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Ischemia induces NF-kappa B activation and DNA synthesis in flow-adapted endothelial cells
I 134 I
I 135 I
CELLULAR RESPONSE TO PHENOLIC ANTIOXIDANTS
HIV-l TAT PROTEIN ACTIVATES ENDOTHELIAL CELL NF-KB IN A MANNER WHICH IS DISTINCT FROM TNF Gisela Vaitaitis, Natalia C. Flores, Ty Cashen and Sonia C. Flores Webb Waring Antioxidant Research Institute
Koii Uchida,t Yuko Naito,t Yasuyoshl Torii,’ Yosbimasa Nakamura,2 Takahito Kondo,3 and Toshihiko Osawa* *Lab. Food & Biodynam., Nagoya Univ. Grad. Sch. Bioagr. Sci., 2Kyoto Univ. Dept. Agr., and 3Nagasaki Univ. Med. Sch., Japan. In the present study, we develop a cell culture system that potently respond to phenolic antioxidants and find a differential regulatory mechanism of GST gene expression. We screened multiple cell lines for inducing GST activity by terr-butylhydroquinone (t-BHQ) and found that only RL34 cells made significant induction. Among phenolic compounds tested, GST activity in RL34 cells was potently induced by hydroquinones and slightly by monophenols and 1,2-diphenols, whereas resorcinol (1,3-diphenol) was inactive. In addition to GST activity, t-BHQ induced enzyme activity of other phase II detoxification enzymes. Immunoblot analysis of GST isozymes demonstrated a significant increase in the level of GSTAl, GSTA3. and GSTPl. The enhanced oroduction of these isozvmes in gene expression was also evident in a drastic elevation of m&A levels. The t-BHQ-induced GST induction was associated with enhanced gene expression of y-glutamylcysteine synthetase, leading to significant increase in GSH level. Pretreatment of the cells with N-acetvlcvsteine comnletelv blocked the tBHQ-induced exnression of GSTAi. but not dSTA3 and GSTPl. sueeestine that ‘GSTAI gene expression may be differentially r&$lateYd from other isozymes. The observations that (i) the redox-active t-BHQ-GSH addicts were detected in the cells, (ii) Hz02 preferentially induced GSTAl. and Ciii‘lt-BHO induced bindine activitv of antioxidant response element’(ARE) of GSTAl suggesyed the i&olvement of an electrophilic signal transduction pathway toward ARE on the gene expression of GSTAl.
1137 1
I 136 I ISCHEMIA SYNTHESIS
INDUCES NF-KAPPA B ACTIVATION IN FLOW-ADAPTED ENDOTHELIAL CELLS
Activation of NF-KB by pro-inflammatory cytokines and/or changes in redox status results in up-regulation of, for example, endothelial cell E-selectin. Because Tat-mediated inhibition of MnSOD results in redox status alterations, we investigated the effects of Tat on the DNA binding activity of endothelial cell NFKB family members. Cells were exposed to Tat and/or TNF and nuclear proteins were extracted and subjected to electrophoretic mobility shift assays. When a consensus KB oligo was used, 3 or 4 nucleoprotein complexes were observed. Supershift analyses demonstrated that while TNF up-regulates all complexes, Tat mostly up-regulates ~50. Treatment with an SOD mimetic abolished Tat-dependent, but not TNF-dependent, p50 upregulation, suggesting that the Tat-mediated changes are redoxsensitive. The kinetics of NF-KB activation differed between Tat and TNF, with TNF resulting in a more persistent response. Only two nucleoprotein complexes, consisting of p52 and ~65, but not ~50, were detected when an oligo containing the authentic KB site of the E-selectin gene was used. While Tat and TNF acted synergistically on DNA binding, Western blot analysis demonstrated that RelB was up-regulated only with TNF. These results suggest that Tat activates NF-KB family members selectively, and with different kinetics from TNF.
AND
Zhihua H. Wei, A. Al-Mehdi, C. Dodia, K. Debolt and A. B. Fisher. for Environ. Med., Uni. Penn Med. Center, Philadelphia, PA 19104
DNA Inst.
INDUCTION OF HUMAN GAMMA-GLUTAMYLCYSTEINE SYNTHETASE SUBUNIT GENES BY PYRROLIDINE DITHIOCARBAMATE
Time and dosedependent increases in the steady state mRNA levels of the genes encoding the catalytic and regulatory subunits of the enzyme gamma-glutamylcysteine synthetase (GCS) were observed in HepG2 human hepatocarcinorna cells after exposure to pyrrolidine dithiocarbamate (PDTC). PDTC was demonstrated to manifest both antioxidant and pro-oxidant properties in HepGP cells, as assessed by the fluorescence of the redox-sensitive dye dihvdrorhodamine 123 and by the oxidation of glutathione,resPe&ely. Attempts to characterize the sienalhne nathwav from PDTC exnosure to the increases in the exupresSio~of the &S catalytic and regulatory subunit genes demonstrated that induction by PDTC could be partiaEy~blocked by treatment with the thiol agent N-acetylcysteine and by the copper chelator bathocuproine dlulfonic add. These flncimgs suggested that the upregulation of the two genes resulted from a PDTC-induced pro-oxidant signal which was partially copper-dependent. Preliminary evaluation of cis elementa within the genes’promoters conferring PDTC responsiveness in HepG2 cells indicated a role for a ureviouslv characterized Antioxidant Resnonse Element (ARE) hthin the 5’-flanking region of the GCS &tlytlc subunit gene and an ARE/AP-1 binding site pair within the promoter of the GCS reeulatorv subunit eene. In summarv, theaestudies demon&ated~that PDTC exposure eli& a cellular response In HepG2 cells characterized by the induction of the genes encoding the two subunits of the enzyme GCS and increased de nova synthesis of the cellular protectant glutathione.
