Grim-19 deficiency promotes decidual macrophage autophagy in recurrent spontaneous abortion.

Dysregulation of decidual macrophages leads to the occurrence of recurrent spontaneous abortion (RSA). However, the role of macrophages in RSA occurrence remains unclear. In this study, we found that the expression of Grim-19 was decreased, and the expression of autophagy related proteins Beclin1, LC3B II/I and BNIP3 was markedly upregulated in decidual macrophages of RSA patients compared with the normal pregnancy group. Furthermore, we demonstrated that downregulation of GRIM-19 increased the expression of autophagy related proteins Beclin1, LC3B II/I, BNIP3 and the proinflammatory cytokines IL1B, IL6 and TNFa in uterine mononuclear cells of GRIM-19+/- mice. The proportion of CD45+CD11b+F4/80+LC3B+ cells in GRIM-19+/- mouse uteri was significantly higher than that in WT mouse uteri. In addition, we confirmed that inhibition of Grim-19 by siRNA enhanced the expression of autophagy related proteins in RAW264.7 cells and THP-1 cells. More importantly, downregulation of Grim-19 in RAW264.7 cells promoted the release of proinflammatory cytokines and promoted phagocytic activity, which could be reversed by autophagy blockade. For THP-1-derived macrophages, the results of RNA-seq suggested that Grim-19 mainly modulates immune and inflammatory-related pathways, leading to cytokine production, and thus contributing to inflammation. Therefore, our data reveal that Grim-19 deficiency influences macrophage function, characterized by enhanced proinflammatory cytokines and phagocytic activity, and this might be regulated by autophagy. This may represent a novel mechanism for the occurrence of RSA.

Mitochondrial GRIM-19 deficiency facilitates gastric cancer metastasis through oncogenic ROS-NRF2-HO-1 axis via a NRF2-HO-1 loop.

BACKGROUND: NRF2, a prime target of cellular defense against oxidative stress, has shown a dark side profile in cancer progression. GRIM-19, an essential subunit of the mitochondrial MRC complex I, was recently identified as a suppressive role in tumorigenesis of human gastric cancer (GC). However, little information is available on the role of GRIM-19 and its cross-talk with NRF2 in GC metastasis. METHODS: Online GC database was used to investigate DNA methylation and survival outcomes of GRIM-19. CRISPR/Cas9 lentivirus-mediated gene editing, metastasis mice models and pharmacological intervention were applied to investigate the role of GRIM-19 deficiency in GC metastasis. Quantitative RT-PCR, FACS, Western blot, IHC, IF and reporter gene assay were performed to explore underlying mechanisms. RESULTS: Low GRIM-19 is correlated with poor survival outcome of GC patients while DNA hypermethylation is associated with GRIM-19 downregulation. GRIM-19 deficiency facilitates GC metastasis and triggers aberrant oxidative stress as well as ROS-dependent NRF2-HO-1 activation. Experimental interventions of specific ROS, NRF2 or HO-1 inhibitor significantly abrogate GRIM-19 deficiency-driven GC metastasis in vitro and in vivo. Moreover, HO-1 inhibition not only reverses GRIM-19 deficiency-driven NRF2 activation, but also feedback blocks NRF2 activator-induced NRF2 signaling, resulting in decreased metastasis-associated genes. CONCLUSIONS: Our data suggest that GRIM-19 deficiency accelerates GC metastasis through the oncogenic ROS-NRF2-HO-1 axis via a positive-feedback NRF2-HO-1 loop. Therefore, this study not only offers novel insights into the role of oncogenic NRF2 in tumor progression, but also provides new strategies to alleviate the dark side of NRF2 by targeting HO-1.

Central Signaling Elements of Intercellular Reactive Oxygen/Nitrogen Species-dependent Induction of Apoptosis in Malignant Cells.

