ABE1844 Sigma-AldrichAnti-NRF-1 Antibody

A key transcriptional regulator of cell metabolism, the peroxisome proliferator-activated receptor γ co-activator 1-α (PPARGC-1-α or PGC-1α), also regulates mitochondrial biogenesis, but its role in antioxidant gene regulation is not well understood. Here, we asked whether genetic heterozygosity of PGC-1α modulates gene expression for the mitochondrial antioxidant enzyme SOD-2 during hepatic inflammatory stress. Using Staphylococcus aureus peritonitis in mice, we found significant Sod2 gene induction in WT mice, whereas PGC-1α heterozygotes (PGC-1α(+/-)) failed to augment Sod2 mRNA and protein levels. Impaired Sod2 regulation in PGC-1α(+/-) mice was accompanied by oxidative stress shown by elevated mitochondrial GSSG/GSH and protein carbonyls. In silico analysis of the mouse proximal Sod2 promoter region revealed consensus binding sites for the Nfe2l2 (Nrf2) transcription factor. Chromatin immunoprecipitation demonstrated diminished Nfe2l2 protein binding to the antioxidant response element promoter site proximal to the Sod2 start site in PGC-1α heterozygous mice, implicating PGC-1α in facilitation of Nfe2l2 DNA binding. Nuclear protein co-immunoprecipitation demonstrated an interaction between hepatic Nfe2l2 and PGC-1α in WT mice that was greatly reduced in PGC-1α(+/-) mice. The data indicate that PGC-1α promotes mitochondrial antioxidant enzyme expression through Nfe2l2-mediated SOD-2 expression in sepsis. The presence of this new PGC-1α-dependent signaling axis indicates that PGC-1α opposes mitochondrial oxidative stress by means of selective induction of one or more antioxidant response element-driven genes. By implication, exploitation of this axis could lead to new pharmacological interventions to improve the antioxidant defenses during oxidative stress-induced mitochondrial damage.

Mitochondrial damage is an important component of multiple organ failure syndrome, a highly lethal complication of severe sepsis that lacks specific therapy. Mitochondrial quality control is regulated in part by the heme oxygenase-1 (HO-1; Hmox1) system through the redox-regulated NF-E2-related factor-2 (Nrf2) transcription factor, but its role in mitochondrial biogenesis in Staphylococcus aureus sepsis is unknown.To test the hypothesis that Nrf2-dependent up-regulation of the HO-1/carbon monoxide (CO) system would preserve mitochondrial biogenesis and rescue mice from lethal S. aureus sepsis.A controlled murine S. aureus peritonitis model with and without inhaled CO was examined for HO-1 and Nrf2 regulation of mitochondrial biogenesis and the resolution of hepatic mitochondrial damage.Sepsis survival was significantly enhanced using inhaled CO (250 ppm once-daily for 1 h), and linked mechanistically to Hmox1 induction and mitochondrial HO activity through Nrf2 transcriptional and Akt kinase activity. HO-1/CO stimulated Nrf2-dependent gene expression and nuclear accumulation of nuclear respiratory factor-1, -2α (Gabpa), and peroxisome proliferator-activated receptor gamma coactivator-1α; increased mitochondrial transcription factor-A and citrate synthase protein levels; and augmented mtDNA copy number. CO enhanced antiinflammatory IL-10 and reduced proinflammatory tumor necrosis factor-α production. By contrast, Nrf2(-/-) and Akt1(-/-) mice lacked CO induction of Hmox1 and mitochondrial biogenesis, and CO rescued neither strain from S. aureus sepsis.We identify an inducible Nrf2/HO-1 regulatory cycle for mitochondrial biogenesis that is prosurvival and counter-inflammatory in sepsis, and describe targeted induction of mitochondrial biogenesis as a potential multiple organ failure therapy.

Erythropoietin (EPO) is often administered to cardiac patients with anemia, particularly from chronic kidney disease, and stimulation of erythropoiesis may stabilize left ventricular and renal function by recruiting protective effects beyond the correction of anemia.We examined the hypothesis that EPO receptor (EpoR) ligand-binding, which activates endothelial NO synthase (eNOS), regulates the prosurvival program of mitochondrial biogenesis in the heart.We investigated the effects of EPO on mitochondrial biogenesis over 14 days in healthy mice. Mice expressing a mitochondrial green fluorescent protein reporter construct demonstrated sharp increases in myocardial mitochondrial density after 3 days of EPO administration that peaked at 7 days and surpassed hepatic or renal effects and anteceded significant increases in blood hemoglobin content. Quantitatively, in wild-type mice, complex II activity, state 3 respiration, and mtDNA copy number increased significantly; also, resting energy expenditure and natural running speed improved, with no evidence of an increase in left ventricular mass index. Mechanistically, EPO activated cardiac mitochondrial biogenesis by enhancement of nuclear respiratory factor-1, PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator 1alpha), and mitochondrial transcription factor-A gene expression in wild-type but not in eNOS(-/-) or protein kinase B (Akt1)(-/-) mice. EpoR was required, because EpoR silencing in cardiomyocytes blocked EPO-mediated nuclear translocation of nuclear respiratory factor-1.These findings support a new physiological and protective role for EPO, acting through its cell surface receptor and eNOS-Akt1 signal transduction, in matching cardiac mitochondrial mass to the convective O(2) transport capacity as erythrocyte mass expands.

Exposure to bacterial lipopolysaccharide (LPS) in vivo damages mitochondrial DNA (mtDNA) and interferes with mitochondrial transcription and oxidative phosphorylation (OXPHOS). Because this damage accompanies oxidative stress and is reversible, we postulated that LPS stimulates mtDNA replication and mitochondrial biogenesis via expression of factors responsive to reactive oxygen species, i.e. nuclear respiratory factor-1 (NRF-1) and mitochondrial transcription factor-A. In testing this hypothesis in rat liver, we found that LPS induces NRF-1 protein expression and activity accompanied by mRNA expression for mitochondrial transcription factor-A, mtDNA polymerase gamma, NRF-2, and single-stranded DNA-binding protein. These events restored the loss in mtDNA copy number and OXPHOS gene expression caused by LPS and increased hepatocyte mitotic index, nuclear cyclin D1 translocation, and phosphorylation of pro-survival kinase, Akt. Thus, NRF-1 was implicated in oxidant-mediated mitochondrial biogenesis to provide OXPHOS for proliferation. This implication was tested in novel mtDNA-deficient cells generated from rat hepatoma cells that overexpress NRF-1. Depletion of mtDNA (rhoo clones) diminished oxidant production and caused loss of NRF-1 expression and growth delay. NRF-1 expression and growth were restored by exogenous oxidant exposure indicating that oxidative stress stimulates biogenesis in part via NRF-1 activation and corresponding to recovery events after LPS-induced liver damage.