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. 2010 Jun-Jul;1797(6-7):1055-65.
doi: 10.1016/j.bbabio.2010.02.008. Epub 2010 Feb 11.

Role of calcineurin, hnRNPA2 and Akt in mitochondrial respiratory stress-mediated transcription activation of nuclear gene targets

Manti Guha  1 , Weigang Tang, Neal Sondheimer, Narayan G Avadhani
Affiliations

Role of calcineurin, hnRNPA2 and Akt in mitochondrial respiratory stress-mediated transcription activation of nuclear gene targets

Manti Guha et al. Biochim Biophys Acta. 2010 Jun-Jul.

Abstract

Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (psim) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca2+ [Ca2+]c as part of this signaling cascade activates Ca2+ responsive phosphatase, calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis is a phenotypic hallmark of C2C12 skeletal muscle cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFkappaB (cRel:p50) pathway, NFAT, CREB and C/EBPdelta. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFkappaB (cRel:p50), C/EBPdelta, CREB, and NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional coactivator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes.

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Figures

Figure 1
Figure 1
Activation of mitochondrial stress factors in mtDNA-depleted C2C12 cells. A. Immunoblot analysis of post nuclear fractions of control, mtDNA-depleted and reverted C2C12 cells. 30 μg protein in each was subjected to immunoblot analysis as described in the Methods section. Blots were quantified using a BioRad imager. B. Calcineurin activity in post nuclear fraction was assayed using a kit from Upstate Biologicals as described in the Methods section. FK506 was added at 10 nM level. Results represent Average ±SEM of three separate assays.
Figure 2
Figure 2
Increased glucose uptake and activation of IGF1R pathway in mtDNA-depleted C2C12 cells. A. 2-deoxyglucose uptake was measured in control, mtDNA-depleted and reverted cells as described in the Methods section. In some cases mitochondrial respiratory stress was induced by adding 25 μM CCCP to control cells as indicated. FK506 was added at 10 nM concentration. B. Effects of CnAα mRNA knock down on Glut4 mRNA level. Realtime PCR analysis of Glut4 mRNA was carried out with total RNA from control and mtDNA-depleted cells with or without expressing siRNA for CnAα mRNA. C. Effects of CnAα mRNA knock down on IGF1R mRNA level. Realtime PCR analysis of IGF1R mRNA from control and mtDNA-depleted cells expressing siRNA for CnAα mRNA or “scrambled” siRNA. D. IGF1R levels under mitochondrial stress. Immunoblot analysis of post-nuclear fractions of control, depleted and reverted C2C12 cells (top panel), A549 (control and mtDNA-depleted), 3T3 and H9C2 cells treated with 25 μM CCCP to induce stress. FK506 was added at 10nM concentration. E. Effects of 1GF1R and IR mRNA knock down on apoptotic cell death was measured using the Nexin assay kit as described in the Methods section. Results (% cell death) show average ±SEM of three separate estimates.
Figure 3
Figure 3
Analysis of minimal promoters of stress target genes and characterization of protein factors involved in transcription regulation. A. Maps of minimal promoters of mouse Cathepsin L, RyR1, human Glut4 promoter and locations of sites for binding to the signature factors. B. Transcriptional activity of the minimal promoter regions of stress response genes in control, mtDNA-depleted and reverted cells. C. Effects of IκBα and IκBβ mRNA depletion on the levels of mRNAs for Glut4, IGF1R and RyR1 genes in mtDNA-depleted cells. D. Characterization of protein factors binding to the minimal promoter DNA of Cathepsin L promoter. Immunoblot of fractions eluted at different salt concentrations were developed using factor specific antibodies as described in the Methods section. E. Top panel: Immunoblot analysis of hnRNPA2 levels in nuclear and cytosolic fractions of control, mtDNA-depleted and reverted cells using hnRNPA2 specific anatibody. Actin was used as loading control for cytosolic protein and p97 was used for nuclear protein. Bottom panel: Real Time PCR analysis of hnRNPA2 mRNA levels in control, mtDNA-depleted and reverted C2C12 cells.
Figure 4
Figure 4
HnRNPA2 binds to the enhanceosome by protein-protein interaction and functions as a transcription coactivator. A. Transcriptional activity of Cathepsin L promoter in control and mtDNA-depleted, and mtDNA-depleted/hnRNPA2 knockdown cells. The promoter construct was co-transfected with indicated cDNAs as described in the Methods section. B. ChIP analysis of the Cathepsin L promoter with hnRNPA2, hnRNPD-L, C/EBPδ, cRel, and CREB antibodies in the indicated cells. C. ChIP analysis of Cathepsin L promoter in control, mtDNA-depleted and reverted cells using hnRNPA2 antibody. In both B and C, preimmune serum was used as a negative antibody control. Real-time PCR analysis using 10% input DNA for normalization. Values are expressed as folds relative to the factor binding in control cells. D. Gel mobility shift analysis using the Cathepsin L minimal promoter DNA and either purified recombinant hnRNPA2 or nuclear extracts from control, mtDNA-depleted and mtDNA-depleted/hnRNPA2 knock down cells (15 μg protein from nuclear extract and 1.0 to 2.5 μg purified hnRNPA2 protein). Unlabeled WT and mutant DNAs (20 fold molar excess) were used for competition.
Figure 5
Figure 5
Role of hnRNPA2 in transcription modulation and in vitro invasive behavior of cells subjected to mitochondrial stress. A. Luciferase activity of Cathepsin L, Glut4 and RyR1 promoters in control, mtDNA-depleted and mtDNA-depleted/hnRNPA2 knockdown cells. Transfection with promoter DNAs and measurement of luciferase activities were described in the Methods. B. 2-Deoxyglucose uptake in control, mtDNA-depleted and mtDNA-depleted/hnRNPA2 knockdown cells. C. Effects of mtDNA-depletion and hnRNPA2 knockdown on cell growth. Number of viable cells was measured using the Guava cell counter. D. Matrigel invasion patterns of control, mtDNA-depleted/mock siRNA, and mtDNA-depleted/hnRNPA2 siRNA cells.
Figure 6
Figure 6
Activation of Akt1 and Akt1 mediated phosphorylation of hnRNPA2 in cells subjected to mitochondrial stress. A. Immunoblot analysis of nuclear protein from control and mtDNA-depleted cells for phosphorylated hnRNPA2 using hnRNPA2 and phospho-Ser antibodies. B. Top panel shows the structural and functional domain of hnRNPA2. The autoradiogram at the bottom shows recombinant Akt1-mediated phosphorylation of purified hnRNPA2. C. Akt1 mRNA quantitation by Real Time PCR in control, mtDNA-depleted, CCCP- treated and reverted cells. D. Akt activity was measured in the nuclear extract of control and mtDNA-depleted cells using an Akt assay kit as described in the Methods section. E. ChIP analysis of Cathepsin L promoter in control, mtDNA-depleted/mock siRNA and mtDNA-depleted/AktsiRNA cells for occupancy of hnRNPA2.
Figure 7
Figure 7
A model for mitochondrial respiratory stress induced activation of nuclear gene expression and altered physiological processes.
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