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.

Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury.

Recognition and clearance of apoptotic cells by phagocytes (also known as efferocytosis), primarily mediated by macrophages, are essential to terminate lung inflammatory responses and promote tissue repair after injury. The Nrf2 transcription factor is crucial for cytoprotection and host defense. Previously, we showed sustained neutrophilic lung inflammation in Nrf2-deficient (Nrf2-/-) mice after hyperoxia-induced lung injury in vivo, but the mechanisms underlying this abnormal phenotype remain unclear. To examine whether Nrf2 regulates apoptotic neutrophil clearance, we used the alveolar macrophages (AMФs) and bone-marrow-derived macrophages (BMDMФs) of wild-type (WT) and Nrf2-/- mice. We found that the efferocytic ability of AMФ was impaired in hyperoxia-exposed mice's lungs, but the effect was more pronounced in Nrf2-/- mice. Importantly, AMФ-mediated efferocytosis remained impaired in Nrf2-/- mice recovering from injury but was restored to the basal state in the wild-type counterparts. Hyperoxia affected apoptotic neutrophil binding, not internalization, in both WT and Nrf2-/- BMDMФs, but the effect was more significant in the latter cells. Augmenting Nrf2 activity restored hyperoxia attenuated efferocytosis in WT, but not in Nrf2-/- macrophages. However, the loss of Nrf2 in neutrophils affected their uptake by WT macrophages. Collectively, these results demonstrate that Nrf2 is required for optimal macrophage-mediated efferocytosis and that activating Nrf2 may provide a physiological way to accelerate apoptotic cell clearance after oxidant injury.

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 Regulates Anti-Inflammatory A20 Deubiquitinase Induction by LPS in Macrophages in Contextual Manner.

The aberrant regulation of inflammatory gene transcription following oxidant and inflammatory stimuli can culminate in unchecked systemic inflammation leading to organ dysfunction. The Nrf2 transcription factor dampens cellular stress and controls inflammation by upregulating antioxidant gene expression and TNFα-induced Protein 3 (TNFAIP3, aka A20) deubiquitinase by controlling NF-kB signaling dampens tissue inflammation. Here, we report that Nrf2 is required for A20 induction by inflammatory stimuli LPS in monocyte/bone marrow derived macrophages (MDMΦs) but not in lung-macrophages (LDMΦs). LPS-induced A20 expression was significantly lower in Nrf2-/- MDMΦs and was not restored by antioxidant supplementation. Nrf2 deficiency markedly impaired LPS-stimulated A20 mRNA expression Nrf2-/- MDMΦs and ChIP assays showed Nrf2 enrichment at the promoter Nrf2-/- MDMΦs upon LPS stimulation, demonstrating that Nrf2 directly regulates A20 expression. Contrary to MDMΦs, LPS-stimulated A20 expression was not largely impaired in Nrf2-/- LDMΦs ex vivo and in vivo and ChIP assays showed lack of increased Nrf2 binding at the A20 promoter in LDMΦ following LPS treatment. Collectively, these results demonstrate a crucial role for Nrf2 in optimal A20 transcriptional induction in macrophages by endotoxin, and this regulation occurs in a contextual manner.

Recent advances in dead cell clearance during acute lung injury and repair.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical syndromes that cause significant mortality in clinical settings and morbidity among survivors accompanied by huge healthcare costs. Lung-resident cell dysfunction/death and neutrophil alveolitis accompanied by proteinous edema are the main pathological features of ALI/ARDS. While understanding of the mechanisms underlying ALI/ARDS pathogenesis is progressing and potential treatments such as statin therapy, nutritional strategies, and mesenchymal cell therapy are emerging, poor clinical outcomes in ALI/ARDS patients persist. Thus, a better understanding of lung-resident cell death and neutrophil alveolitis and their mitigation and clearance mechanisms may provide new therapeutic strategies to accelerate lung repair and improve outcomes in critically ill patients. Macrophages are required for normal tissue development and homeostasis as well as regulating tissue injury and repair through modulation of inflammation and other cellular processes. While macrophages mediate various functions, here we review recent dead cell clearance (efferocytosis) mechanisms mediated by these immune cells for maintaining tissue homeostasis after infectious and non-infectious lung injury.

