Hagir B. Suliman

Heme Oxygenase-1 Regulates Cardiac Mitochondrial Biogenesis via Nrf2-Mediated Transcriptional Control of Nuclear Respiratory Factor-1

Heme oxygenase (HO)-1 is a protective antioxidant enzyme that prevents cardiomyocyte apoptosis, for instance, during progressive cardiomyopathy. Here we identify a fundamental aspect of the HO-1 protection mechanism by demonstrating that HO-1 activity in mouse heart stimulates the bigenomic mitochondrial biogenesis program via induction of NF-E2–related factor (Nrf)2 gene expression and nuclear translocation. Nrf2 upregulates the mRNA, protein, and activity for HO-1 as well as mRNA and protein for nuclear respiratory factor (NRF)-1. Mechanistically, in cardiomyocytes, endogenous carbon monoxide (CO) generated by HO-1 overexpression stimulates superoxide dismutase-2 upregulation and mitochondrial H2O2 production, which activates Akt/PKB. Akt deactivates glycogen synthase kinase-3&bgr;, which permits Nrf2 nuclear translocation and occupancy of 4 antioxidant response elements (AREs) in the NRF-1 promoter. The ensuing accumulation of nuclear NRF-1 protein leads to gene activation for mitochondrial biogenesis, which opposes apoptosis and necrosis caused by the cardio-toxic anthracycline chemotherapeutic agent, doxorubicin. In cardiac cells, Akt silencing exacerbates doxorubicin-induced apoptosis, and in vivo CO rescues wild-type but not Akt1−/− mice from doxorubicin cardiomyopathy. These findings consign HO-1/CO signaling through Nrf2 and Akt to the myocardial transcriptional program for mitochondrial biogenesis, provide a rationale for targeted mitochondrial CO therapy, and connect cardiac mitochondrial volume expansion with the inducible network of xenobiotic and antioxidant cellular defenses.

Extracellular superoxide dismutase in the airways of transgenic mice reduces inflammation and attenuates lung toxicity following hyperoxia

Extracellular superoxide dismutase (EC-SOD, or SOD3) is the major extracellular antioxidant enzyme in the lung. To study the biologic role of EC-SOD in hyperoxic-induced pulmonary disease, we created transgenic (Tg) mice that specifically target overexpression of human EC-SOD (hEC-SOD) to alveolar type II and nonciliated bronchial epithelial cells. Mice heterozygous for the hEC-SOD transgene showed threefold higher EC-SOD levels in the lung compared with wild-type (Wt) littermate controls. A significant amount of hEC-SOD was present in the epithelial lining fluid layer. Both Tg and Wt mice were exposed to normobaric hyperoxia (>99% oxygen) for 48, 72, and 84 hours. Mice overexpressing hEC-SOD in the airways attenuated the hyperoxic lung injury response, showed decreased morphologic evidence of lung damage, had reduced numbers of recruited inflammatory cells, and had a reduced lung wet/dry ratio. To evaluate whether reduced numbers of neutrophil infiltration were directly responsible for the tolerance to oxygen toxicity observed in the Tg mice, we made Wt and Tg mice neutropenic using anti-neutrophil antibodies and subsequently exposed them to 72 hours of hyperoxia. Both Wt and Tg neutrophil-depleted (ND) mice have less severe lung injury compared with non-ND animals, thus providing direct evidence that neutrophils recruited to the lung during hyperoxia play a distinct role in the resultant acute lung injury. We conclude that oxidative and inflammatory processes in the extracellular lung compartment contribute to hyperoxic-induced lung damage and that overexpression of hEC-SOD mediates a protective response to hyperoxia, at least in part, by attenuating the neutrophil inflammatory response.

Survival in critical illness is associated with early activation of mitochondrial biogenesis.

