T Cell Nrf2/Keap1 Gene Editing Using CRISPR/Cas9 and Experimental Kidney Ischemia-Reperfusion Injury.

Aims: T cells play pathophysiologic roles in kidney ischemia-reperfusion injury (IRI), and the nuclear factor erythroid 2-related factor 2/kelch-like ECH-associated protein 1 (Nrf2/Keap1) pathway regulates T cell responses. We hypothesized that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated Keap1-knockout (KO) augments Nrf2 antioxidant potential of CD4+ T cells, and that Keap1-KO CD4+ T cell immunotherapy protects from kidney IRI. Results: CD4+ T cell Keap1-KO resulted in significant increase of Nrf2 target genes NAD(P)H quinone dehydrogenase 1, heme oxygenase 1, glutamate-cysteine ligase catalytic subunit, and glutamate-cysteine ligase modifier subunit. Keap1-KO cells displayed no signs of exhaustion, and had significantly lower levels of interleukin 2 (IL2) and IL6 in normoxic conditions, but increased interferon gamma in hypoxic conditions in vitro. In vivo, adoptive transfer of Keap1-KO CD4+ T cells before IRI improved kidney function in T cell-deficient nu/nu mice compared with mice receiving unedited control CD4+ T cells. Keap1-KO CD4+ T cells isolated from recipient kidneys 24 h post IR were less activated compared with unedited CD4+ T cells, isolated from control kidneys. Innovation: Editing Nrf2/Keap1 pathway in murine T cells using CRISPR/Cas9 is an innovative and promising immunotherapy approach for kidney IRI and possibly other solid organ IRI. Conclusion: CRISPR/Cas9-mediated Keap1-KO increased Nrf2-regulated antioxidant gene expression in murine CD4+ T cells, modified responses to in vitro hypoxia and in vivo kidney IRI. Gene editing targeting the Nrf2/Keap1 pathway in T cells is a promising approach for immune-mediated kidney diseases.

Effect of time-restricted eating on sex hormone levels in premenopausal and postmenopausal females.

OBJECTIVE: Concerns have been raised regarding the impact of time-restricted eating (TRE) on sex hormones in females. This study examined how TRE affects sex steroids in premenopausal and postmenopausal females. METHODS: This is a secondary analysis of an 8-week TRE study (4- to 6-hour eating window) conducted in adults with obesity. Men and perimenopausal females were excluded. Females were classified into two groups based on menstrual status: premenopausal (n = 12) or postmenopausal (n = 11). RESULTS: After 8 weeks, body weight decreased in premenopausal females (-3% ± 2%) and postmenopausal females (-4% ± 2%) (main effect of time, p < 0.001), with no difference between groups (no group × time interaction). Circulating levels of testosterone, androstenedione, and sex hormone binding globulin (SHBG) did not change in either group (no group × time interaction). Dehydroepiandrosterone (DHEA) concentrations decreased (p < 0.05) in premenopausal (-14% ± 32%) and postmenopausal females (-13% ± 34%; main effect of time, p = 0.03), with no difference between groups. Estradiol, estrone, and progesterone were measured only in postmenopausal females, and they remained unchanged. CONCLUSIONS: In premenopausal females, androgens and SHBG remained unchanged during TRE, whereas DHEA decreased. In postmenopausal females, estrogens, progesterone, androgens, and SHBG did not change, but DHEA was reduced.

Role of Lysocardiolipin Acyltransferase in Cigarette Smoke-Induced Lung Epithelial Cell Mitochondrial ROS, Mitochondrial Dynamics, and Apoptosis.

Cigarette smoke is the primary cause of Chronic Obstructive Pulmonary Disorder (COPD). Cigarette smoke extract (CSE)-induced oxidative damage of the lungs results in mitochondrial dysfunction and apoptosis of epithelium. Mitochondrial cardiolipin (CL) present in the inner mitochondrial membrane plays an important role in mitochondrial function, wherein its fatty acid composition is regulated by lysocardiolipin acyltransferase (LYCAT). In this study, we investigated the role of LYCAT expression and activity in mitochondrial oxidative stress, mitochondrial dynamics, and lung epithelial cell apoptosis. LYCAT expression was increased in human lung specimens from smokers, and cigarette smoke-exposed-mouse lung tissues. Cigarette smoke extract (CSE) increased LYCAT mRNA levels and protein expression, modulated cardiolipin fatty acid composition, and enhanced mitochondrial fission in the bronchial epithelial cell line, BEAS-2B in vitro. Inhibition of LYCAT activity with a peptide mimetic, attenuated CSE-mediated mitochondrial (mt) reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis, while MitoTEMPO attenuated CSE-induced MitoROS, mitochondrial fission and apoptosis of BEAS-2B cells. Collectively, these findings suggest that increased LYCAT expression promotes MitoROS, mitochondrial dynamics and apoptosis of lung epithelial cells. Given the key role of LYCAT in mitochondrial cardiolipin remodeling and function, strategies aimed at inhibiting LYCAT activity and ROS may offer an innovative approach to minimize lung inflammation caused by cigarette smoke.

