Michitaka Ozaki

Role of STAT-3 in regulation of hepatic gluconeogenic genes and carbohydrate metabolism in vivo.

The transcription factor, signal transducer and activator of transcription-3 (STAT-3) contributes to various physiological processes. Here we show that mice with liver-specific deficiency in STAT-3, achieved using the Cre-loxP system, show insulin resistance associated with increased hepatic expression of gluconeogenic genes. Restoration of hepatic STAT-3 expression in these mice, using adenovirus-mediated gene transfer, corrected the metabolic abnormalities and the alterations in hepatic expression of gluconeogenic genes. Overexpression of STAT-3 in cultured hepatocytes inhibited gluconeogenic gene expression independently of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), an upstream regulator of gluconeogenic genes. Liver-specific expression of a constitutively active form of STAT-3, achieved by infection with an adenovirus vector, markedly reduced blood glucose, plasma insulin concentrations and hepatic gluconeogenic gene expression in diabetic mice. Hepatic STAT-3 signaling is thus essential for normal glucose homeostasis and may provide new therapeutic targets for diabetes mellitus.

Rac1 inhibits TNF-α-induced endothelial cell apoptosis: dual regulation by reactive oxygen species

Reactive oxygen species (ROS) have been implicated as mediators of tumor necrosis factor‐alpha (TNF) ‐induced apoptosis. In addition to leading to cell death, ROS can also promote cell growth and/or survival. We investigated these two roles of ROS in TNF‐induced endothelial cell apoptosis. Human umbilical vein endothelial cells (HUVECs) stimulated with TNF produced an intracellular burst of ROS. Adenoviral‐mediated gene transfer of a dominant negative form of the small GTPase Rac1 (Rac1N17) partially suppressed the TNF‐induced oxidative burst without affecting TNF‐induced mitochondrial ROS production. HUVECs were protected from TNF‐induced apoptosis. Expression of Rac1N17 blocked TNF‐induced activation of nuclear factor‐kappa B (NF‐κB), increased activity of caspase‐3, and markedly augmented endothelial cell susceptibility to TNF‐induced apoptosis. Direct inhibition of NF‐κB through adenoviral expression of the super repressor form of inhibitor of kBα (I‐κB S32/36A) also increased susceptibility of HUVECs to TNF‐induced apoptosis. Rotenone, a mitochondrial electron transport chain inhibitor, suppressed TNF‐induced mitochondrial ROS production, proteolytic cleavage of procaspase‐3, and apoptosis. These findings show that Rac1 is an important regulator of TNF‐induced ROS production in endothelial cells. Moreover, they suggest that Rac1‐dependent ROS, directly or indirecly, lead to protection against TNF‐induced death, whereas mitochondrial‐derived ROS promote TNF‐induced apoptosis.—Deshpande, S. S., Angkeow, P., Huang, J., Ozaki, M., Irani, K. Rac1 inhibits TNF‐α‐induced endothelial cell apoptosis: dual regulation by reactive oxygen species. FASEB J. 14, 1705–1714 (2000)

Stat3 protects against Fas-induced liver injury by redox-dependent and -independent mechanisms

Signal transducer and activator of transcription-3 (Stat3) is one of the most important molecules involved in the initiation of liver development and regeneration. In order to investigate the hepatoprotective effects of Stat3, we examined whether Stat3 protects against Fas-mediated liver injury in the mouse. A constitutively activated form of Stat3 (Stat3-C) was adenovirally overexpressed in mouse liver by intravenous injection, and then a nonlethal dose of Fas agonist (Jo2) was injected intraperitoneally into the mouse (0.3 microg/g body wt). Stat3-C dramatically suppressed both apoptosis and necrosis induced by Jo2. In contrast, liver-specific Stat3-knockout mice failed to survive following Jo2 injection. Stat3-C upregulated expression of FLICE inhibitor protein (FLIP), Bcl-xL, and Bcl-2, and accordingly downregulated activities of FLICE and caspase-3 that were redox-independent. Interestingly, Stat3-C also upregulated the redox-associated protein redox factor-1 (Ref-1) and reduced apoptosis in liver following Jo2 injection by suppressing oxidative stress and redox-sensitive caspase-3 activity. These findings indicate that Stat3 activation protects against Fas-mediated liver injury by inhibiting caspase activities in redox-dependent and -independent mechanisms.

Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion-induced necrosis and apoptosis in vivo

Reperfusion of ischemic tissue results in the generation of reactive oxygen species that contribute to tissue injury. The sources of reactive oxygen species in reperfused tissue are not fully characterized. We hypothesized that the small GTPase Rac1 mediates the oxidative burst in reperfused tissue and thereby contributes to reperfusion injury. In an in vivo model of mouse hepatic isch‐emia/reperfusion injury, recombinant adenoviral expression of a dominant negative Rac1 (Rac1N17) completely suppressed the ischemia/reperfusion‐induced production of reactive oxygen species and lipid peroxides, activation of nuclear factor‐kappa B, and resulted in a significant reduction of acute liver necrosis. Expression of Rac1N17 also suppressed ischemia/reperfusion‐induced acute apoptosis. The protection offered by Rac1N17 was also evident in knockout mice deficient for the gp91phox component of the phagocyte NADPH oxidase. This work demonstrates the crucial role of a Rac1‐regulated oxidase in mediating the production of injurious reactive oxygen species, which contribute to acute necrotic and apoptotic cell death induced by isch‐emia/reperfusion in vivo. Targeted inhibition of this oxidase, which is distinct from the phagocyte NADPH oxidase, should provide a new avenue for in vivo therapy aimed at protecting organs at risk from ischemia/reperfusion injury.—Ozaki, M., Deshpande, S. S., Angkeow, P., Bellan, J., Lowenstein, C. J., Dinauer, M. C., Goldschmidt‐Clermont, P. J., Irani, K. Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion‐induced necrosis and apoptosis in vivo. FASEB J. 14, 418—429 (2000)

Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis

The rac1 GTPase promotes oxidative stress through reactive oxygen species (ROS) production, whereas the DNA repair enzyme and transcriptional regulator redox factor-1 (ref-1) protects against cell death due to oxidative stimuli. However, the function of ref-1 in regulating intracellular oxidative stress, particularly that induced by rac1, has not been defined. We examined the role of ref-1 in vascular endothelial cell oxidative stress and apoptosis. Ref-1 was expressed in both the cytoplasm and nuclei of resting endothelial cells. Cytoplasmic ref-1 translocated to the nucleus with the oxidative trigger hypoxia/reoxygenation (H/R). Forced cytoplasmic overexpression of ref-1 suppressed H/R-induced oxidative stress (H2O2 production), NF-κB activation, and apoptosis, and also mitigated rac1-regulated H2O2 production and NF-κB transcriptional activity. We conclude that inhibition of oxidative stress is another mechanism by which ref-1 protects against apoptosis, and that this is achieved through modulation of cytoplasmic rac1-regulated ROS generation. This suggests a novel extra-nuclear function of ref-1.

Inhibition of hypoxia/reoxygenation-induced oxidative stress in HGF-stimulated antiapoptotic signaling: role of PI3-K and Akt kinase upon rac1

AbstractRac1-regulated reactive oxygen species (ROS) production has been implicated in apoptosis. In contrast, pleiotropic protein kinase Akt protects against apoptosis. However, the pro- and antiapoptotic mechanisms of rac1 and Akt, respectively, and the intersection between these mechanisms are incompletely understood. In a model of oxidative stress and apoptosis induced by hypoxia/reoxygenation (H/R) in primary hepatocytes, activation of the PI3–K Akt axis by the prosurvival hepatocyte growth factor (HGF) inhibited H/R-stimulated rac1 activation and intracellular ROS production, and suppressed apoptosis. Suppression of PI3-K or Akt activity abrogated the inhibitory effect of HGF on rac1 activity and rac1-regulated oxidative stress. Furthermore, constitutive activation of Akt or PI3-K in the absence of HGF was sufficient to phosphorylate rac1, inhibit rac1 activation, and suppress rac1-regulated ROS production. These findings demonstrate that growth factor-stimulated activation of PI3-K–Akt is necessary and sufficient to suppress intracellular oxidative stress and apoptosis by inhibiting activation of pro-apoptotic, prooxidative rac1 GTPase.

Reactive oxygen species are essential mediators in antigen presentation by Kupffer cells

Kupffer cells (KC) act as APC in the liver and play a major role in the clearance of gut‐derived antigens and pathogens entering the liver with portal venous blood. Antigen presentation by KC has been implicated in regulation of the local and systemic immune responses. In this study, modulation of KC antigen presentation by antioxidants and the role of reactive oxygen species (ROS) as essential mediators of antigen presentation in KC were investigated. Co‐culture of KC with ovalbumin (OVA) antigens resulted in upstream intracellular endogenous ROS generation and increased expression of MHC class II and costimulator molecules, and consequent OVA‐specific CD4+ T‐cell proliferation in response to antigen presentation by KC. Scavenging of KC ROS by antioxidants, or blocking of KC ROS generation by specific inhibitors of NADPH oxidase and/or xanthine oxidase, or by specific inhibitors of the mitochondrial electron transport chain, significantly decreased OVA‐specific T‐cell proliferation in response to antigen presentation by KC. Increased expression of MHC class II and costimulatory molecules in KC pulsed with OVA antigens was blocked by inhibiting ROS generation enzymatically. Intracellular endogenous ROS generation during antigen processing may therefore provide essential secondary signalling for KC antigen presentation.