Intercellular reactive oxygen/reactive nitrogen species-(ROS/RNS)-dependent induction of apoptosis in malignant cells is discussed as a potential control step during oncogenesis. In previous studies, the mechanism of intercellular apoptosis-inducing signaling was mainly established through the use of specific inhibitors and scavengers. Here, a detailed analysis was carried out based on small interfering ribonucleic acid (siRNA)-mediated knockdown of central players of intercellular ROS/RNS signaling and of the mitochondrial and the FAS receptor-dependent pathway of apoptosis. The data show that transforming growth factor ß1, transforming growth factor ß receptor, NADPH oxidase-1 (NOX1), NOX1 organizer, and NOX1 activator control the HOCl and the NO/peroxynitrite signaling pathways. Dual oxidase-1 (DUOX1) is specifically involved in HOCl signaling, and NO synthase in NO/peroxynitrite signaling. Both pathways utilize intracellular signal transduction through protein kinase C zeta, sphingomyelinase and central elements of the mitochondrial pathway of apoptosis, whereas the FAS receptor and FAS ligand do not seem to play a role.

The Cytosolic NADPH Oxidase Subunit NoxO1 Promotes an Endothelial Stalk Cell Phenotype.

OBJECTIVE: Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. APPROACH AND RESULTS: A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1(-/-) when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1(-/-) lung endothelial cells (LECs) and a more tip-cell-like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1(-/-) LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1(-/-) LECs when compared with wild-type LECs. CONCLUSIONS: NoxO1 stimulates α-secretase activity probably through reactive oxygen species-mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.

Differential effects of NOX4 and NOX1 on immune cell-mediated inflammation in the aortic sinus of diabetic ApoE-/- mice.

Oxidative stress and inflammation are central mediators of atherosclerosis particularly in the context of diabetes. The potential interactions between the major producers of vascular reactive oxygen species (ROS), NADPH oxidase (NOX) enzymes and immune-inflammatory processes remain to be fully elucidated. In the present study we investigated the roles of the NADPH oxidase subunit isoforms, NOX4 and NOX1, in immune cell activation and recruitment to the aortic sinus atherosclerotic plaque in diabetic ApoE(-/-) mice. Plaque area analysis showed that NOX4- and NOX1-derived ROS contribute to atherosclerosis in the aortic sinus following 10 weeks of diabetes. Immunohistochemical staining of the plaques revealed that NOX4-derived ROS regulate T-cell recruitment. In addition, NOX4-deficient mice showed a reduction in activated CD4(+) T-cells in the draining lymph nodes of the aortic sinus coupled with reduced pro-inflammatory gene expression in the aortic sinus. Conversely, NOX1-derived ROS appeared to play a more important role in macrophage accumulation. These findings demonstrate distinct roles for NOX4 and NOX1 in immune-inflammatory responses that drive atherosclerosis in the aortic sinus of diabetic mice.

Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis.

OBJECTIVE: Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. APPROACH AND RESULTS: NOX1(-/Y) platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2(-/-) platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1(-/Y) platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2(-/-) platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2(-/-) mice. Wild-type thrombocytopenic mice injected with NOX2(-/-) platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. CONCLUSIONS: NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.

Unchanged NADPH Oxidase Activity in Nox1-Nox2-Nox4 Triple Knockout Mice: What Do NADPH-Stimulated Chemiluminescence Assays Really Detect?

NADPH oxidases of the Nox family are considered important sources of cellular reactive oxygen species (ROS) production. This conclusion is, in part, based on the ability of NADPH to elicit a chemiluminescence signal in tissue/cell homogenates or membrane preparations in the presence of enhancers such as lucigenin, luminol, or L012. However, the ability of these particular assays to specifically detect Nox activity and Nox-derived ROS has not been proven. In this study, we demonstrate that combined knockout of the three main Nox enzymes of the mouse (Nox1-Nox2-Nox4 triple knockout) had no impact on NADPH-stimulated chemiluminescence signals in the aorta, heart, and kidney homogenates. In the NADPH-stimulated membrane assays, no effect of in vivo angiotensin II pretreatment or deletion of Nox enzymes was observed. In in vitro studies in HEK293 cells, the overexpression of Nox5 or Nox4 markedly increased ROS production in intact cells, whereas overexpression of Nox5 or Nox4 had no influence on the signal in membrane assays. In contrast, overexpression of nitric oxide synthase or cytochrome P450 enzymes resulted in an increased chemiluminescence signal in isolated membranes. On the basis of these observations, we propose the hypothesis that NADPH-stimulated chemiluminescence-based membrane assays, as currently used, do not reflect Nox activity.