RATIONALE We previously reported outcome-associated decreases in muscle energetic status and mitochondrial dysfunction in septic patients with multiorgan failure. We postulate that survivors have a greater ability to maintain or recover normal mitochondrial functionality. OBJECTIVES To determine whether mitochondrial biogenesis, the process promoting mitochondrial capacity, is affected in critically ill patients. METHODS Muscle biopsies were taken from 16 critically ill patients recently admitted to intensive care (average 1-2 d) and from 10 healthy, age-matched patients undergoing elective hip surgery. MEASUREMENTS AND MAIN RESULTS Survival, mitochondrial morphology, mitochondrial protein content and enzyme activity, mitochondrial biogenesis factor mRNA, microarray analysis, and phosphorylated (energy) metabolites were determined. Ten of 16 critically ill patients survived intensive care. Mitochondrial size increased with worsening outcome, suggestive of swelling. Respiratory protein subunits and transcripts were depleted in critically ill patients and to a greater extent in nonsurvivors. The mRNA content of peroxisome proliferator-activated receptor γ coactivator 1-α (transcriptional coactivator of mitochondrial biogenesis) was only elevated in survivors, as was the mitochondrial oxidative stress protein manganese superoxide dismutase. Eventual survivors demonstrated elevated muscle ATP and a decreased phosphocreatine/ATP ratio. CONCLUSIONS Eventual survivors responded early to critical illness with mitochondrial biogenesis and antioxidant defense responses. These responses may partially counteract mitochondrial protein depletion, helping to maintain functionality and energetic status. Impaired responses, as suggested in nonsurvivors, could increase susceptibility to mitochondrial damage and cellular energetic failure or impede the ability to recover normal function. Clinical trial registered with clinical trials.gov (NCT00187824).

The CO/HO system reverses inhibition of mitochondrial biogenesis and prevents murine doxorubicin cardiomyopathy

The clinical utility of anthracycline anticancer agents, especially doxorubicin, is limited by a progressive toxic cardiomyopathy linked to mitochondrial damage and cardiomyocyte apoptosis. Here we demonstrate that the post-doxorubicin mouse heart fails to upregulate the nuclear program for mitochondrial biogenesis and its associated intrinsic antiapoptosis proteins, leading to severe mitochondrial DNA (mtDNA) depletion, sarcomere destruction, apoptosis, necrosis, and excessive wall stress and fibrosis. Furthermore, we exploited recent evidence that mitochondrial biogenesis is regulated by the CO/heme oxygenase (CO/HO) system to ameliorate doxorubicin cardiomyopathy in mice. We found that the myocardial pathology was averted by periodic CO inhalation, which restored mitochondrial biogenesis and circumvented intrinsic apoptosis through caspase-3 and apoptosis-inducing factor. Moreover, CO simultaneously reversed doxorubicin-induced loss of DNA binding by GATA-4 and restored critical sarcomeric proteins. In isolated rat cardiac cells, HO-1 enzyme overexpression prevented doxorubicin-induced mtDNA depletion and apoptosis via activation of Akt1/PKB and guanylate cyclase, while HO-1 gene silencing exacerbated doxorubicin-induced mtDNA depletion and apoptosis. Thus doxorubicin disrupts cardiac mitochondrial biogenesis, which promotes intrinsic apoptosis, while CO/HO promotes mitochondrial biogenesis and opposes apoptosis, forestalling fibrosis and cardiomyopathy. These findings imply that the therapeutic index of anthracycline cancer chemotherapeutics can be improved by the protection of cardiac mitochondrial biogenesis.

Mitochondrial transcription factor A induction by redox activation of nuclear respiratory factor 1.