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.

Isoquinoline Kelch-like ECH-Associated Protein 1-Nuclear Factor (Erythroid-Derived 2)-like 2 (KEAP1-NRF2) Inhibitors with High Metabolic Stability.

Pharmacological activation of NRF2 (nuclear factor erythroid 2-related factor 2) arises from blocking the interaction of NRF2 with its negative regulator, KEAP1 (Kelch-like ECH-associated protein 1). We previously reported an isoquinoline-based NRF2 activator, but this compound showed negative logD7.4 and a -2 charge at physiological pH, which may have limited its membrane permeability. In this work, we report potent, metabolically stable analogs that result from replacing a carboxymethyl group at the 4-position with a fluoroalkyl group.

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.

Replacement of a Naphthalene Scaffold in Kelch-like ECH-Associated Protein 1 (KEAP1)/Nuclear Factor (Erythroid-derived 2)-like 2 (NRF2) Inhibitors.

Activators of nuclear factor-erythroid 2-related factor 2 (NRF2) could lead to promising therapeutics for prevention and treatment of oxidative stress and inflammatory disorders. Ubiquitination and subsequent degradation of the transcription factor NRF2 is mediated by Kelch-like ECH-associated protein-1 (KEAP1). Inhibition of the KEAP1/NRF2 interaction with small molecules leads to NRF2 activation. Previously, we and others described naphthalene-based NRF2 activators, but the 1,4-diaminonaphthalene scaffold may not represent a drug-like scaffold. Paying particular attention to aqueous solubility, metabolic stability, potency, and mutagenicity, we modified a previously known, naphthalene-based nonelectrophilic NRF2 activator to give a series of non-naphthalene and heterocyclic scaffolds. We found that, compared to previously reported naphthalene-based compounds, a 1,4-isoquinoline scaffold provides a better mutagenic profile without sacrificing potency, stability, or solubility.

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.

Lysocardiolipin acyltransferase regulates TGF-ß mediated lung fibroblast differentiation.

Lysocardiolipin acyltransferase (LYCAT), a cardiolipin remodeling enzyme, plays a key role in mitochondrial function and vascular development. We previously reported that reduced LYCAT mRNA levels in peripheral blood mononuclear cells correlated with poor pulmonary function outcomes and decreased survival in IPF patients. Further LYCAT overexpression reduced lung fibrosis, and LYCAT knockdown accentuated experimental pulmonary fibrosis. NADPH Oxidase 4 (NOX4) expression and oxidative stress are known to contribute to lung fibroblast differentiation and progression of fibrosis. In this study, we investigated the role of LYCAT in TGF-ß mediated differentiation of human lung fibroblasts to myofibroblasts, and whether this occurred through mitochondrial superoxide and NOX4 mediated hydrogen peroxide (H2O2) generation. Our data indicated that LYCAT expression was up-regulated in primary lung fibroblasts isolated from IPF patients and bleomycin-challenged mice, compared to controls. In vitro, siRNA-mediated SMAD3 depletion inhibited TGF-ß stimulated LYCAT expression in human lung fibroblasts. ChIP immunoprecipitation assay revealed TGF-ß stimulated SMAD2/3 binding to the endogenous LYCAT promoter, and mutation of the SMAD2/3 binding sites (-179/-183 and -540/-544) reduced TGF-ß-stimulated LYCAT promoter activity. Overexpression of LYCAT attenuated TGF-ß-induced mitochondrial and intracellular oxidative stress, NOX4 expression and differentiation of human lung fibroblasts. Further, pretreatment with Mito-TEMPO, a mitochondrial superoxide scavenger, blocked TGF-ß-induced mitochondrial superoxide, NOX4 expression and differentiation of human lung fibroblasts. Treatment of human lung fibroblast with NOX1/NOX4 inhibitor, GKT137831, also attenuated TGF-ß induced fibroblast differentiation and mitochondrial oxidative stress. Collectively, these results suggest that LYCAT is a negative regulator of TGF-ß-induced lung fibroblast differentiation by modulation of mitochondrial superoxide and NOX4 dependent H2O2 generation, and this may serve as a potential therapeutic target for human lung fibrosis.

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.