Inhibition of aberrant tissue remodelling by mesenchymal stromal cells singly coated with soft gels presenting defined chemomechanical cues.

The precise understanding and control of microenvironmental cues could be used to optimize the efficacy of cell therapeutics. Here, we show that mesenchymal stromal cells (MSCs) singly coated with a soft conformal gel presenting defined chemomechanical cues promote matrix remodelling by secreting soluble interstitial collagenases in response to the presence of tumour necrosis factor alpha (TNF-α). In mice with fibrotic lung injury, treatment with the coated MSCs maintained normal collagen levels, fibre density and microelasticity in lung tissue, and the continuous presentation of recombinant TNF-α in the gel facilitated the reversal of aberrant tissue remodelling by the cells when inflammation subsided in the host. Gel coatings with predefined chemomechanical cues could be used to tailor cells with specific mechanisms of action for desired therapeutic outcomes.

Nrf2 mediates hypoxia-inducible HIF1α activation in kidney tubular epithelial cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2) and hypoxia-inducible factor-1α (HIF1α) transcription factors protect against ischemic acute kidney injury (AKI) by upregulating metabolic and cytoprotective gene expression. In this study, we tested the hypothesis that Nrf2 is required for HIF1α-mediated hypoxic responses using Nrf2-sufficient (wild-type) and Nrf2-deficient (Nrf2-/-) primary murine renal/kidney tubular epithelial cells (RTECs) and human immortalized tubular epithelial cells (HK2 cells) with HIF1 inhibition and activation. The HIF1 pathway inhibitor digoxin blocked hypoxia-stimulated HIF1α activation and heme oxygenase (HMOX1) expression in HK2 cells. Hypoxia-mimicking cobalt (II) chloride-stimulated HMOX1 expression was significantly lower in Nrf2-/- RTECs than in wild-type counterparts. Similarly, hypoxia-stimulated HIF1α-dependent metabolic gene expression was markedly impaired in Nrf2-/- RTECs. Nrf2 deficiency impaired hypoxia-induced HIF1α stabilization independent of increased prolyl 4-hydroxylase gene expression. We found decreased HIF1α mRNA levels in Nrf2-/- RTECs under both normoxia and hypoxia-reoxygenation conditions. In silico analysis and chromatin immunoprecipitation assays demonstrated Nrf2 binding to the HIF1α promoter in normoxia, but its binding decreased in hypoxia-exposed HK2 cells. However, Nrf2 binding at the HIF1α promoter was enriched following reoxygenation, demonstrating that Nrf2 maintains constitutive HIF1α expression. Consistent with this result, we found decreased levels of Nrf2 in hypoxia and that were restored following reoxygenation. Inhibition of mitochondrial complex I prevented hypoxia-induced Nrf2 downregulation and also increased basal Nrf2 levels. These results demonstrate a crucial role for Nrf2 in optimal HIF1α activation in hypoxia and that mitochondrial signaling downregulates Nrf2 levels in hypoxia, whereas reoxygenation restores it. Nrf2 and HIF1α interact to provide optimal metabolic and cytoprotective responses in ischemic AKI.

Preconditioning the immature lung with enhanced Nrf2 activity protects against oxidant-induced hypoalveolarization in mice.