The nuclear expression of mitochondrial transcription factor A (Tfam), which is required for mitochondrial DNA (mtDNA) transcription and replication, must be linked to cellular energy needs. Because respiration generates reactive oxygen species as a side-product, we tested the idea that reactive oxygen species regulate Tfam expression through phosphorylation of nuclear respiratory factor (NRF-1) and binding to the Tfam promoter. In mitochondriarich rat hepatoma cells that overexpress NRF-1, basal and oxidant-induced increases were found in Tfam expression and mtDNA content. Specific binding of NRF-1 to Tfam promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. NRF-1-Tfam binding was augmented under pro-oxidant conditions. NRF-1 gene silencing produced 1:1 knockdown of Tfam expression and decreased mtDNA content. To evaluate oxidation-reduction (redox) regulation of NRF-1 in Tfam expression, blockade of upstream phosphatidylinositol 3-kinase was used to demonstrate loss of oxidant stimulation of NRF-1 phosphorylation and Tfam expression. The oxidant response was also abrogated by specific inhibition of Akt/protein kinase B. Examination of the NRF-1 amino acid sequence revealed an Akt phosphorylation consensus at which site-directed mutagenesis abolished NRF-1 phosphorylation by Akt. Finally, Akt phosphorylation and NRF-1 translocation predictably lacked oxidant regulation in a cancer line having no PTEN tumor suppressor (HCC1937 cells). This study discloses novel redox regulation of NRF-1 phosphorylation and nuclear translocation by phosphatidylinositol 3,4,5-triphosphate kinase/Akt signaling in controlling Tfam induction by an anti-oxidant pro-survival network.

Lipopolysaccharide Stimulates Mitochondrial Biogenesis via Activation of Nuclear Respiratory Factor-1

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 γ, 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 (ρo 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.

Transient hypoxia stimulates mitochondrial biogenesis in brain subcortex by a neuronal nitric oxide synthase-dependent mechanism.

The adaptive mechanisms that protect brain metabolism during and after hypoxia, for instance, during hypoxic preconditioning, are coordinated in part by nitric oxide (NO). We tested the hypothesis that acute transient hypoxia stimulates NO synthase (NOS)-activated mechanisms of mitochondrial biogenesis in the hypoxia-sensitive subcortex of wild-type (Wt) and neuronal NOS (nNOS) and endothelial NOS (eNOS)-deficient mice. Mice were exposed to hypobaric hypoxia for 6 h, and changes in immediate hypoxic transcriptional regulation of mitochondrial biogenesis was assessed in relation to mitochondrial DNA (mtDNA) content and mitochondrial density. There were no differences in cerebral blood flow or hippocampal PO2 responses to acute hypoxia among these strains of mice. In Wt mice, hypoxia increased mRNA levels for peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1 α), nuclear respiratory factor-1, and mitochondrial transcription factor A. After 24 h, new mitochondria, localized in reporter mice expressing mitochondrial green fluorescence protein, were seen primarily in hippocampal neurons. eNOS−/− mice displayed lower basal levels but maintained hypoxic induction of these transcripts. In contrast, nuclear transcriptional regulation of mitochondrial biogenesis in nNOS−/− mice was normal at baseline but did not respond to hypoxia. After hypoxia, subcortical mtDNA content increased in Wt and eNOS−/− mice but not in nNOS−/− mice. Hypoxia stimulated PGC-1α protein expression and phosphorylation of protein kinase A and cAMP response element binding (CREB) protein in Wt mice, but CREB only was activated in eNOS−/− mice and not in nNOS−/− mice. These findings demonstrate that hypoxic preconditioning elicits subcortical mitochondrial biogenesis by a novel mechanism that requires nNOS regulation of PGC-1α and CREB.

Heme Oxygenase-1 Couples Activation of Mitochondrial Biogenesis to Anti-inflammatory Cytokine Expression