Rac1 regulates stress-induced, redox-dependent heat shock factor activation.

The signaling pathway by which environmental stresses activate heat shock factors (HSFs) is not completely understood. We show that the small GTPase rac1, and Rac1-regulated reactive oxygen species (ROS) play an important role in stress-stimulated heat shock response. A dominant-negative allele of Rac1 (Rac1N17) inhibits the hypoxia/reoxygenation and sodium arsenite-induced transcriptional activity of HSF-1 and the transcription of heat shock protein 70. Rac1N17 also suppresses the production of intracellular ROS induced by hypoxia/reoxygenation or sodium arsenite. Moreover, direct suppression of intracellular ROS levels by antioxidants decreases stress-stimulated HSF activity. However, expression of a constitutively active mutant of Rac1 (Rac1V12) in the absence of extracellular stresses does not increase intracellular ROS levels or induce the heat shock response. These results show that Rac1 is a necessary but insufficient component of the stress-induced signaling pathway that leads to ROS production, activation of HSFs, and transcription of heat shock proteins.

The survival pathways phosphatidylinositol-3 kinase (PI3-K)/phosphoinositide-dependent protein kinase 1 (PDK1)/Akt modulate liver regeneration through hepatocyte size rather than proliferation.

Liver regeneration comprises a series of complicated processes. The current study was designed to investigate the roles of phosphoinositide‐dependent protein kinase 1 (PDK1)‐associated pathways in liver regeneration after partial hepatectomy (PH) using liver‐specific Pdk1‐knockout (L‐Pdk1KO) and Pdk1/STAT3 double KO (L‐DKO) mice. There was no liver regeneration, and 70% PH was lethal in L‐Pdk1KO mice. Liver regeneration was severely impaired equally in L‐Pdk1KO and L‐DKO mice, even after nonlethal 30% PH. There was no cell growth (measured as increase of cell size) after hepatectomy in L‐Pdk1KO mice, although the post‐PH mitotic response was the same as in controls. As expected, hepatectomy did not induce hepatic Akt‐phosphorylation (Thr308) in L‐Pdk1KO mice, and post‐PH phosphorylation of Akt, mammalian target of rapamycin (mTOR), p70 ribosomal S6 kinase (p70S6K), and S6 were also reduced. To examine the specific role of PDK1‐associated signals, a “pif‐pocket” mutant of PDK1, which allows PDK1 only to phosphorylate Akt, was used. Liver regeneration was recovered in L‐Pdk1KO mice with a “pif‐pocket” mutant of PDK1. This re‐activated Akt in L‐Pdk1KO mice liver and induced post‐PH cell growth, without affecting cell proliferation. Further deletion of STAT3 (L‐DKO mice) did not further deteriorate liver regeneration, although this certainly reduced post‐PH mitotic response. These findings indicate that PDK1/Akt contribute to liver regeneration by regulating cell size. Regarding phosphatidylinositol‐3 kinase (PI3‐K), immediate upstream signal of PDK1, activation of PI3‐K induced cell proliferation via STAT3 activation in the liver of L‐Pdk1KO mice but did not improve impaired liver regeneration. This confirmed the pivotal role of PDK1 in liver regeneration and cell growth. Conclusion: PDK1/Akt‐mediated responsive cell growth is essential for normal liver regeneration after PH, especially when cell proliferation is impaired. (HEPATOLOGY 2009;49:204‐214.)

Redox factor-1/APE suppresses oxidative stress by inhibiting the rac1 GTPase

Oxidative stress triggered by many environmental and clinical insults results in cellular injury and death. The small GTPase rac1 promotes oxidative stress via the production of reactive oxygen species (ROS). In turn, the homeostatic response to such stress includes up‐regulation of the dual function reducing protein/DNA repair enzyme APE/redox factor‐1(ref‐1). In this report we explore the function and relationship between ref‐1 and rac1 in the setting of oxidative stress triggered by re‐oxygenation/reperfusion. In a model of mouse hepatic ischemia/reperfusion (I/R), recombinant adenoviral overexpression of ref‐1 resulted in suppression of reperfusion‐stimulated oxidative stress, NF‐κB induction, apoptosis, and acute injury, whereas down‐regulation of endogenous ref‐1 by adenoviral expression of antisense ref‐1 led to an increase in these reperfusion‐induced parameters. Ref‐1 also mitigated ROS production induced by adenoviral expression of an active form of rac1. Finally, overexpression of ref‐1 in primary hepatocytes suppressed reoxygenation‐stimulated rac1 activity. This work demonstrates a novel function of ref‐1 in inhibition of rac1 activity, and rac1‐mediated oxidative stress and injury.