Role of neuronal NADPH oxidase 1 in the peri-infarct regions after stroke.

The molecular mechanism underlying the selective vulnerability of neurons to oxidative damage caused by ischemia-reperfusion (I/R) injury remains unknown. We sought to determine the role of NADPH oxidase 1 (Nox1) in cerebral I/R-induced brain injury and survival of newborn cells in the ischemic injured region. Male Wistar rats were subjected to 90 min middle cerebral artery occlusion (MCAO) followed by reperfusion. After reperfusion, infarction size, level of superoxide and 8-hydroxy-2'-deoxyguanosine (8-oxo-2dG), and Nox1 immunoreactivity were determined. RNAi-mediated knockdown of Nox1 was used to investigate the role of Nox1 in I/R-induced oxidative damage, neuronal death, motor function recovery, and ischemic neurogenesis. After I/R, Nox1 expression and 8-oxo-2dG immunoreactivity was increased in cortical neurons of the peri-infarct regions. Both infarction size and neuronal death in I/R injury were significantly reduced by adeno-associated virus (AAV)-mediated transduction of Nox1 short hairpin RNA (shRNA). AAV-mediated Nox1 knockdown enhanced functional recovery after MCAO. The level of survival and differentiation of newborn cells in the peri-infarct regions were increased by Nox1 inhibition. Our data suggest that Nox-1 may be responsible for oxidative damage to DNA, subsequent cortical neuronal degeneration, functional recovery, and regulation of ischemic neurogenesis in the peri-infarct regions after stroke.

NOX1 is responsible for cell death through STAT3 activation in hyperoxia and is associated with the pathogenesis of acute respiratory distress syndrome.

Reactive oxygen species (ROS) contribute to alveolar cell death in acute respiratory distress syndrome (ARDS) and we previously demonstrated that NOX1-derived ROS contributed to hyperoxia-induced alveolar cell death in mice. The study investigates whether NOX1 expression is modulated in epithelial cells concomitantly to cell death and associated to STAT3 signaling in the exudative phase of ARDS. In addition, the role of STAT3 activation in NOX1-dependent epithelial cell death was confirmed by using a lung epithelial cell line and in mice exposed to hyperoxia. NOX1 expression, cell death and STAT3 staining were evaluated in the lungs of control and ARDS patients by immunohistochemistry. In parallel, a stable NOX1-silenced murine epithelial cell line (MLE12) and NOX1-deficient mice were used to characterize signalling pathways. In the present study, we show that NOX1 is detected in alveolar epithelial cells of ARDS patients in the exudative stage. In addition, increased alveolar epithelial cell death and phosphorylated STAT3 are observed in ARDS patients and associated with NOX1 expression. Phosphorylated STAT3 is also correlated with TUNEL staining. We also confirmed that NOX1-dependent STAT3 activation participates to alveolar epithelial cell death. Silencing and acute inhibition of NOX1 in MLE12 led to decreased cell death and cleaved-caspase 3 induced by hyperoxia. Additionally, hyperoxia-induced STAT3 phosphorylation is dependent on NOX1 expression and associated with cell death in MLE12 and mice. This study demonstrates that NOX1 is involved in human ARDS pathophysiology and is responsible for the damage occurring in alveolar epithelial cells at least in part via STAT3 signalling pathways.

Interaction of NADPH oxidase 1 with Toll-like receptor 2 induces migration of smooth muscle cells.

