Akira Moh

Function of Mitochondrial Stat3 in Cellular Respiration

Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3–/– cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the proteins function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.

Cell Type–Dependent Pro- and Anti-Inflammatory Role of Signal Transducer and Activator of Transcription 3 in Alcoholic Liver Injury

BACKGROUND & AIMS Signal transducer and activator of transcription 3 (STAT3) is known to be activated in human alcoholic liver disease, but its role in the pathogenesis of alcoholic liver injury remains obscure. METHODS The role of STAT3 in alcoholic liver injury was investigated in hepatocyte-specific STAT3 knockout (H-STAT3KO) mice and macrophage/neutrophil-specific STAT3 KO (M/N-STAT3KO) mice. Alcoholic liver injury was achieved by feeding mice a liquid diet containing 5% ethanol for up to 8 weeks. RESULTS Compared with wild-type mice, feeding H-STAT3KO mice with an ethanol-containing diet induced greater hepatic steatosis, hypertriglyceridemia, and hepatic expression of lipogenic genes (sterol regulatory element-binding protein, fatty acid synthase, acetyl-CoA carboxylase-1, and stearoyl-CoA desaturase 1), but less inflammation and lower expression of hepatic proinflammatory cytokines. In contrast, ethanol-fed M/N-STAT3KO mice showed more hepatic inflammation, worse injury, and increased hepatic expression of proinflammatory cytokines compared with wild-type mice. Kupffer cells isolated from ethanol-fed H-STAT3KO mice produced similar amounts of reactive oxygen species and tumor necrosis factor alpha, whereas Kupffer cells from M/N-STAT3KO mice produced more reactive oxygen species and tumor necrosis factor alpha compared with wild-type controls. CONCLUSIONS These findings suggest that STAT3 regulates hepatic inflammation in a cell type-dependent manner during alcoholic liver injury: STAT3 in hepatocytes promotes whereas STAT3 in macrophages/Kupffer cells suppresses inflammation. In addition, activation of hepatocellular STAT3 ameliorates alcoholic fatty liver via inhibition of sterol regulatory element-binding protein 1c expression.

Ablation of the Galnt3 Gene Leads to Low-Circulating Intact Fibroblast Growth Factor 23 (Fgf23) Concentrations and Hyperphosphatemia Despite Increased Fgf23 Expression

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generated mice lacking the Galnt3 gene, which developed hyperphosphatemia without apparent calcifications. In response to hyperphosphatemia, Galnt3-deficient mice had markedly increased Fgf23 expression in bone. However, compared with wild-type and heterozygous littermates, homozygous mice had only about half of circulating intact Fgf23 levels and higher levels of C-terminal Fgf23 fragments in bone. Galnt3-deficient mice also exhibited an inappropriately normal 1,25-dihydroxyvitamin D level and decreased alkaline phosphatase activity. Furthermore, renal expression of sodium-phosphate cotransporters and Kl were elevated in Galnt3-deficient mice. Interestingly, there were sex-specific phenotypes; only Galnt3-deficient males showed growth retardation, infertility, and significantly increased bone mineral density. In summary, ablation of Galnt3 impaired secretion of intact Fgf23, leading to decreased circulating Fgf23 and hyperphosphatemia, despite increased Fgf23 expression. Our findings indicate that Galnt3-deficient mice have a biochemical phenotype of tumoral calcinosis and provide in vivo evidence that Galnt3 plays an essential role in proper secretion of Fgf23 in mice.

Cyclic di-GMP is Essential for the Survival of the Lyme Disease Spirochete in Ticks

Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogens life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogens survival in its vector host.

STAT5 programs a distinct subset of GM-CSF-producing T helper cells that is essential for autoimmune neuroinflammation

