NADPH Oxidase 1 in Liver Macrophages Promotes Inflammation and Tumor Development in Mice.
ABSTRACT: BACKGROUND & AIMS:Although there are associations among oxidative stress, reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX) activation, and hepatocellular carcinoma (HCC) development, it is not clear how NOX contributes to hepatocarcinogenesis. We studied the functions of different NOX proteins in mice after administration of a liver carcinogen. METHODS:Fourteen-day-old Nox1-/- mice, Nox4-/- mice, Nox1-/-Nox4-/- (double-knockout) mice, and wild-type (WT) C57BL/6 mice were given a single intraperitoneal injection of diethylnitrosamine (DEN) and liver tumors were examined at 9 months. We also studied the effects of DEN in mice with disruption of Nox1 specifically in hepatocytes (Nox1?Hep), hepatic stellate cells (Nox1?Hep), or macrophages (Nox1?Mac). Some mice were also given injections of the NOX1-specific inhibitor ML171. To study the acute effects of DEN, 8-12-week-old mice were given a single intraperitoneal injection, and liver and serum were collected at 72 hours. Liver tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and immunoblots. Hepatocytes and macrophages were isolated from WT and knockout mice and analyzed by immunoblots. RESULTS:Nox4-/- mice and WT mice developed liver tumors within 9 months after administration of DEN, whereas Nox1-/- mice developed 80% fewer tumors, which were 50% smaller than those of WT mice. Nox1?Hep and Nox1?HSC mice developed liver tumors of the same number and size as WT mice, whereas Nox1?Mac developed fewer and smaller tumors, similar to Nox1-/- mice. After DEN injection, levels of tumor necrosis factor, interleukin 6 (IL6), and phosphorylated signal transducer and activator of transcription 3 were increased in livers from WT, but not Nox1-/- or Nox1?Mac, mice. Conditioned medium from necrotic hepatocytes induced expression of NOX1 in cultured macrophages, followed by expression of tumor necrosis factor, IL6, and other inflammatory cytokines; this medium did not induce expression of IL6 or cytokines in Nox1?Mac macrophages. WT mice given DEN followed by ML171 developed fewer and smaller liver tumors than mice given DEN followed by vehicle. CONCLUSIONS:In mice given injections of a liver carcinogen (DEN), expression of NOX1 by macrophages promotes hepatic tumorigenesis by inducing the production of inflammatory cytokines. We propose that upon liver injury, damage-associated molecular patterns released from dying hepatocytes activate liver macrophages to produce cytokines that promote tumor development. Strategies to block NOX1 or these cytokines might be developed to slow hepatocellular carcinoma progression.
Project description:Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) generates reactive oxygen species (ROS) in hepatic stellate cells (HSCs) during liver fibrosis. In response to fibrogenic agonists, such as angiotensin II (Ang II), the NOX1 components form an active complex, including Ras-related botulinum toxin substrate 1 (Rac1). Superoxide dismutase 1 (SOD1) interacts with the NOX-Rac1 complex to stimulate NOX activity. NOX4 is also induced in activated HSCs/myofibroblast by increased gene expression. Here, we investigate the role of an enhanced activity SOD1 G37R mutation (SODmu) and the effects of GKT137831, a dual NOX1/4 inhibitor, on HSCs and liver fibrosis. To induce liver fibrosis, wild-type (WT) and SOD1mu mice were treated with CCl(4) or bile duct ligation (BDL). Then, to address the role of NOX-SOD1-mediated ROS production in HSC activation and liver fibrosis, mice were treated with a NOX1/4 inhibitor. Fibrosis and ROS generation was assessed by histology and measurement of thiobarbituric acid reactive substances and NOX-related genes. Primary cultured HSCs isolated from WT, SODmu, and NOX1 knockout (KO) mice were assessed for ROS production, Rac1 activity, and NOX gene expression. Liver fibrosis was increased in SOD1mu mice, and ROS production and Rac1 activity were increased in SOD1mu HSCs. The NOX1/4 inhibitor, GKT137831, attenuated liver fibrosis and ROS production in both SOD1mu and WT mice as well as messenger RNA expression of fibrotic and NOX genes. Treatment with GKT137831 suppressed ROS production and NOX and fibrotic gene expression, but not Rac1 activity, in SOD1mut and WT HSCs. Both Ang II and tumor growth factor beta up-regulated NOX4, but Ang II required NOX1.SOD1mu induces excessive NOX1 activation through Rac1 in HSCs, causing enhanced NOX4 up-regulation, ROS generation, and liver fibrosis. Treatment targeting NOX1/4 may be a new therapy for liver fibrosis.