Bronchopulmonary dysplasia (BPD) is a chronic disease of preterm babies with poor clinical outcomes. Nrf2 transcription factor is crucial for cytoprotective response, whereas Keap1-an endogenous inhibitor of Nrf2 signaling-dampens these protective responses. Nrf2-sufficient (wild type) newborn mice exposed to hyperoxia develop hypoalveolarization, which phenocopies human BPD, and Nrf2 deficiency worsens it. In this study, we used PND1 pups bearing bearing hypomorphic Keap1 floxed alleles (Keap1f/f) with increased levels of Nrf2 to test the hypothesis that constitutive levels of Nrf2 in the premature lung are insufficient to mitigate hyperoxia-induced hypoalveolarization. Both wildtype and Keap1f/f pups at PND1 were exposed to hyperoxia for 72 h and then allowed to recover at room air for two weeks (at PND18), sacrificed, and lung hypoalveolarization and inflammation assessed. Hyperoxia-induced lung hypoalveolarization was remarkably lower in Keap1f/f pups than in wildtype counterparts (28.9% vs 2.4%, wildtype vs Keap1f/f). Likewise, Keap1f/f pups were protected against prolonged (96 h) hyperoxia-induced hypoalveolarization. However, there were no differences in hyperoxia-induced lung inflammatory response immediately after exposure or at PND18. Lack of hypoalveolarization in Keap1f/f pups was accompanied by increased levels of expression of antioxidant genes and GSH as assessed immediately following hyperoxia. Keap1 knockdown resulted in upregulation of lung cell proliferation postnatally but had opposing effects following hyperoxia. Collectively, our study demonstrates that augmenting endogenous Nrf2 activation by targeting Keap1 may provide a physiological way to prevent hypoalveolarization associated with prematurity.

Lysocardiolipin acyltransferase regulates NSCLC cell proliferation and migration by modulating mitochondrial dynamics.

Lysocardiolipin acyltransferase (LYCAT), a cardiolipin (CL)-remodeling enzyme, is crucial for maintaining normal mitochondrial function and vascular development. Despite the well-characterized role for LYCAT in the regulation of mitochondrial dynamics, its involvement in lung cancer, if any, remains incompletely understood. In this study, in silico analysis of TCGA lung cancer data sets revealed a significant increase in LYCAT expression, which was later corroborated in human lung cancer tissues and immortalized lung cancer cell lines via indirect immunofluorescence and immunoblotting, respectively. Stable knockdown of LYCAT in NSCLC cell lines not only reduced CL and increased monolyso-CL levels but also reduced in vivo tumor growth, as determined by xenograft studies in athymic nude mice. Furthermore, blocking LYCAT activity using a LYCAT mimetic peptide attenuated cell migration, suggesting a novel role for LYCAT activity in promoting NSCLC. Mechanistically, the pro-proliferative effects of LYCAT were mediated by an increase in mitochondrial fusion and a G1/S cell cycle transition, both of which are linked to increased cell proliferation. Taken together, these results demonstrate a novel role for LYCAT in promoting NSCLC and suggest that targeting LYCAT expression or activity in NSCLC may provide new avenues for the therapeutic treatment of lung cancer.

Keap1-Nrf2 System Plays an Important Role in Invariant Natural Killer T Cell Development and Homeostasis.

Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) proteins work in concert to regulate the levels of reactive oxygen species (ROS). The Keap1-Nrf2 antioxidant system also participates in T cell differentiation and inflammation, but its role in innate T cell development and functions remains unclear. We report that T cell-specific deletion of Keap1 results in defective development and reduced numbers of invariant natural killer T (NKT) cells in the thymus and the peripheral organs in a cell-intrinsic manner. The frequency of NKT2 and NKT17 cells increases while NKT1 decreases in these mice. Keap1-deficient NKT cells show increased rates of proliferation and apoptosis, as well as increased glucose uptake and mitochondrial function, but reduced ROS, CD122, and Bcl2 expression. In NKT cells deficient in Nrf2 and Keap1, all these phenotypic and metabolic defects are corrected. Thus, the Keap1-Nrf2 system contributes to NKT cell development and homeostasis by regulating cell metabolism.

PAR2-Mediated cAMP Generation Suppresses TRPV4-Dependent Ca2+ Signaling in Alveolar Macrophages to Resolve TLR4-Induced Inflammation.

