DJ-1 interacts with and regulates paraoxonase-2, an enzyme critical for neuronal survival in response to oxidative stress.

Loss-of-function mutations in DJ-1 (PARK7) gene account for about 1% of all familial Parkinson's disease (PD). While its physiological function(s) are not completely clear, DJ-1 protects neurons against oxidative stress in both in vitro and in vivo models of PD. The molecular mechanism(s) through which DJ-1 alleviates oxidative stress-mediated damage remains elusive. In this study, we identified Paraoxonase-2 (PON2) as an interacting target of DJ-1. PON2 activity is elevated in response to oxidative stress and DJ-1 is crucial for this response. Importantly, we showed that PON2 deficiency hypersensitizes neurons to oxidative stress induced by MPP+ (1-methyl-4-phenylpyridinium). Conversely, over-expression of PON2 protects neurons in this death paradigm. Interestingly, PON2 effectively rescues DJ-1 deficiency-mediated hypersensitivity to oxidative stress. Taken together, our data suggest a model by which DJ-1 exerts its antioxidant activities, at least partly through regulation of PON2

AMP-Activated Protein Kinase-Regulated Activation of the PGC-1α Promoter in Skeletal Muscle Cells

The mechanisms by which PGC-1α gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1α using AICAR, an activator of AMPK, that is known to increase PGC-1α expression. A 2.2 kb fragment of the human PGC-1α promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-κB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1α promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at −495 within the PGC-1α promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1α promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1α promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1α promoter activity. The USF-1-mediated increase in PGC-1α promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1α gene expression. This could represent a potential therapeutic target to control PGC-1α expression in skeletal muscle

Effect of GATA-4 and USF-1 overexpression on PGC-1α promoter activity and mRNA expression.

<p>A. Representative western blots of protein extracts made from C₂C₁₂ cells transfected with either 2 or 4 µg of GATA-4 or USF-1 or an empty vector (EV) control. B. USF-1 and GATA-4 were co-transfected with the pGL3 (EV; 500ng) or the p851 PGC-1α promoter reporter construct (500ng) along with the appropriate empty vector controls. Relative luciferase activities were measured 48 hours after transfection and are plotted as the fold change above empty vector. Values are means±SEM, (n = 8); * p<0.05 versus p851-EV and #, p<0.05 versus p851-USF-1 or p851-GATA-4. C. Cells were transfected with 4 µg of USF-1 or an empty vector (EV) control. EtBr-stained DNA gel of PGC-1α amplified by PCR from EV- and USF-1 transfected cells. s12rRNA was used to verify equal loading. Data are representative of one experiment with conditions repeated in duplicate (AU = arbitrary units).</p

The human PGC-1α promoter.

<p>The nucleotide sequence +28 to −2190 corresponding to the proximal 2-kb hPGC-1α promoter is shown. The arrows indicate the transcription start sites, which have been previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003614#pone.0003614-Akimoto2" target="_blank">[14]</a>. Putative binding sites for transcription factors are either <i>underlined</i> or <i>overlined</i>. Also included are binding sites for transcription factors that have previously been characterized [12;13;19]. Numbers enclosed in circles represent the 5′- deletions of the PGC-1α promoter reporter constructs p 2215, p 1164, p 851, p501, p 191 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003614#pone-0003614-g002" target="_blank">Fig. 2</a>.</p

AMPK activation induces PGC-1α mRNA expression and transcriptionally activates the PGC-1α promoter.

<p>C₂C₁₂ cells were treated with either AICAR (1 mM) or Vehicle for 24 hrs. A. Representative Western Blot probed with a Phospho-AMPKα (Thr172), stripped and then re-probed with total AMPKα for loading control (upper panel). Summary of repeated experiments of the effect of AICAR on AMPK activation is shown (lower panel; n = 4). B. upper panel, EtBr-stained DNA gel of PGC-1α amplified by PCR from vehicle- and AICAR-treated cells. GAPDH was also amplified by PCR and used to verify equal loading. Lower panel: A summary of repeated experiments of the effects of AICAR on PGC-1α mRNA expression (n = 3). C. AICAR-induced transcriptional regulation of the PGC-1α promoter. Relative luciferase activity of the PGC-1α promoter constructs in vehicle- or AICAR-treated cells is shown (n = 4–6). D. The AICAR-responsive region (ARR) from −473 to −821 was cloned into the pGL4.23 minimal promoter vector and AICAR-induced transcriptional regulation of this region was assessed (n = 3). For all data, values are means±S.E.M, *, p<0.05 versus Vehicle-treated control; §, p<005 versus pGL4.23.</p

AMPK activation increases GATA/EBox DNA binding.

<p>A. Representative EMSAs of nuclear extracts from Vehicle− (−AIC) and AICAR− (+AIC) treated cells that were incubated with radiolabeled oligonucleotides corresponding to the EBox, SRE and IRS sequences found within the AICAR-responsive region of the PGC-1α promoter (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003614#pone-0003614-g001" target="_blank">Fig. 1</a>). B. A representative EMSA (<i>Left panel)</i> and a summary of multiple experiments (<i>Right panel)</i> showing the effect of AICAR (A) on GATA/EBox-DNA binding. Values are represented as means±S.E.M (n = 8) relative to vehicle-treated (V) cells. C. Representative EMSAs of nuclear extracts that were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt. Vehicle-treated cells were incubated with radiolabeled oligonucleotides corresponding to GATA/EBox wt as well as antibodies against MyoD, USF-1 and c-Myc which are known to bind to the EBox sequence. FP: free probe, 25×, 50×, 100× CO: 25-fold, 50-fold, 100-fold molar excess of cold oligo, No Ab: No Antibody, SRF: SRF antibody, FKHR: Forkhead antibody, GATA-4: GATA-4 antibody, c-Myc: c-Myc Antibody, USF-1: USF-1 antibody. **The representative blot of IRS/DNA binding was made from parts of the same gel. D. Representative chromatin immunoprecipitation from cells treated with or without 1 mM AICAR for 24 hours. Protein/DNA complexes were immunoprecipitated with USF-1 antibody, or with non-specific IgG. Primers encompassing the region between −473 and −823 were used to analyze USF-1 binding to the PGC-1α promoter. The representative blot on the left was made from parts of the same gel. At right is a graphical summary of repeated experiments. Values are representative of means±S.E.M (n = 6).</p