Project description:In higher concentrations, the blood pressure regulating hormone angiotensin II leads to vasoconstriction, hypertension, and oxidative stress by activating NADPH oxidases which are a major enzymatic source of reactive oxygen species (ROS). With the help of knockout animals, the impact of the three predominant NADPH oxidases present in the kidney, i.e., Nox1, Nox2 and Nox4 on angiotensin II-induced oxidative damage was studied. Male wildtype (WT) C57BL/6 mice, Nox1-, Nox2- and Nox4-deficient mice were equipped with osmotic minipumps, delivering either vehicle (PBS) or angiotensin II, for 28 days. Angiotensin II increased blood pressure and urinary albumin levels significantly in all treated mouse strains. In Nox1 knockout mice these increases were significantly lower than in WT, or Nox2 knockout mice. In WT mice, angiotensin II also raised systemic oxidative stress, ROS formation and DNA lesions in the kidney. A local significantly increased ROS production was also found in Nox2 and Nox4 knockout mice but not in Nox1 knockout mice who further had significantly lower systemic oxidative stress and DNA damage than WT animals. Nox2 and Nox4 knockout mice had increased basal DNA damage, concealing possible angiotensin II-induced increases. In conclusion, in the kidney, Nox1 seemed to play a role in angiotensin II-induced DNA damage.
Project description:The increased production of reactive oxygen species (ROS) has been postulated to play a key role in the progression of nonalcoholic fatty liver disease (NAFLD). However, the source of ROS and mechanisms underlying the development of NAFLD have yet to be established. We observed a significant up-regulation of a minor isoform of NADPH oxidase, NOX1, in the liver of nonalcoholic steatohepatitis (NASH) patients as well as of mice fed a high-fat and high-cholesterol (HFC) diet for 8 weeks. In mice deficient in Nox1 (Nox1KO), increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 demonstrated in HFC diet-fed wild-type mice (WT) were significantly attenuated. Concomitantly, increased protein nitrotyrosine adducts, a marker of peroxynitrite-induced injury detected in hepatic sinusoids of WT, were significantly suppressed in Nox1KO. The expression of NOX1 mRNA was much higher in the fractions of enriched liver sinusoidal endothelial cells (LSECs) than in those of hepatocytes. In primary cultured LSECs, palmitic acid (PA) up-regulated the mRNA level of NOX1, but not of NOX2 or NOX4. The production of nitric oxide by LSECs was significantly attenuated by PA-treatment in WT but not in Nox1KO. When the in vitro relaxation of TWNT1, a cell line that originated from hepatic stellate cells, was assessed by the gel contraction assay, the relaxation of stellate cells induced by LSECs was attenuated by PA treatment. In contrast, the relaxation effect of LSECs was preserved in cells isolated from Nox1KO. Taken together, the up-regulation of NOX1 in LSECs may elicit peroxynitrite-mediated cellular injury and impaired hepatic microcirculation through the reduced bioavailability of nitric oxide. ROS derived from NOX1 may therefore constitute a critical component in the progression of NAFLD.
Project description:Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN bioactivation and higher levels of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development.
Project description:Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV)-induced pathogenesis. Studies have suggested that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increases the generation of superoxide and hydrogen peroxide close to the hepatocyte nucleus and that this source of ROS is reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 4 (Nox4). Huh7 human hepatoma cells and telomerase-reconstituted primary human hepatocytes, transfected or infected with virus-producing HCV strains of genotypes 2a and 1b, were examined for messenger RNA (mRNA), protein, and subcellular localization of Nox proteins along with the human liver. We found that genotype 2a HCV induced persistent elevations of Nox1 and Nox4 mRNA and proteins in Huh7 cells. HCV genotype 1b likewise elevated the levels of Nox1 and Nox4 in telomerase-reconstituted primary human hepatocytes. Furthermore, Nox1 and Nox4 proteins were increased in HCV-infected human liver versus uninfected liver samples. Unlike Nox1, Nox4 was prominent in the nuclear compartment of these cells as well as the human liver, particularly in the presence of HCV. HCV-induced ROS and nuclear nitrotyrosine could be decreased with small interfering RNAs to Nox1 and Nox4. Finally, HCV increased the level of transforming growth factor beta 1 (TGFbeta1). TGFbeta1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGFbeta1.HCV induced a persistent elevation of Nox1 and Nox4 and increased nuclear localization of Nox4 in hepatocytes in vitro and in the human liver. Hepatocyte Nox proteins are likely to act as a persistent, endogenous source of ROS during HCV-induced pathogenesis.