Alveolar macrophages (AMs), upon sensing pathogens, trigger host defense by activating toll-like receptor 4 (TLR4), but the counterbalancing mechanisms that deactivate AM inflammatory signaling and prevent lethal edema, the hallmark of acute lung injury (ALI), remain unknown. Here, we demonstrate the essential role of AM protease-activating receptor 2 (PAR2) in rapidly suppressing inflammation to prevent long-lasting injury. We show that thrombin, released during TLR4-induced lung injury, directly activates PAR2 to generate cAMP, which abolishes Ca2+ entry through the TRPV4 channel. Deletion of PAR2 and thus the accompanying cAMP generation augments Ca2+ entry via TRPV4, causing sustained activation of the transcription factor NFAT to produce long-lasting TLR4-mediated inflammatory lung injury. Rescuing thrombin-sensitive PAR2 expression or blocking TRPV4 activity in PAR2-null AMs restores their capacity to resolve inflammation and reverse lung injury. Thus, activation of the thrombin-induced PAR2-cAMP cascade in AMs suppresses TLR4 inflammatory signaling to reinstate tissue integrity.

FOSL1 Promotes Kras-induced Lung Cancer through Amphiregulin and Cell Survival Gene Regulation.

The FOSL1/AP-1 transcription factor regulates gene expression, thereby controlling various pathophysiological processes. It is a major effector of RAS-ERK1/2 signaling and is activated in human lung epithelia by tumorigenic stimuli. Recent evidence shows an inverse correlation between FOSL1 expression and the survival of patients with lung cancer and adenocarcinomas; however, its role in lung tumorigenesis remains elusive. In this work, we sought to determine the role of FOSL1 in Kras-induced lung adenocarcinoma in vivo and its downstream effector mechanisms. We used mice expressing the Kras oncogene in the lung with concomitant Fosl1 deletion, Kras-activated murine alveolar epithelial cells (mAECs) with Fosl1 deletion, and KRAS mutant human lung adenocarcinoma (HLAC) cells with FOSL1 deficiency, and performed cell proliferation and gene expression analyses. Mutant Kras induced Fosl1 expression in vitro (mAECs) and in vivo (lung tissue), and mice with Fosl1 deletion showed reduced levels of mutant Kras-induced lung tumorigenesis and survived longer than Fosl1-sufficient mice. Studies with mutant Kras-activated mAECs and KRAS-mutant HLAC cells revealed that FOSL1 regulates mutant KRAS-induced gene expression, thereby controlling cell proliferation and survival. In contrast, FOSL1 depletion in non-KRAS-mutant HLAC cells and nonmalignant human lung epithelia had no effect. Our data support the notion that FOSL1-mediated expression of amphiregulin and apoptotic and antioxidative genes plays a role in regulating HLAC cell proliferation and survival. FOSL1 is a determinant of lung cancer in vivo and regulates HLAC cell proliferation and survival, largely in the context of KRAS mutations. Activation of FOSL1 in adenocarcinomas may be a prognostic marker and potential target for human lung cancer with KRAS mutations.

Expression profiling of genes regulated by sphingosine kinase1 signaling in a murine model of hyperoxia induced neonatal bronchopulmonary dysplasia.

BACKGROUND: Sphingosine- 1-Phosphate (S1P) is a bioactive lipid and an intracellular as well as an extracellular signaling molecule. S1P ligand specifically binds to five related cell surface G-protein-coupled receptors (S1P1-5). S1P levels are tightly regulated by its synthesis catalyzed by sphingosine kinases (SphKs) 1 & 2 and catabolism by S1P phosphatases, lipid phosphate phosphatases and S1P lyase. We previously reported that knock down of SphK1 (Sphk1 -/- ) in a neonatal mouse BPD model conferred significant protection against hyperoxia induced lung injury. To better understand the underlying molecular mechanisms, genome-wide gene expression profiling was performed on mouse lung tissue using Affymetrix MoGene 2.0 array. RESULTS: Two-way ANOVA analysis was performed and differentially expressed genes under hyperoxia were identified using Sphk1 -/- mice and their wild type (WT) equivalents. Pathway (PW) enrichment analyses identified several signaling pathways that are likely to play a key role in hyperoxia induced lung injury in the neonates. These included signaling pathways that were anticipated such as those involved in lipid signaling, cell cycle regulation, DNA damage/apoptosis, inflammation/immune response, and cell adhesion/extracellular matrix (ECM) remodeling. We noted hyperoxia induced downregulation of the expression of genes related to mitotic spindle formation in the WT which was not observed in Sphk1 -/- neonates. Our data clearly suggests a role for SphK1 in neonatal hyperoxic lung injury through elevated inflammation and apoptosis in lung tissue. Further, validation by RT-PCR on 24 differentially expressed genes showed 83% concordance both in terms of fold change and vectorial changes. Our findings are in agreement with previously reported human BPD microarray data and completely support our published in vivo findings. In addition, the data also revealed a significant role for additional unanticipitated signaling pathways involving Wnt and GADD45. CONCLUSION: Using SphK1 knockout mice and differential gene expression analysis, we have shown here that S1P/SphK1 signaling plays a key role in promoting hyperoxia induced DNA damage, inflammation, apoptosis and ECM remodeling in neonatal lungs. It also appears to suppress pro-survival cellular responses involved in normal lung development. We therefore propose SphK1 as a therapeutic target for the development drugs to combat BPD.