AIMS: NADPH oxidase (Nox) isozymes that generate intracellular reactive oxygen species (ROS) and Toll-like receptor 2 (TLR2), an inflammatory mediator, are both involved in the development of atherosclerotic lesions. To identify the molecular connection between TLR2 and Nox isozymes in vascular remodelling, we analysed generation of ROS and pro-inflammatory cytokines in aortic smooth muscle cells from Nox1-deficient mice in response to the synthetic triacylated lipoprotein Pam3CSK, a TLR2 agonist. METHODS AND RESULTS: We showed that TLR2 signalling stimulates progression of the pro-inflammatory phenotype in mouse aortic smooth muscle cells (MASMCs) through activation of Nox1. We demonstrated the interaction of TLR2 with Nox1 using yeast two-hybrid and co-immunoprecipitation assays. MASMCs from Nox1-deficient mice failed to generate of ROS in response to Pam3CSK4, indicating that Nox1 is essential for TLR2-dependent production of ROS. We also found that Pam3CSK4 stimulated migration of MASMCs from wild-type mice in a Transwell system, but MASMCs from Nox1-deficient mice failed to show this response. Wild-type MASMCs produced matrix metalloprotease 2 in response to Pam3CSK4, whereas Nox1-deficient MASMCs failed to generate this protease. Moreover, stimulation of MASMCs with Pam3CSK4 resulted in increased expression of the pro-inflammatory cytokine macrophage inflammatory protein 2 in a Nox1-dependent manner, leading to enhanced monocyte-endothelial cell adhesion and trans-endothelial migration of U937 cells. CONCLUSION: These data suggest that Nox1 plays an important role in TLR2-mediated intracellular H2O2 generation, activation of matrix metalloprotease 2, and secretion of pro-inflammatory cytokines, which in turn stimulate MASMC migration and vascular remodelling.

NADH oxidase functions as an adhesin in Streptococcus pneumoniae and elicits a protective immune response in mice.

The initial event in disease caused by S. pneumoniae is adhesion of the bacterium to respiratory epithelial cells, mediated by surface expressed molecules including cell-wall proteins. NADH oxidase (NOX), which reduces free oxygen to water in the cytoplasm, was identified in a non-lectin enriched pneumococcal cell-wall fraction. Recombinant NOX (rNOX) was screened with sera obtained longitudinally from children and demonstrated age-dependent immunogenicity. NOX ablation in S. pneumoniae significantly reduced bacterial adhesion to A549 epithelial cells in vitro and their virulence in the intranasal or intraperitoneal challenge models in mice, compared to the parental strain. Supplementation of Δnox WU2 with the nox gene restored its virulence. Saturation of A549 target cells with rNOX or neutralization of cell-wall residing NOX using anti-rNOX antiserum decreased adhesion to A549 cells. rNOX-binding phages inhibited bacterial adhesion. Moreover, peptides derived from the human proteins contactin 4, chondroitin 4 sulfotraferase and laminin5, homologous to the insert peptides in the neutralizing phages, inhibited bacterial adhesion to the A549 cells. Furthermore, rNOX immunization of mice elicited a protective immune response to intranasal or intraperitoneal S. pneumoniae challenge, whereas pneumococcal virulence was neutralized by anti-rNOX antiserum prior to intraperitoneal challenge. Our results suggest that in addition to its enzymatic activity, NOX contributes to S. pneumoniae virulence as a putative adhesin and thus peptides derived from its target molecules may be considered for the treatment of pneumococcal infections. Finally, rNOX elicited a protective immune response in both aerobic and anaerobic environments, which renders NOX a candidate for future pneumococcal vaccine.

Nox4 is a protective reactive oxygen species generating vascular NADPH oxidase.

RATIONALE: The function of Nox4, a source of vascular H(2)O(2), is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction. OBJECTIVE: We determined the function of Nox4 in situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4(-/-) mice. METHODS AND RESULTS: Nox4 was highly expressed in the endothelium and contributed to H(2)O(2) formation. Nox4(-/-) mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4(-/-) lung endothelial cells (LECs) was attenuated and restored by low concentrations of H(2)O(2,) whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs. CONCLUSION: Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.

Down-regulation of tumor-associated NADH oxidase, tNOX (ENOX2), enhances capsaicin-induced inhibition of gastric cancer cell growth.

Gastric cancer is a common human malignancy and a major contributor to cancer-related deaths worldwide. Unfortunately, the prognosis of most gastric cancer patients is poor because they are generally diagnosed at a late stage after the cancer has already metastasized. Most current research, therefore, emphasizes selective targeting of cancer cells by apoptosis-inducing agents. One such therapeutic agent is capsaicin, a component of chili peppers that has been shown to possess anti-growth activity against various cancer cell lines. Here, we examined the effect of capsaicin on SNU-1 and TMC-1 gastric cancer cells and found differing outcomes between the two cell lines. Our results show that capsaicin induced significant cytotoxicity with increases in oxidative stress, PARP cleavage, and apoptosis in sensitive SNU-1 cells. In contrast, TMC-1 cells were much less sensitive to capsaicin, exhibiting low cytotoxicity and very little apoptosis in response to capsaicin treatment. Capsaicin-induced apoptosis in SNU-1 cells was associated with down-regulation of tumor-associated NADH oxidase (tNOX) mRNA and protein. On the contrary, tNOX expression was scarcely affected by capsaicin in TMC-1 cells. We further showed that tNOX-knockdown sensitized TMC-1 cells to capsaicin-induced apoptosis and G1 phase accumulation, and led to decreased cell growth, demonstrating that tNOX is essential for cancer cell growth. Collectively, these results indicate that capsaicin induces divergent effects of the growth of gastric cancer cells that parallel its effects on tNOX expression, and demonstrate that forced tNOX down-regulation restored capsaicin-induced growth inhibition in TMC-1 cells.