T helper (TH)-cell subsets, such as TH1 and TH17, mediate inflammation in both peripheral tissues and central nervous system. Here we show that STAT5 is required for T helper-cell pathogenicity in autoimmune neuroinflammation but not in experimental colitis. Although STAT5 promotes regulatory T cell generation and immune suppression, loss of STAT5 in CD4+ T cells resulted in diminished development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Our results showed that loss of encephalitogenic activity of STAT5-deficient autoreactive CD4+ T cells was independent of IFN-γ or interleukin 17 (IL-17) production, but was due to the impaired expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), a crucial mediator of T-cell pathogenicity. We further showed that IL-7-activated STAT5 promotes the generation of GM-CSF-producing CD4+ T cells, which were preferentially able to induce more severe EAE than TH17 or TH1 cells. Consistent with GM-CSF-producing cells being a distinct subset of TH cells, the differentiation program of these cells was distinct from that of TH17 or TH1 cells. We further found that IL-3 was secreted in a similar pattern as GM-CSF in this subset of TH cells. In conclusion, the IL-7-STAT5 axis promotes the generation of GM-CSF/IL-3-producing TH cells. These cells display a distinct transcriptional profile and may represent a novel subset of T helper cells which we designate as TH-GM.

Survivin mediates aberrant hematopoietic progenitor cell proliferation and acute leukemia in mice induced by internal tandem duplication of Flt3

Internal tandem duplication mutations in the Flt3 tyrosine kinase gene (ITD-Flt3) and overexpression of Survivin are frequently found in patients with acute myeloid leukemia (AML). We investigated whether Survivin mediates the enhanced survival of primary hematopoietic progenitor cells (HPCs) resulting from ITD-Flt3 signaling. Ectopic ITD-Flt3 mutants increased Survivin expression in Ba/F3 cells downstream of PI3-kinase/Akt. Treatment of ITD-Flt3(+) human MV4-11 leukemia cells with the ITD-Flt3 inhibitor SU5416 reduced Survivin expression and inhibited cell proliferation. ITD-Flt3 dramatically increased the number of primary mouse marrow c-kit(+), Sca-1(+), Lin(Neg) cells and colony-forming unit granulocyte-macrophages (CFU-GMs) able to proliferate in the absence of growth factors, whereas Survivin deletion significantly reduced growth factor-independent proliferation and increased apoptosis, which was further accentuated by SU5416. Ectopic ITD-Flt3 reduced differentiation of Lin(Neg) marrow cells cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus stem cell factor, which was partially blocked by Survivin deletion. In addition, Survivin deletion decreased secondary colony formation induced by ITD-Flt3. Dominant-negative (dn)-Survivin delayed development of acute leukemia in mice that received a transplant of Ba/F3 cells expressing ITD-Flt3. These results suggest that Survivin regulates expansion of ITD-Flt3-transformed HPCs with self-renewal capability and development of ITD-Flt3(+) acute leukemia and that antagonizing Survivin may provide therapeutic benefit for patients with acute leukemia expressing ITD-Flt3.

Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

Noonan syndrome (NS) is an autosomal dominant congenital disorder characterized by multiple birth defects including heart defects and myeloproliferative disease (MPD). Approximately 50% of NS patients have germline gain-of-function mutations in PTPN11, which encodes the protein-tyrosine phosphatase, Shp2. We provide evidence that conditional ablation of Stat3 in hematopoietic cells and cardiac valvular tissues leads to myeloid progenitor hyperplasia and pulmonary stenosis due to the leaflet thickening, respectively. Consistently, STAT3 activation is significantly compromised in peripheral blood cells from NS patients bearing Shp2-activating mutations. Biochemical and functional analyses demonstrate that activated Shp2 is able to down-regulate Tyr(P)-Stat3 and that constitutively active Stat3 rescues activating mutant Shp2-induced granulocyte-macrophage colony-stimulating factor hypersensitivity in bone marrow cells. Collectively, our work demonstrates that Stat3 is an essential signaling component potentially contributing to the pathogenesis of NS and juvenile myelomonocytic leukemia caused by PTPN11 gain-of-function mutations.

Anti‐Inflammatory and Anti‐Apoptotic Roles of Endothelial Cell STAT3 in Alcoholic Liver Injury

