Stress Signaling and Activation of the Transcriptional Coactivator Heterogeneous Ribonucleoprotein A2

This article shows that mitochondrial respiratory dysfunction activates a stress signaling that induces Akt1 activation. Akt1 activation occurs through calcineurin-mediated IGF1R/PI3-K pathway. Akt1-mediated phosphorylation of hnRNPA2 is a key requirement for the propagation of stress signaling and activation of nuclear target genes

Mitochondria-targeted heme oxygenase-1 induces oxidative stress and mitochondrial dysfunction in macrophages, kidney fibroblasts and in chronic alcohol hepatotoxicity

The inducible form of Heme Oxygenase-1 (HO-1), a major endoplasmic reticulum (ER) associated heme protein, is known to play important roles in protection against oxidative and chemical stress by degrading free heme released from degradation of heme proteins. In this study we show that induced expression of HO-1 by subjecting macrophage RAW-264.7 cells to chemical or physiological hypoxia resulted in significant translocation of HO-1 protein to mitochondria. Transient transfection of COS-7 cells with cloned cDNA also resulted in mitochondrial translocation of HO-1. Deletion of N-terminal ER targeting domain increased mitochondrial translocation under the transient transfection conditions. Mitochondrial localization of both intact HO-1 and N-terminal truncated HO-1 caused loss of heme aa-3 and cytochrome c oxidase (CcO) activity in COS-7 cells. The truncated protein, which localizes to mitochondria at higher levels, induced substantially steeper loss of CcO activity and reduced heme aa3 content. Furthermore, cells expressing mitochondria targeted HO-1 also induced higher ROS production. Consistent with dysfunctional state of mitochondria induced by HO-1, the mitochondrial recruitment of autophagy markers LC-3 and Drp-1 was also increased in these cells. Chronic ethanol feeding in rats also caused an increase in mitochondrial HO-1 and decrease in CcO activity. These results show that as opposed to the protective effect of the ER associated HO-1, mitochondria targeted HO-1 under normoxic conditions induces mitochondrial dysfunction

Defects in cytochrome c oxidase expression induce a metabolic shift to glycolysis and carcinogenesis

Mitochondrial metabolic dysfunction is often seen in cancers. This paper shows that the defect in a mitochondrial electron transport component, the cytochrome c oxidase (CcO), leads to increased glycolysis and carcinogenesis. Using whole genome microarray expression analysis we show that genetic silencing of the CcO subunit Cox4i1 in mouse C2C12 myoblasts resulted in metabolic shift to glycolysis, activated a retrograde stress signaling, and induced carcinogenesis. In the knockdown cells, the expression of Cox4i1 was less than 5% of the control and the expression of the irreversible glycolytic enzymes (Hk1, Pfkm and Pkm) increased two folds, facilitating metabolic shift to glycolysis. The expression of Ca2+ sensitive Calcineurin (Ppp3ca) and the expression of PI3-kinase (Pik3r4 and Pik3cb) increased by two folds. This Ca2+/Calcineurin/PI3K retrograde stress signaling induced the up-regulation of many nuclear genes involved in tumor progression. Overall, we found 1047 genes with 2-folds expression change (with p-value less than 0.01) between the knockdown and the control, among which were 35 up-regulated genes in pathways in cancer (enrichment p-value less than 10−5). Functional analysis revealed that the up-regulated genes in pathways in cancer were dominated by genes in signal transduction, regulation of transcription and PI3K signaling pathway. These results suggest that a defect in CcO complex initiates a retrograde signaling which can induce tumor progression. Physiological studies of these cells and esophageal tumors from human patients support these results. GEO accession number = GSE68525

Multiple isoforms of mitochondrial glutathione S-transferases and their differential induction under oxidative stress.

The mitochondrial respiratory chain, which consumes approx. 85-90% of the oxygen utilized by cells, is a major source of reactive oxygen species (ROS). Mitochondrial genetic and biosynthetic systems are highly susceptible to ROS toxicity. Intramitochondrial glutathione (GSH) is a major defence against ROS. In the present study, we have investigated the nature of the glutathione S-transferase (GST) pool in mouse liver mitochondria, and have purified three distinct forms of GST: GSTA1-1 and GSTA4-4 of the Alpha family, and GSTM1-1 belonging to the Mu family. The mitochondrial localization of these multiple GSTs was confirmed using a combination of immunoblot analysis, protease protection assay, enzyme activity, N-terminal amino acid sequencing, peptide mapping and confocal immunofluorescence analysis. Additionally, exogenously added 4-hydroxynonenal (HNE), a reactive byproduct of lipid peroxidation, to COS cells differentially affected the cytosolic and mitochondrial GSH pools in a dose- and time-dependent manner. Our results show that HNE-mediated mitochondrial oxidative stress caused a decrease in the GSH pool, increased membrane lipid peroxidation, and increased levels of GSTs, glutathione peroxidase and Hsp70 (heat-shock protein 70). The HNE-induced oxidative stress persisted for longer in the mitochondrial compartment, where the recovery of GSH pool was slower than in the cytosolic compartment. Our study, for the first time, demonstrates the presence in mitochondria of multiple forms of GSTs that show molecular properties similar to those of their cytosolic counterparts. Our results suggest that mitochondrial GSTs may play an important role in defence against chemical and oxidative stress