Superoxide production by macrophages and T cells is critical for the induction of autoreactivity and type 1 diabetes.

OBJECTIVE: The role of reactive oxygen species (ROS) and their dissipation in type 1 diabetes pathogenesis have garnered considerable controversy. Our recent work has demonstrated the importance of NADPH oxidase (NOX) activity for type 1 diabetes development and modulating T-cell autoreactivity. We previously linked decreased monocyte ROS with diabetes resistance in the alloxan-resistant mouse, and NOD-Ncf1(m1J) mice with a genetic ablation of NOX activity had reduced and delayed type 1 diabetes compared with NOD mice. RESEARCH DESIGN AND METHODS: To determine the required cellular sources of ROS that are necessary for type 1 diabetes initiation, we used antibody depletion and adoptive transfer experiments into NOD and NOD-Scid females, respectively. After receiving treatment, female mice were monitored for hyperglycemia and overt diabetes. RESULTS: Depletion of macrophages and neutrophils fully protected NOD mice from type 1 diabetes. However, elimination of neutrophils alone showed no significant reduction or delay. Type 1 diabetes induction in NOD-Scid mice by adoptive transfer with NOD-Ncf1(m1J) splenocytes was significantly delayed compared with NOD splenocytes, suggesting macrophage ROS and modulation of effector responses are critical for diabetes. The adaptive immune response was also altered by the absence of NOX activity, as purified T cells from NOD-Ncf1(m1J) mice exhibited delayed transfer kinetics. Cotransfer experiments demonstrated the defect was intrinsic to NOX-deficient CD8(+) T cells. After stimulation, cytotoxic T cells exhibited decreased effector function in the absence of superoxide production. CONCLUSIONS: These data demonstrate that the impaired autoreactive response of NOX-deficient NOD-Ncf1(m1J) immune system results from an alteration in the antigen-presenting cell-T-cell axis rather than failure of neutrophils to act as effector cells and that ROS signaling is important for the initiation of ß-cell-directed autoimmunity by T cells.

GRIM-19 and p16(INK4a) synergistically regulate cell cycle progression and E2F1-responsive gene expression.

GRIM-19 (Gene associated with Retinoid-IFN-induced Mortality-19) was originally isolated as a growth suppressor in a genome-wide knockdown screen with antisense libraries. Like classical tumor suppressors, mutations, and/or loss of GRIM-19 expression occur in primary human tumors; and it is inactivated by viral gene products. Our search for potential GRIM-19-binding proteins, using mass spectrometry, that permit its antitumor actions led to the inhibitor of cyclin-dependent kinase 4, CDKN2A. The GRIM-19/CDKN2A synergistically suppressed cell cycle progression via inhibiting E2F1-driven gene expression. The N terminus of GRIM-19 and the fourth ankyrin repeat of CDKN2A are crucial for their interaction. The biological relevance of these interactions is underscored by observations that GRIM-19 promotes the inhibitory effect of CDKN2A on CDK4; and mutations from primary tumors disrupt its ability to interact with GRIM-19 and suppress E2F1-driven gene expression.

Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death.

