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Activation of extracellular signal-regulated kinases by EGF and oxidants in lung epithelial cells
I 176
11771
A CRITICAL APPRAISAL OF HYDROGEN PEROXIDE AND SUPEROXIDE AS SIGNALING MOLECULES USING THE JWSAPK MAP KINASE PATHWAY D. R. Moellering, Y.-M. Go, M. C. Maland, R. P. Patel, H. Jo, and V. M. Darley-Usmar. Department of Pathology, University Of Alabama at Birmingham, Birmingham, AL. 35294 The c-Jun NHrterminal protein kinases (JNKs), also known as stress activated protein kinases (SAPK), are redox sensitive signaling molecules activated by a number of stimuli, including reactive oxygen or nitrogen species. It has been suggested that JNK activation can be elicited by hydrogen peroxide (HZ02) or superoxide (Oz. ) but the high non-physiological concentrations of ROS this requires cast doubt on the biological relevance of these findings. Indeed, we found that bolus addition of 25C-500 )IM HZOZwas required to induce JNK activation in bovine aortic endothelial cells (BAEC). However, intracellular generation of ROS in signaling domains may be far more effective in activating JNK and this was addressed using 2,bDimethoxyThis compound is a non1,6naphthoquinone (DMNQ). alkylating redox cycling agent that specifically generates 02-. In the cell this will also be converted to hydrogen peroxide by At non -toxic catalase and glutathione peroxidases concentrations DMNQ (5 *M) activated JNK in a time and concentration-dependent manner. JNK activation was transient with a maximal stimulation at 15 minutes (2.5 fold), while These levels of returning to baseline within 60 minutes. activation are similar to those found with bolus addition of H202. Rates of productton of O< and H202 in the presence of DMNQ indicate activation in the FM/min concentration range; far lower than bolus additions and physiologically plausible.
EFFECTS OF NITROGLYCERIN TREATMENT ON NOS III GENE EXPRESSION AND SUPEROXIDE PRODUCTION T MwI;PL U. Hink, H. Li, H. Mollnnu, U. Forstermann. University Hospital HornburR
20246
GERMANY.
Chronic nitroglycerin (NTG) treatment has been shown to cause cross tolerance to endothelium dependent vasodilators, which may involve increased production of superoxide (02- ). It remains to be elucidated, to what extent chronic treatment with NTG may alter the activity and expression of the nitric oxide synthase (NOS III) gene and whether 02- may influence the bioactivity of NO in the setting of nitrate tolerance. We therefore studied the influence of chronic NTG-treatment on NOS III-expression assessed by Western blot and RNase protection assay. Superoxide (OZ- ) as well as NO formation were quantified using lucigenin- and CLA-enhanced chemiluminescence (CL) as well as electron spin resonance (ESR) spectroscopy. Treatment of Wistar rats with NTG for 3d caused marked tolerance, cross-tolerance to the endothelium dependent vasodilator acetylcholine and a significant increase in lucigenin and CLA-CL. Tolerance was associated with a significant increase in NOS III mRNA by 150*3% and NOS III protein by 139+17%. In control vessels, the inhibitor of the nitric oxide synthase NG-nitro-L-arginine (L-NNA) increased vascular Oowhile in the setting of tolerance L-NNA decreased steady state 02--levels compatible with NOS III-mediated 02- formation. Vascular NO-bioavailability assessed with ESR-spectroscopy revealed a strong reduction in vascular NO in the setting of nitrate tolerance. These findings suggest that NTG treatment increases expression of a dysfunctional NOS III leading to decreased vascular NO-bioavailability and to increased formation of 02..
564
The OxyR transcription factor is directly sensitive to oxidation and activates the expression of antioxidant genes such as katG (cat&se), oxyS (a small regulatory RNA) in response to hydrogen peroxide in Escherichia coli. OxyR is activated through the formation of a disulfide bond (between Cys199 and Cys208) and is deactivated by enzymatic reduction with glutaredoxin 1. The redox potential of OxyR (-185mV) ensures that OxyR is reduced in absence of stress. To gain insight into the specificity of OxyR sensing we compared the effects of hydrogen peroxide, diamide, and different NO-producing compounds on OxyR activation of the oxyS target gene. Hydrogen peroxide lead to the highest induction of the oxyS (25-fold) followed by diamide @l-fold), GSNO (S-fold), acidified NaN02(7-fold), SNOCys (2-fold), acidified NaN03(no effect) and sodium nitroprusside (SNI’, no effect). In contrast, the expression of hrnp (flavohaemoglobin), an NO regulated gene, was induced 5-fold by GSNO followed by SNP (4-fold), NaN02 (3.7-fold), SNOCys (1.7-fold), diamide (1.5-fold), NaN03 (1.3-fold) and hydrogen peroxide (no effect). To probe the contributions of the conserved residues surrounding Cl99 and C208 (194-205) to the activation mechanism, we replaced each of the residues with alanine. Strains expressing the OxyR derivatives H198D, H198E, R201A, D196A and D202A were hypersensitive to hydrogen peroxide. Thus we suggest that the basic residues are involved in lowering the pKa of the C199-SH group or in stabilizing the thiolate form of Cl99 and the acidic residues are involved in protonating the leaving group (-OH) of hydrogen peroxide. We are now further investigating the exact contributions of each of the residues by analyzing the kinetics of C199-C208 disulfide bond formation.
