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Staphylococcus aureus sepsis and mitochondrial accrual of the 8-oxoguanine DNA glycosylase DNA repair enzyme in mice.

ABSTRACT: Damage to mitochondrial DNA (mtDNA) by the production of reactive oxygen species during inflammatory states, such as sepsis, is repaired by poorly understood mechanisms.To test the hypothesis that the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), contributes to mtDNA repair in sepsis.Using a well-characterized mouse model of Staphylococcus aureus sepsis, we analyzed molecular markers for mitochondrial biogenesis and OGG1 translocation into liver mitochondria as well as OGG1 mRNA expression at 0, 24, 48, and 72 hours after infection. The effects of OGG1 RNA silencing on mtDNA content were determined in control, tumor necrosis factor-?, and peptidoglycan-exposed rat hepatoma cells. Based on in situ analysis of the OGG1 promoter region, chromatin immunoprecipitation assays were performed for nuclear respiratory factor (NRF)-1 and NRF-2? GA-binding protein (GABP) binding to the promoter of OGG1.Mice infected with 10(7) cfu S. aureus intraperitoneally demonstrated hepatic oxidative mtDNA damage and significantly lower hepatic mtDNA content as well as increased mitochondrial OGG1 protein and enzyme activity compared with control mice. The infection also caused increases in hepatic OGG1 transcript levels and NRF-1 and NRF-2? transcript and protein levels. A bioinformatics analysis of the Ogg1 gene locus identified several promoter sites containing NRF-1 and NRF-2? DNA binding motifs, and chromatin immunoprecipitation assays confirmed in situ binding of both transcription factors to the Ogg1 promoter within 24 hours of infection.These studies identify OGG1 as an early mitochondrial response protein during sepsis under regulation by the NRF-1 and NRF-2? transcription factors that regulate mitochondrial biogenesis.

SUBMITTER: Bartz RR

PROVIDER: S-EPMC3040391 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Staphylococcus aureus sepsis and mitochondrial accrual of the 8-oxoguanine DNA glycosylase DNA repair enzyme in mice.

Bartz Raquel R RR Suliman Hagir B HB Fu Ping P Welty-Wolf Karen K Carraway Martha Sue MS MacGarvey Nancy Chou NC Withers Crystal M CM Sweeney Timothy E TE Piantadosi Claude A CA

American journal of respiratory and critical care medicine 20100823 2

<h4>Rationale</h4>Damage to mitochondrial DNA (mtDNA) by the production of reactive oxygen species during inflammatory states, such as sepsis, is repaired by poorly understood mechanisms.<h4>Objectives</h4>To test the hypothesis that the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), contributes to mtDNA repair in sepsis.<h4>Methods</h4>Using a well-characterized mouse model of Staphylococcus aureus sepsis, we analyzed molecular markers for mitochondrial biogenesis and OGG1 translocatio ...[more]

PMID: 20732986

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Project description:Purpose: The aim of this study is to test whether global gene expression induced by multiple challenges with OGG1-BER product 8-oxoG in mouse airways is linked to airway remodeling. Methods: RNAs extracted from individual mouse lungs (experimental group: n=5) were pooled and a total 1 ?g RNA was used for Next-Generation Sequencing (NGS) analyses on an Illumina HiSeq 1000 sequencing system. Sequence analyses were performed in duplicate. First- and second-strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq Sample Preparation Kit as recommended by the manufacturer (Illumina). Library quailty was evaluated by using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Library DNA templates were quantitated by qPCR using known reference standards. Cluster formation of the library of DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation. Paired-end, 50-base sequencing was performed with a TruSeq SBS kit v3 (Illumina) on the Illumina HiSeq 1000 by protocols defined by the manufacturer. Base call conversion to sequence reads was performed using CASAVA-1.8.2. Sequence data were analyzed with the Bowtie2, Tophat2 and GFOLD programs. Processed data are presented as reads per kilobase transcript per million (RPKM), normalized to the experimental control (RNA from saline challenged lungs) and reported as fold change (test/control). Results: We mapped an average of 31.41 million sequence reads per sample and identified 23,337 transcripts in total RNA extracted from lungs of Balb/cJ mice as described in Methods. Approximately 14% of the transcripts showed differential expression between the saline-challenged control and 8-oxoguanine-challeged mouse lungs, with a fold change ?3.0. We validated the expression changes of 18 selected EMT-related genes of interest for our studies by qRT-PCR. Hierarchical clustering followed by Protein ANalysis THrough Evolutionary Relationships database (PANTHER) analysis of differentially expressed genes was done using GENE-E online software from Broad Institute (http://www.broadinstitute.org/cancer/software/GENE-E/). Results from PANTHER analysis of upregulated transcripts (fold change ?3.0) showed overrepresentation of various biological functions (GO terms) including developmental process (GO:0032502, P=4.58E-33), system development (GO:0048731, P=9.16E-33), cellular process (GO:0009987, P= 5.52E-31), cell adhesion (GO:0007155, P= 8.63E-28) among others. Pathway analysis (PANTHER) indicated that the most overrepresented pathways were: cadherin signaling (P00012, P=6.62E-07), wnt signaling (P00057, P= 5.81E-06), integrin signaling (P00034, P= 1.09E-05) among others. In addition to gene expression analysis, we confirmed airway remodeling by histological examination (Hematoxylin and Eosin, Masson's trichrome staining) of lung sections at seven days from the last challenge (day 11). Conclusions: This is the first study showing a link between gene expression at whole-transcriptome level induced by chronic OGG1-BER (mimicked by multiple challenges with 8-oxoG) and airway remodeling, supported by histological structural changes in lungs. Balb/cJ mice (5 per group) were intranasally challenged with 8-oxoguanine (1 µM, 60 µl) for three times at days 0, 2 and 4. Control group mice were intranasally challenged with saline (60 µl). At 30, 60 and 120 min after the third challenge (day 4), mice were sacrificed and lungs were processed for RNA extraction. RNAs from individual mice within the same group were pooled and subjected to deep-sequencing analysis in duplicate using NSG on an Illumina HiSeq 1000 sequencing system. After alignment and processing, the resulting RPKM from treatment groups (8-oxoG-challenged) were normalized to control group (saline-challenged).

Dataset Information

Staphylococcus aureus sepsis and mitochondrial accrual of the 8-oxoguanine DNA glycosylase DNA repair enzyme in mice.

Publications

Staphylococcus aureus sepsis and mitochondrial accrual of the 8-oxoguanine DNA glycosylase DNA repair enzyme in mice.

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