Combination of retinoic acids (RAs) and interferons (IFNs) has synergistic apoptotic effects and is used in cancer treatment. However, the underlying mechanisms remain unknown. Here, we demonstrate that mitochondrial respiratory chain (MRC) plays an essential role in the IFN-beta/RA-induced cancer cell death. We found that IFN-beta/RA upregulates the expression of MRC complex subunits. Mitochondrial-nuclear translocation of these subunits was not observed, but overproduction of reactive oxygen species (ROS), which causes loss of mitochondrial function, was detected upon IFN-beta/RA treatment. Knockdown of GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) and NDUFS3 (NADH dehydrogenase (ubiquinone) Fe-S protein 3), two subunits of MRC complex I, by siRNA in two cancer cell lines conferred resistance to IFN-beta/RA-induced apoptosis and reduced ROS production. In parallel, expression of late genes induced by IFN-beta/RA that are directly involved in growth inhibition and cell death was also repressed in the knockdown cells. Our data suggest that the MRC regulates IFN-beta/RA-induced cell death by modulating ROS production and late gene expression.

Vav proteins control MyD88-dependent oxidative burst.

The importance of reactive oxygen intermediate (ROI) production in antimicrobial responses is demonstrated in human patients who suffer from chronic granulomatous disease (CGD) due to defective NADPH oxidase function. Exactly how bacterial products activating Toll-like receptors (TLRs) induce oxidative burst is unknown. Here, we identify the Vav family of Rho guanine nucleotide exchange factors (GEFs) as critical mediators of LPS-induced MyD88-dependent activation of Rac2, NADPH oxidase, and ROI production using mice deficient in Vav1, Vav2, and Vav3. Vav proteins are also required for p38 MAPK activation and for normal regulation of proinflammatory cytokine production, but not for other MyD88-controlled effector pathways such as those involving JNK, COX2, or iNOS and the production of reactive nitrogen intermediates (RNIs). Thus, our data indicate that Vav specifically transduces a subset of signals emanating from MyD88.

GRIM-19, a cell death regulatory protein, is essential for assembly and function of mitochondrial complex I.

Mitochondria play essential roles in cellular energy production via the oxidative phosphorylation system (OXPHOS) consisting of five multiprotein complexes and also in the initiation of apoptosis. NADH:ubiquinone oxidoreductase (complex I) is the largest complex that catalyzes the first step of electron transfer in the OXPHOS system. GRIM-19 was originally identified as a nuclear protein with apoptotic nature in interferon (IFN)- and all-trans-retinoic acid (RA)-induced tumor cells. To reveal its biological role, we generated mice deficient in GRIM-19 by gene targeting. Homologous deletion of GRIM-19 causes embryonic lethality at embryonic day 9.5. GRIM-19(-/-) blastocysts show retarded growth in vitro and, strikingly, display abnormal mitochondrial structure, morphology, and cellular distribution. We reexamined the cellular localization of GRIM-19 in various cell types and found its primary localization in the mitochondria. Furthermore, GRIM-19 is detected in the native form of mitochondrial complex I. Finally, we show that elimination of GRIM-19 destroys the assembly and electron transfer activity of complex I and also influences the other complexes in the mitochondrial respiratory chain. Our result demonstrates that GRIM-19, a gene product with a specific role in IFN-RA-induced cell death, is a functional component of mitochondrial complex I and is essential for early embryonic development.

Mitochondrial complex I is deficient in renal oncocytomas.

Renal oncocytomas are benign tumors characterized by dense accumulation of mitochondria the cause of which remains unknown so far. Consistently, mitochondrial DNA content and the amounts and catalytic activities of several oxidative phosphorylation (OXPHOS) complexes were known to be increased in these tumors, but it was not ascertained that the OXPHOS system was functional. Here we investigated mitochondrial complex I and found that its NADH dehydrogenase activity and protein content were specifically decreased in oncocytomas, in stark contrast with the parallel decrease of all respiratory chain complexes in other, malignant, renal tumors. We conclude that deficiency of complex I in oncocytomas might be the early event causing the increased mitochondrial biogenesis, attempting to compensate for the loss of OXPHOS function. Since other tumors were found to be linked to mitochondrial deficiencies like genetic alterations of fumarate hydratase or succinate dehydrogenase, oncocytoma could be the third type of benign tumor associated with impairment of mitochondrial ATP production in an oxidative, quiescent tissue. Besides, complex I enzyme activity was moderately decreased in the vicinity of oncocytomas, when compared with normal tissue adjacent to other renal tumors. This suggested that oncocytomas are the result of at least two serial modifications altering the mitochondrial respiratory chain.