11791
11781 Epyendorf,
THE MECHANISM OF REDOX SENSING BY THE OXYR TRANSCRIPTION FACTOR PARTHA MUKHOPADHYAYnnd GlSELA STORZ. CBMB/NICHDINlH. Rcthesdn,MD 20892.
OXYGEN
ACTIVATION OF EXTRACELLULAR SIGNAL-REGULATED KINASES BY EGF AND OXIDANTS IN LUNG EPITHELIAL CELLS. 43thy I Palmer. Sylke Buder-Ho mm Douglas ‘. Mom& Deportment of Pntho Pqy, Unmersm,
I. Tnntjes, and Brooke T. Burlin@m, VT.
of Vermont,
The Extracellular Signal-Regulated Kinase (ERK) pathway is induced by cytokines and oxidative stress and initiates transcriptional events resulting in proliferation and/or apoptosis. In studies here, we examined, using Western blot analyses and immunocytochemistry, the patterns of expression of phosphorylated ERK l/2 proteins (I’-ERK) induced by the agents, epidermal growth factor (EGF, 5 ng/ml), hydrogen peroxide (100-300 uM) and asbestos (5 ug/cm’) in murine alveolar type II epithelial cells (Cl0 line). In addition, we used laser scanning cytometry (LSC) to determine the time frame of nuclear translocation of I’-ERK and its relationship to cell cycle alterations, proliferation, and apoptosis by these agents. EGF, an agent inducing proliferation, caused increases (pc.05) in P-ERK which peaked at 15 minutes and were elevated for 4 hrs. LSC also showed rapid (15 minutes) increases in nuclear localization of phosphorylated ERKs. In contrast, hydrogen peroxide-exposed cells, which demonstrated dose-related development of apoptosis, exhibited similar patterns of increased I’-ERK by Western blot analyses but more protracted nuclear localization, i.e. 2 hours, as detected by LSC. Addition of asbestos caused apoptosis and more protracted increases in phosphorylated ERKs which were elevated (pc.05) from l-24 hours. However, the time frame of nuclear translocation of P-ERK was similar to that observed with hydrogen peroxide. These studies indicate that the kinetics of ERK phosphorylation, nuclear translocation, and transcriptional activation of target genes may differ with various stimuli. Supported by grants T32ES0712, ROlES/HL09213 and ROlHL39469.
’ 9
9
11771
A CRITICAL APPRAISAL OF HYDROGEN PEROXIDE AND SUPEROXIDE AS SIGNALING MOLECULES USING THE JWSAPK MAP KINASE PATHWAY D. R. Moellering, Y.-M. Go, M. C. Maland, R. P. Patel, H. Jo, and V. M. Darley-Usmar. Department of Pathology, University Of Alabama at Birmingham, Birmingham, AL. 35294 The c-Jun NHrterminal protein kinases (JNKs), also known as stress activated protein kinases (SAPK), are redox sensitive signaling molecules activated by a number of stimuli, including reactive oxygen or nitrogen species. It has been suggested that JNK activation can be elicited by hydrogen peroxide (HZ02) or superoxide (Oz. ) but the high non-physiological concentrations of ROS this requires cast doubt on the biological relevance of these findings. Indeed, we found that bolus addition of 25C-500 )IM HZOZwas required to induce JNK activation in bovine aortic endothelial cells (BAEC). However, intracellular generation of ROS in signaling domains may be far more effective in activating JNK and this was addressed using 2,bDimethoxyThis compound is a non1,6naphthoquinone (DMNQ). alkylating redox cycling agent that specifically generates 02-. In the cell this will also be converted to hydrogen peroxide by At non -toxic catalase and glutathione peroxidases concentrations DMNQ (5 *M) activated JNK in a time and concentration-dependent manner. JNK activation was transient with a maximal stimulation at 15 minutes (2.5 fold), while These levels of returning to baseline within 60 minutes. activation are similar to those found with bolus addition of H202. Rates of productton of O< and H202 in the presence of DMNQ indicate activation in the FM/min concentration range; far lower than bolus additions and physiologically plausible.
EFFECTS OF NITROGLYCERIN TREATMENT ON NOS III GENE EXPRESSION AND SUPEROXIDE PRODUCTION T MwI;PL U. Hink, H. Li, H. Mollnnu, U. Forstermann. University Hospital HornburR
20246
GERMANY.