BACKGROUND It is generally believed that the hepatoprotective effect of interleukin-6 (IL-6) is mediated via activation of signal transducer and activator of transcription 3 (STAT3) in hepatocytes. IL-6-deficient mice are more susceptible to alcohol-induced hepatocyte apoptosis and steatosis and elevation of serum alanine transaminase (ALT); however, whereas hepatocyte-specific STAT3 knockout mice are more susceptible to alcohol-induced hepatic steatosis, they have similar hepatocyte apoptosis and serum ALT after alcohol feeding compared with wild-type mice. This suggests that the hepatoprotective effect of IL-6 in alcoholic liver injury may be mediated via activation of STAT3-independent signals in hepatocytes, activation of STAT3 in nonparenchymal cells, or both. We have previously shown that IL-6 also activates STAT3 in sinusoidal endothelial cells (SECs). Thus, the purpose of this study was to investigate whether STAT3 in endothelial cells also plays a protective role in alcoholic liver injury. METHODS Wild-type and endothelial cell-specific STAT3 knockout (STAT3(E-/-)) mice were pair-fed and fed ethanol containing diet for 4 weeks. Liver injury and inflammation were determined. RESULTS Feeding mice with ethanol-containing diet for 4 weeks induced greater hepatic injury (elevation of serum ALT) and liver weight in STAT3(E-/-) mice than wild-type control groups. In addition, ethanol-fed STAT3(E-/-) mice displayed greater hepatic inflammation and substantially elevated serum and hepatic levels of IL-6 and TNF-alpha compared with wild-type mice. Furthermore, ethanol-fed STAT3(E-/-) mice displayed a greater abundance of apoptotic SECs and higher levels of serum hyaluronic acid than wild-type controls. CONCLUSIONS These data suggest that endothelial cell STAT3 plays important dual functions of attenuating hepatic inflammation and SEC death during alcoholic liver injury.

STAT3 Sensitizes Insulin Signaling by Negatively Regulating Glycogen Synthase Kinase-3β

OBJECTIVE— Glucose homeostasis is achieved by triggering regulation of glycogen synthesis genes in response to insulin when mammals feed, but the underlying molecular mechanism remains largely unknown. The aim of our study was to examine the role of the signal transducers and activators of transcription 3 (STAT3) in insulin signaling. RESEARCH DESIGN AND METHODS— We generated a strain of mice carrying a targeted disruption of Stat3 gene in the liver (L-Stat3−/− mice). Hepatocytes of the L-Stat3−/− mice were isolated to establish cell lines for mechanistic studies. Nuclear translocation and DNA-protein interaction of STAT3 was analyzed with immunofluorescent and chromatin immunoprecipitation methods, respectively. Levels of glucose, insulin, leptin, and glucagon were profiled, and putative downstream molecules of STAT3 were examined in the presence of various stimuli in L-Stat3−/− and control mice. RESULTS— STAT3 was found to sensitize the insulin signaling through suppression of GSK-3β, a negative regulator of insulin signaling pathway. During feeding, both mRNA and protein levels of GSK-3β decreased in Stat3f/+ mice, which reflected the need of hepatocytes for insulin to induce glycogen synthesis. In contrast, the L-Stat3−/− mice lost this control and showed a monophasic increase in the GSK-3β level in response to insulin. Administration of GSK-3β inhibitors lithium chloride and L803-mts restored glucose homeostasis and rescued the glucose intolerance and impaired insulin response in L-Stat3−/− mice. CONCLUSIONS— These data indicate that STAT3 sensitizes insulin signaling by negatively regulating GSK-3β. Inactivation of STAT3 in the liver contributes significantly to the pathogenesis of insulin resistance.

Osteoblast/osteocyte-specific inactivation of Stat3 decreases load-driven bone formation and accumulates reactive oxygen species.

Signal transducers and activators of transcription 3 (Stat3) is a transcription factor expressed in many cell types including osteoblasts, osteocytes, and osteoclasts. STAT3 mutations cause a rare human immunodeficiency disease that presents reduced bone mineral density and recurrent pathological fractures. To investigate the role of Stat3 in load-driven bone metabolism, two strains of osteoblast/osteocyte-selective Stat3 knockout (KO) mice were generated. Compared to age-matched littermate controls, this selective inactivation of Stat3 significantly lowered bone mineral density (7-12%, p<0.05) as well as ultimate force (21-34%, p<0.01). In ulna loading (2.50-2.75N with 120 cycles/day at 2Hz for 3 consecutive days), Stat3 KO mice were less responsive than littermate controls as indicated by reduction in relative mineralizing surface (rMS/BS, 47-59%, p<0.05) and relative bone formation rate (rBFR/BS, 64-75%, p<0.001). Furthermore, inactivation of Stat3 suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Taken together, the results support the notion that the loss-of-function mutation of Stat3 in osteoblasts and osteocytes diminishes load-driven bone formation and impairs the regulation of oxidative stress in mitochondria.