K-homology splicing regulatory protein (KSRP) promotes post-transcriptional destabilization of Spry4 transcripts in non-small cell lung cancer.

AU-rich element-binding proteins (ARE-BPs) offer post-transcriptional regulation of gene expression via physical interaction and recruitment of RNA decay machinery to the AU-rich elements within the 3'-UTR of the target transcripts. However, the role of ARE-BPs in lung cancer remains poorly understood. In this study, we have identified that K-homology splicing regulatory protein (KSRP), an ARE-BP, is robustly up-regulated in human lung cancer. Importantly, Kaplan-Meier survival analysis indicated that elevated KSRP expression was correlated with poor overall survival of lung cancer patients. Furthermore, cigarette smoke, a leading risk factor for lung cancer, was also identified to be an important contributor to increased KSRP expression. Remarkably, silencing of KSRP decreased cell proliferation, reversed anchorage-independent growth, and reduced migration/invasion, suggesting an oncogenic role for KSRP in lung cancer. Finally, we provide mechanistic evidence that KSRP promotes the down-regulation of Spry4 by a previously unidentified mechanism, i.e. post-transcriptional mRNA regulation.

Nrf2-AKT interactions regulate heme oxygenase 1 expression in kidney epithelia during hypoxia and hypoxia-reoxygenation.

Ischemia-reperfusion (IR)-induced kidney injury is a major clinical problem, but its underlying mechanisms remain unclear. The transcription factor known as nuclear factor, erythroid 2-like 2 (NFE2L2 or Nrf2) is crucial for protection against oxidative stress generated by pro-oxidant insults. We have previously shown that Nrf2 deficiency enhances susceptibility to IR-induced kidney injury in mice and that its upregulation is protective. Here, we examined Nrf2 target antioxidant gene expression and the mechanisms of its activation in both human and murine kidney epithelia following acute (2 h) and chronic (12 h) hypoxia and reoxygenation conditions. We found that acute hypoxia modestly stimulates and chronic hypoxia strongly stimulates Nrf2 putative target HMOX1 expression, but not that of other antioxidant genes. Inhibition of AKT1/2 or ERK1/2 signaling blocked this induction; AKT1/2 but not ERK1/2 inhibition affected Nrf2 levels in basal and acute hypoxia-reoxygenation states. Unexpectedly, chromatin immunoprecipitation assays revealed reduced levels of Nrf2 binding at the distal AB1 and SX2 enhancers and proximal promoter of HMOX1 in acute hypoxia, accompanied by diminished levels of nuclear Nrf2. In contrast, Nrf2 binding at the AB1 and SX2 enhancers significantly but differentially increased during chronic hypoxia and reoxygenation, with reaccumulation of nuclear Nrf2 levels. Small interfering-RNA-mediated Nrf2 depletion attenuated acute and chronic hypoxia-inducible HMOX1 expression, and primary Nrf2-null kidney epithelia showed reduced levels of HMOX1 induction in response to both acute and chronic hypoxia. Collectively, our data demonstrate that Nrf2 upregulates HMOX1 expression in kidney epithelia through a distinct mechanism during acute and chronic hypoxia reoxygenation, and that both AKT1/2 and ERK1/2 signaling are required for this process.

c-Jun Is Required for Nuclear Factor-κB-Dependent, LPS-Stimulated Fos-Related Antigen-1 Transcription in Alveolar Macrophages.