Chronic nitroglycerin (NTG) treatment has been shown to cause cross tolerance to endothelium dependent vasodilators, which may involve increased production of superoxide (02- ). It remains to be elucidated, to what extent chronic treatment with NTG may alter the activity and expression of the nitric oxide synthase (NOS III) gene and whether 02- may influence the bioactivity of NO in the setting of nitrate tolerance. We therefore studied the influence of chronic NTG-treatment on NOS III-expression assessed by Western blot and RNase protection assay. Superoxide (OZ- ) as well as NO formation were quantified using lucigenin- and CLA-enhanced chemiluminescence (CL) as well as electron spin resonance (ESR) spectroscopy. Treatment of Wistar rats with NTG for 3d caused marked tolerance, cross-tolerance to the endothelium dependent vasodilator acetylcholine and a significant increase in lucigenin and CLA-CL. Tolerance was associated with a significant increase in NOS III mRNA by 150*3% and NOS III protein by 139+17%. In control vessels, the inhibitor of the nitric oxide synthase NG-nitro-L-arginine (L-NNA) increased vascular Oowhile in the setting of tolerance L-NNA decreased steady state 02--levels compatible with NOS III-mediated 02- formation. Vascular NO-bioavailability assessed with ESR-spectroscopy revealed a strong reduction in vascular NO in the setting of nitrate tolerance. These findings suggest that NTG treatment increases expression of a dysfunctional NOS III leading to decreased vascular NO-bioavailability and to increased formation of 02..
564
The OxyR transcription factor is directly sensitive to oxidation and activates the expression of antioxidant genes such as katG (cat&se), oxyS (a small regulatory RNA) in response to hydrogen peroxide in Escherichia coli. OxyR is activated through the formation of a disulfide bond (between Cys199 and Cys208) and is deactivated by enzymatic reduction with glutaredoxin 1. The redox potential of OxyR (-185mV) ensures that OxyR is reduced in absence of stress. To gain insight into the specificity of OxyR sensing we compared the effects of hydrogen peroxide, diamide, and different NO-producing compounds on OxyR activation of the oxyS target gene. Hydrogen peroxide lead to the highest induction of the oxyS (25-fold) followed by diamide @l-fold), GSNO (S-fold), acidified NaN02(7-fold), SNOCys (2-fold), acidified NaN03(no effect) and sodium nitroprusside (SNI’, no effect). In contrast, the expression of hrnp (flavohaemoglobin), an NO regulated gene, was induced 5-fold by GSNO followed by SNP (4-fold), NaN02 (3.7-fold), SNOCys (1.7-fold), diamide (1.5-fold), NaN03 (1.3-fold) and hydrogen peroxide (no effect). To probe the contributions of the conserved residues surrounding Cl99 and C208 (194-205) to the activation mechanism, we replaced each of the residues with alanine. Strains expressing the OxyR derivatives H198D, H198E, R201A, D196A and D202A were hypersensitive to hydrogen peroxide. Thus we suggest that the basic residues are involved in lowering the pKa of the C199-SH group or in stabilizing the thiolate form of Cl99 and the acidic residues are involved in protonating the leaving group (-OH) of hydrogen peroxide. We are now further investigating the exact contributions of each of the residues by analyzing the kinetics of C199-C208 disulfide bond formation.
11791
11781 Epyendorf,
THE MECHANISM OF REDOX SENSING BY THE OXYR TRANSCRIPTION FACTOR PARTHA MUKHOPADHYAYnnd GlSELA STORZ. CBMB/NICHDINlH. Rcthesdn,MD 20892.
OXYGEN
ACTIVATION OF EXTRACELLULAR SIGNAL-REGULATED KINASES BY EGF AND OXIDANTS IN LUNG EPITHELIAL CELLS. 43thy I Palmer. Sylke Buder-Ho mm Douglas ‘. Mom& Deportment of Pntho Pqy, Unmersm,
I. Tnntjes, and Brooke T. Burlin@m, VT.
of Vermont,
The Extracellular Signal-Regulated Kinase (ERK) pathway is induced by cytokines and oxidative stress and initiates transcriptional events resulting in proliferation and/or apoptosis. In studies here, we examined, using Western blot analyses and immunocytochemistry, the patterns of expression of phosphorylated ERK l/2 proteins (I’-ERK) induced by the agents, epidermal growth factor (EGF, 5 ng/ml), hydrogen peroxide (100-300 uM) and asbestos (5 ug/cm’) in murine alveolar type II epithelial cells (Cl0 line). In addition, we used laser scanning cytometry (LSC) to determine the time frame of nuclear translocation of I’-ERK and its relationship to cell cycle alterations, proliferation, and apoptosis by these agents. EGF, an agent inducing proliferation, caused increases (pc.05) in P-ERK which peaked at 15 minutes and were elevated for 4 hrs. LSC also showed rapid (15 minutes) increases in nuclear localization of phosphorylated ERKs. In contrast, hydrogen peroxide-exposed cells, which demonstrated dose-related development of apoptosis, exhibited similar patterns of increased I’-ERK by Western blot analyses but more protracted nuclear localization, i.e. 2 hours, as detected by LSC. Addition of asbestos caused apoptosis and more protracted increases in phosphorylated ERKs which were elevated (pc.05) from l-24 hours. However, the time frame of nuclear translocation of P-ERK was similar to that observed with hydrogen peroxide. These studies indicate that the kinetics of ERK phosphorylation, nuclear translocation, and transcriptional activation of target genes may differ with various stimuli. Supported by grants T32ES0712, ROlES/HL09213 and ROlHL39469.
’ 9
9