Project description:Metallothioneins (MT) are potent scavengers of free radicals that are silenced in primary hepatocellular carcinomas (HCC) of human and rodent origin. To examine whether loss of MT promotes hepatocarcinogenesis, male Mt-1 and Mt-2 double knockout (MTKO) and wild-type (WT) mice were exposed to diethylnitrosamine (DEN) and induction of HCC was monitored at 23 and 33 weeks. The size and number of liver tumors, the ratio between liver and body weight, and liver damage were markedly elevated in the MTKO mice at both time points compared with the WT mice. At 23 weeks, MTKO mice developed HCC whereas WT mice developed only preneoplastic nodules suggesting that loss of MT accelerates hepatocarcinogenesis. MTKO tumors also exhibited higher superoxide anion levels. Although NF-?B activity increased in the liver nuclear extracts of both genotypes after DEN exposure, the complex formed in MTKO mice was predominantly p50/65 heterodimer (transcriptional activator) as opposed to p50 homodimer (transcriptional repressor) in WT mice. Phosphorylation of p65 at Ser276 causing its activation was also significantly augmented in DEN-exposed MTKO livers. NF-?B targets that include early growth response genes and proinflammatory cytokines were significantly upregulated in MTKO mice. Concurrently, there was a remarkable increase (?100-fold) in Pai-1 expression; significant increase in c-Jun, c-Fos, c-Myc, Ets2, and ATF3 expressions; and growth factor signaling that probably contributed to the increased tumor growth in MTKO mice. Taken together, these results demonstrate that MTs protect mice from hepatocarcinogen-induced liver damage and carcinogenesis, underscoring their potential therapeutic application against hepatocellular cancer.
Project description:Angiotensin II (ANG II) stimulates production of superoxide (O(2)(-)) by NADPH oxidase (NOX) in medullary thick ascending limbs (TALs). There are three isoforms of the catalytic subunit (NOX1, 2, and 4) known to be expressed in the kidney. We hypothesized that NOX2 mediates ANG II-induced O(2)(-) production by TALs. To test this, we measured NOX1, 2, and 4 mRNA and protein by RT-PCR and Western blot in TAL suspensions from rats and found three catalytic subunits expressed in the TAL. We measured O(2)(-) production using a lucigenin-based assay. To assess the contribution of NOX2, we measured ANG II-induced O(2)(-) production in wild-type and NOX2 knockout mice (KO). ANG II increased O(2)(-) production by 346 relative light units (RLU)/mg protein in the wild-type mice (n = 9; P < 0.0007 vs. control). In the knockout mice, ANG II increased O(2)(-) production by 290 RLU/mg protein (n = 9; P < 0.007 vs. control). This suggests that NOX2 does not contribute to ANG II-induced O(2)(-) production (P < 0.6 WT vs. KO). To test whether NOX4 mediates the effect of ANG II, we selectively decreased NOX4 expression in rats using an adenovirus that expresses NOX4 short hairpin (sh)RNA. Six to seven days after in vivo transduction of the kidney outer medulla, NOX4 mRNA was reduced by 77%, while NOX1 and NOX2 mRNA was unaffected. In control TALs, ANG II stimulated O(2)(-) production by 96%. In TALs transduced with NOX4 shRNA, ANG II-stimulated O(2)(-) production was not significantly different from the baseline. We concluded that NOX4 is the main catalytic isoform of NADPH oxidase that contributes to ANG II-stimulated O(2)(-) production by TALs.
Project description:Matrilin-2 (Matn2) is a multidomain adaptor protein which plays a role in the assembly of extracellular matrix (ECM). It is produced by oval cells during stem cell-driven liver regeneration. In our study, the impact of Matn2 on hepatocarcinogenesis was investigated in Matn2(-/-) mice comparing them with wild-type (WT) mice in a diethylnitrosamine (DEN) model. The liver tissue was analyzed macroscopically, histologically and immunohistochemically, at protein level by Proteome Profiler Arrays and Western blot analysis. Matn2(-/-) mice exhibited higher susceptibility to hepatocarcinogenesis compared to wild-type mice. In the liver of Matn2(-/-) mice, spontaneous microscopic tumor foci were detected without DEN treatment. After 15 ?g/g body weight DEN treatment, the liver of Matn2(-/-) mice contained macroscopic tumors of both larger number and size than the WT liver. In contrast with the WT liver, spontaneous phosphorylation of EGFR, Erk1/2 GSK-3?/? and retinoblastoma protein (p-Rb), decrease in p21/CIP1 level, and increase in ?-Catenin protein expression were detected in Matn2(-/-) livers. Focal Ki-67 positivity of these samples provided additional support to our presumption that the lack of Matn2 drives the liver into a pro-proliferatory state, making it prone to tumor development. This enhanced proliferative capacity was further increased in the tumor nodules of DEN-treated Matn2(-/-) livers. Our study suggests that Matn2 functions as a tumor suppressor in hepatocarcinogenesis, and in this process activation of EGFR together with that of Erk1/2, as well as inactivation of GSK-3?, play strategic roles.