Previously, we have reported that Fos-related antigen-1 (Fra-1) transcription factor promotes LPS-induced acute lung injury and mortality, and that LPS-induced Fra-1 expression in the lung occurs predominantly in alveolar macrophages. Nuclear factor-κB (NF-κB) and c-Jun transcription factors play key roles in modulating inflammatory and immune responses induced by infectious and non-infectious insults. Here, we report that NF-κB and c-Jun coregulate Fra-1 induction by LPS in alveolar macrophages and that this regulation occurs through both the NF-κB and the extracellular signal-regulated protein kinase (ERK) signaling pathways. Transient transfections with Fra-1 promoter-reporter constructs and inhibitor studies revealed that the transcriptional activation of Fra-1 by LPS in alveolar macrophages is mediated by NF-κB and ERK1/2 signaling. Importantly, chromatin immunoprecipitation assays revealed the recruitment of c-Jun and NF-κB to the endogenous Fra-1 promoter after LPS stimulation. We found that inhibition of ERK1/2 signaling reduced LPS-stimulated c-Jun and NF-κB recruitment to the promoter. Likewise, NF-κB inhibitor blocked LPS-induced NF-κB and c-Jun binding to the promoter. ERK1/2 inhibition had no effect on c-Jun activation but suppressed LPS-stimulated NF-κB phosphorylation. Finally, functional assays showed reduced levels of LPS-stimulated NF-κB regulated proinflammatory IL-1ß and macrophage inflammatory protein-1α expression and increased antiinflammatory IL-10 expression in lung alveolar macrophages of Fra-1-null mice in vivo. Thus, our studies indicate that NF-κB and c-Jun coregulate LPS-induced Fra-1 transcription and that Fra-1 selectively modulates LPS-stimulated inflammatory cytokine expression in lung alveolar macrophages during inflammatory lung injury.

ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells.

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression.

Sirtuin 1 Promotes Hyperoxia-Induced Lung Epithelial Cell Death Independent of NF-E2-Related Factor 2 Activation.

Lung epithelial cell damage accompanied by death is a cardinal feature of toxicant- and prooxidant-induced acute lung injury. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NEF2L2 or NRF2) activates several antioxidant enzymes (AOEs) and prosurvival genes in response to oxidant stress, and its deficiency enhances susceptibility to hyperoxic lung injury and other oxidant-induced lung pathologies. Sirtuin 1 (SIRT1) regulates cell growth and survival in response to both physiological and pathological stresses by selectively deacetylating multiple proteins required for chromatin remodeling and transcription; therefore, we sought to examine potential SIRT1-NRF2 cross-talk in the regulation of AOE expression during hyperoxia-induced lung epithelial cell death. Unexpectedly, pharmacological inhibition or small interfering RNA-mediated depletion of SIRT1 caused a reduction in cell death, accompanied by reduced levels of NRF2-dependent AOE expression in chronic hyperoxia. NRF2 acetylation was markedly and transiently higher in cells exposed to acute (6 h) hyperoxia. Sirtinol blocked this acute effect, but NRF2 acetylation was low or undetectable in cells exposed to chronic hyperoxia (24-36 h) both with and without sirtinol. SIRT1 activation by resveratrol augmented hyperoxia-induced death in cells with NRF2 deficiency. SIRT1 inhibition or depletion led to a reduced activation of the cell-death executioner caspase 3, whereas caspase inhibition prevented death. Consistent with these results, sirtinol attenuated hyperoxia-induced lung alveolar permeability and toxicity in vivo. Collectively, these results reveal that, in chronic hyperoxia, SIRT1 promotes hyperoxia-induced lung epithelial cell damage and death by altering pro- and antiapoptotic balance, not by dampening optimal NRF2-dependent AOE expression.

T Lymphocyte-Specific Activation of Nrf2 Protects from AKI.

T lymphocytes are established mediators of ischemia reperfusion (IR)-induced AKI, but traditional immune principles do not explain their mechanism of early action in the absence of alloantigen. Nrf2 is a transcription factor that is crucial for cytoprotective gene expression and is generally thought to have a key role in dampening IR-induced AKI through protective effects on epithelial cells. We proposed an alternative hypothesis that augmentation of Nrf2 in T cells is essential to mitigate oxidative stress during IR-induced AKI. We therefore generated mice with genetically amplified levels of Nrf2 specifically in T cells and examined the effect on antioxidant gene expression, T cell activation, cytokine production, and IR-induced AKI. T cell-specific augmentation of Nrf2 significantly increased baseline antioxidant gene expression. These mice had a high frequency of intrarenal CD25(+)Foxp3(+) regulatory T cells and decreased frequencies of CD11b(+)CD11c(+) and F4/80(+) cells. Intracellular levels of TNF-α, IFN-γ, and IL-17 were significantly lower in CD4(+) T cells with high Nrf2 expression. Mice with increased T cell expression of Nrf2 were significantly protected from functional and histologic consequences of AKI. Furthermore, adoptive transfer of high-Nrf2 T cells protected wild-type mice from IR injury and significantly improved their survival. These data demonstrate that T cell-specific activation of Nrf2 protects from IR-induced AKI, revealing a novel mechanism of tissue protection during acute injury responses.