The induction of heme oxygenase-1 (HO-1; Hmox1) by inflammation, for instance in sepsis, is associated both with an anti-inflammatory response and with mitochondrial biogenesis. Here, we tested the idea that HO-1, acting through the Nfe2l2 (Nrf2) transcription factor, links anti-inflammatory cytokine expression to activation of mitochondrial biogenesis. HO-1 induction after LPS stimulated anti-inflammatory IL-10 and IL-1 receptor antagonist (IL-1Ra) expression in mouse liver, human HepG2 cells, and mouse J774.1 macrophages but blunted tumor necrosis factor-α expression. This was accompanied by nuclear Nfe2l2 accumulation and led us to identify abundant Nfe2l2 and other mitochondrial biogenesis transcription factor binding sites in the promoter regions of IL10 and IL1Ra compared with pro-inflammatory genes regulated by NF-κΒ. Mechanistically, HO-1, through its CO product, enabled these transcription factors to bind the core IL10 and IL1Ra promoters, which for IL10 included Nfe2l2, nuclear respiratory factor (NRF)-2 (Gabpa), and MEF2, and for IL1Ra, included NRF-1 and MEF2. In cells, Hmox1 or Nfe2l2 RNA silencing prevented IL-10 and IL-1Ra up-regulation, and HO-1 induction failed post-LPS in Nfe2l2-silenced cells and post-sepsis in Nfe2l2−/− mice. Nfe2l2−/− mice compared with WT mice, showed more liver damage, higher mortality, and ineffective CO rescue in sepsis. Nfe2l2−/− mice in sepsis also generated higher hepatic TNF-α mRNA levels, lower NRF-1 and PGC-1α mRNA levels, and no enhancement of anti-inflammatory Il10, Socs3, or bcl-xL gene expression. These findings disclose a highly structured transcriptional network that couples mitochondrial biogenesis to counter-inflammation with major implications for immune suppression in sepsis.

Lung EC-SOD overexpression attenuates hypoxic induction of Egr-1 and chronic hypoxic pulmonary vascular remodeling

Although production of reactive oxygen species (ROS) such as superoxide (O(2)(.-)) has been implicated in chronic hypoxia-induced pulmonary hypertension (PH) and pulmonary vascular remodeling, the transcription factors and gene targets through which ROS exert their effects have not been completely identified. We used mice overexpressing the extracellular antioxidant enzyme extracellular superoxide dismutase (EC-SOD TG) to test the hypothesis that O(2)(.-) generated in the extracellular compartment under hypoxic conditions contributes to PH through the induction of the transcription factor, early growth response-1 (Egr-1), and its downstream gene target, tissue factor (TF). We found that chronic hypoxia decreased lung EC-SOD activity and protein expression in wild-type mice, but that EC-SOD activity remained five to seven times higher in EC-SOD TG mice under hypoxic conditions. EC-SOD overexpression attenuated chronic hypoxic PH, and vascular remodeling, measured by right ventricular systolic pressures, proliferation of cells in the vessel wall, muscularization of small pulmonary vessels, and collagen deposition. EC-SOD overexpression also prevented the early hypoxia-dependent upregulation of the redox-sensitive transcription factor Egr-1 and the procoagulant protein TF. These data provide the first evidence that EC-SOD activity is disrupted in chronic hypoxia, and increased EC-SOD activity can attenuate chronic hypoxic PH by limiting the hypoxic upregulation of redox-sensitive genes.

Redox regulation of mitochondrial biogenesis

The cell renews, adapts, or expands its mitochondrial population during episodes of cell damage or periods of intensified energy demand by the induction of mitochondrial biogenesis. This bigenomic program is modulated by redox-sensitive signals that respond to physiological nitric oxide (NO), carbon monoxide (CO), and mitochondrial reactive oxygen species production. This review summarizes our current ideas about the pathways involved in the activation of mitochondrial biogenesis by the physiological gases leading to changes in the redox milieu of the cell, with an emphasis on the responses to oxidative stress and inflammation. The cells energy supply is protected from conditions that damage mitochondria by an inducible transcriptional program of mitochondrial biogenesis that operates in large part through redox signals involving the nitric oxide synthase and the heme oxygenase-1/CO systems. These redox events stimulate the coordinated activities of several multifunctional transcription factors and coactivators also involved in the elimination of defective mitochondria and the expression of counterinflammatory and antioxidant genes, such as IL10 and SOD2, as part of a unified damage-control network. The redox-regulated mechanisms of mitochondrial biogenesis schematically outlined in the graphical abstract link mitochondrial quality control to an enhanced capacity to support the cells metabolic needs while improving its resistance to metabolic failure and avoidance of cell death during periods of oxidative stress.