Project description:The NADPH oxidase (NOX) isoform NOX4 has been linked with diabetic kidney disease (DKD). However, a mechanistic understanding of the downstream effects of NOX4 remains to be established. We report that podocyte-specific induction of NOX4 in vivo was sufficient to recapitulate the characteristic glomerular changes noted with DKD, including glomerular hypertrophy, mesangial matrix accumulation, glomerular basement membrane thickening, albuminuria, and podocyte dropout. Intervention with a NOX1/NOX4 inhibitor reduced albuminuria, glomerular hypertrophy, and mesangial matrix accumulation in the F1 Akita model of DKD. Metabolomic analyses from these mouse studies revealed that tricarboxylic acid (TCA) cycle-related urinary metabolites were increased in DKD, but fumarate levels were uniquely reduced by the NOX1/NOX4 inhibitor. Expression of fumarate hydratase (FH), which regulates urine fumarate accumulation, was reduced in the diabetic kidney (in mouse and human tissue), and administration of the NOX1/NOX4 inhibitor increased glomerular FH levels in diabetic mice. Induction of Nox4 in vitro and in the podocyte-specific NOX4 transgenic mouse led to reduced FH levels. In vitro, fumarate stimulated endoplasmic reticulum stress, matrix gene expression, and expression of hypoxia-inducible factor-1? (HIF-1?) and TGF-?. Similar upregulation of renal HIF-1? and TGF-? expression was observed in NOX4 transgenic mice and diabetic mice and was attenuated by NOX1/NOX4 inhibition in diabetic mice. In conclusion, NOX4 is a major mediator of diabetes-associated glomerular dysfunction through targeting of renal FH, which increases fumarate levels. Fumarate is therefore a key link connecting metabolic pathways to DKD pathogenesis, and measuring urinary fumarate levels may have application for monitoring renal NOX4 activity.
Project description:Hepatocellular carcinoma (HCC) is a typical inflammation-associated cancer, but may also provoke antitumour immune responses whose significance and underlying mechanisms are incompletely understood.To characterise immune responses in the diethylnitrosamine (DEN)-liver cancer mouse model.Tumour development and immune cell functions upon DEN treatment were compared between C57BL/6 wild-type (WT), chemokine scavenging receptor D6-deficient, B cell- (Igh6), CD4 T cell- (MHC-II) and T-/B cell-deficient (Rag1) mice. Relevance for human HCC was tested by comparing gene array results from 139 HCC tissues.The induction of premalignant lesions after 24 weeks and of HCC-like tumours after 42 weeks by DEN in mice was accompanied by significant leucocyte infiltration in the liver and upregulation of distinct intrahepatic chemokines (CCL2, CCL5, CXCL9). Macrophages and CD8 (cytotoxic) T cells were most prominently enriched in tumour-bearing livers, similar to samples from human HCC. Myeloid-derived suppressor cells (MDSC) increased in extrahepatic compartments of DEN-treated mice (bone marrow, spleen). The contribution of immune cell subsets for DEN-induced hepatocarcinogenesis was functionally dissected. In D6(-/-) mice, which lack the chemokine scavenging receptor D6, hepatic macrophage infiltration was significantly increased, but tumour formation and progression did not differ from that of WT mice. In contrast, progression of hepatic tumours (numbers, diameters, tumour load) was strikingly enhanced in T-/B cell-deficient Rag1(-/-) mice upon DEN treatment. When mice deficient for B cells (Igh6(-/-), ?MT) or major histocompatibility complex II were used, the data indicated that T cells prevent initial tumour formation, while B cells critically limit growth of established tumours. Accordingly, in tumour-bearing mice antibody production against liver-related model antigen was enhanced, indicating tumour-associated B cell activation. In agreement, T and B cell pathways were differentially regulated in gene array analyses from 139 human HCC tissues and significantly associated with patients' survival.Distinct axes of the adaptive immune system, which are also prognostic in human HCC, actively suppress DEN-induced hepatocarcinogenesis by controlling tumour formation and progression.