Visualization of Fra-1/AP-1 activation during LPS-induced inflammatory lung injury using fluorescence optical imaging.

Inappropriate lung inflammatory response following oxidant and toxicant exposure can lead to abnormal repair and disease pathogenesis, including fibrosis. Thus early detection of molecular and cellular processes and mediators promoting lung inflammation is necessary to develop better strategies for therapeutic intervention and disease management. Previously, we have shown that transcription factor Fra-1/AP-1 plays key roles in lung inflammatory response, as Fra-1-null mice are less susceptible than wild-type mice to LPS-induced lung injury and mortality. Herein, we developed a transgenic reporter mouse model expressing tdTomato under the control of FRA-1 (human) promoter (referred to as FRA-1(TdTg) mice) to monitor its activation during inflammatory lung injury using fluorescence protein-based optical imaging and molecular analysis in vivo and ex vivo. A higher red fluorescent signal was observed in the lungs of LPS-treated FRA-1(TdTg) mice compared with vehicle controls, and Western blot and qRT-PCR analyses revealed a significant correlation with the FRA-1-tdTomato reporter expression. Immunocolocalization demonstrated expression of FRA-1-tdTomato largely in lung alveolar macrophages and to some extent in epithelial cells. Moreover, we validated these results with a second reporter mouse model that expressed green fluorescent protein upon activation of endogenous Fra-1 promoter. Additionally, we demonstrated increased expression of FRA-1 in alveolar macrophages in human lung instilled with Escherichia coli ex vivo. Collectively, our data obtained from two independent reporter mouse models and from human samples underscore the significance of Fra-1 activation in alveolar macrophages during inflammatory lung injury and may aid in developing strategies to target this transcription factor in lung injury and repair.

PI3K-AKT Signaling via Nrf2 Protects against Hyperoxia-Induced Acute Lung Injury, but Promotes Inflammation Post-Injury Independent of Nrf2 in Mice.

Lung epithelial and endothelial cell death accompanied by inflammation contributes to hyperoxia-induced acute lung injury (ALI). Impaired resolution of ALI can promote and/or perpetuate lung pathogenesis, including fibrosis. Previously, we have shown that the transcription factor Nrf2 induces cytoprotective gene expression and confers protection against hyperoxic lung injury, and that Nrf2-mediated signaling is also crucial for the restoration of lung homeostasis post-injury. Although we have reported that PI3K/AKT signaling is required for Nrf2 activation in lung epithelial cells, significance of the PI3K/AKT-Nrf2 crosstalk during hyperoxic lung injury and repair remains unclear. Thus, we evaluated this aspect using Nrf2 knockout (Nrf2(-/-)) and wild-type (Nrf2(+/+)) mouse models. Here, we show that pharmacologic inhibition of PI3K/AKT signaling increased lung inflammation and alveolar permeability in Nrf2(+/+) mice, accompanied by decreased expression of Nrf2-target genes such as Nqo1 and Hmox1. PI3K/AKT inhibition dampened hyperoxia-stimulated Nqo1 and Hmox1 expression in lung epithelial cells and alveolar macrophages. Contrasting with its protective effects, PI3K/AKT inhibition suppressed lung inflammation in Nrf2(+/+) mice during post-injury. In Nrf2(-/-) mice exposed to room-air, PI3K/AKT inhibition caused lung injury and inflammation, but it did not exaggerate hyperoxia-induced ALI. During post-injury, PI3K/AKT inhibition did not augment, but rather attenuated, lung inflammation in Nrf2(-/-) mice. These results suggest that PI3K/AKT-Nrf2 signaling is required to dampen hyperoxia-induced lung injury and inflammation. Paradoxically, the PI3K/AKT pathway promotes lung inflammation, independent of Nrf2, during post-injury.