OXYGEN
’ 9
8
s53
I 135 I
CELLULAR RESPONSE TO PHENOLIC ANTIOXIDANTS
HIV-l TAT PROTEIN ACTIVATES ENDOTHELIAL CELL NF-KB IN A MANNER WHICH IS DISTINCT FROM TNF Gisela Vaitaitis, Natalia C. Flores, Ty Cashen and Sonia C. Flores Webb Waring Antioxidant Research Institute
Koii Uchida,t Yuko Naito,t Yasuyoshl Torii,’ Yosbimasa Nakamura,2 Takahito Kondo,3 and Toshihiko Osawa* *Lab. Food & Biodynam., Nagoya Univ. Grad. Sch. Bioagr. Sci., 2Kyoto Univ. Dept. Agr., and 3Nagasaki Univ. Med. Sch., Japan. In the present study, we develop a cell culture system that potently respond to phenolic antioxidants and find a differential regulatory mechanism of GST gene expression. We screened multiple cell lines for inducing GST activity by terr-butylhydroquinone (t-BHQ) and found that only RL34 cells made significant induction. Among phenolic compounds tested, GST activity in RL34 cells was potently induced by hydroquinones and slightly by monophenols and 1,2-diphenols, whereas resorcinol (1,3-diphenol) was inactive. In addition to GST activity, t-BHQ induced enzyme activity of other phase II detoxification enzymes. Immunoblot analysis of GST isozymes demonstrated a significant increase in the level of GSTAl, GSTA3. and GSTPl. The enhanced oroduction of these isozvmes in gene expression was also evident in a drastic elevation of m&A levels. The t-BHQ-induced GST induction was associated with enhanced gene expression of y-glutamylcysteine synthetase, leading to significant increase in GSH level. Pretreatment of the cells with N-acetvlcvsteine comnletelv blocked the tBHQ-induced exnression of GSTAi. but not dSTA3 and GSTPl. sueeestine that ‘GSTAI gene expression may be differentially r&$lateYd from other isozymes. The observations that (i) the redox-active t-BHQ-GSH addicts were detected in the cells, (ii) Hz02 preferentially induced GSTAl. and Ciii‘lt-BHO induced bindine activitv of antioxidant response element’(ARE) of GSTAl suggesyed the i&olvement of an electrophilic signal transduction pathway toward ARE on the gene expression of GSTAl.
1137 1
I 136 I ISCHEMIA SYNTHESIS
INDUCES NF-KAPPA B ACTIVATION IN FLOW-ADAPTED ENDOTHELIAL CELLS
Activation of NF-KB by pro-inflammatory cytokines and/or changes in redox status results in up-regulation of, for example, endothelial cell E-selectin. Because Tat-mediated inhibition of MnSOD results in redox status alterations, we investigated the effects of Tat on the DNA binding activity of endothelial cell NFKB family members. Cells were exposed to Tat and/or TNF and nuclear proteins were extracted and subjected to electrophoretic mobility shift assays. When a consensus KB oligo was used, 3 or 4 nucleoprotein complexes were observed. Supershift analyses demonstrated that while TNF up-regulates all complexes, Tat mostly up-regulates ~50. Treatment with an SOD mimetic abolished Tat-dependent, but not TNF-dependent, p50 upregulation, suggesting that the Tat-mediated changes are redoxsensitive. The kinetics of NF-KB activation differed between Tat and TNF, with TNF resulting in a more persistent response. Only two nucleoprotein complexes, consisting of p52 and ~65, but not ~50, were detected when an oligo containing the authentic KB site of the E-selectin gene was used. While Tat and TNF acted synergistically on DNA binding, Western blot analysis demonstrated that RelB was up-regulated only with TNF. These results suggest that Tat activates NF-KB family members selectively, and with different kinetics from TNF.
AND
Zhihua H. Wei, A. Al-Mehdi, C. Dodia, K. Debolt and A. B. Fisher. for Environ. Med., Uni. Penn Med. Center, Philadelphia, PA 19104
DNA Inst.
INDUCTION OF HUMAN GAMMA-GLUTAMYLCYSTEINE SYNTHETASE SUBUNIT GENES BY PYRROLIDINE DITHIOCARBAMATE
Time and dosedependent increases in the steady state mRNA levels of the genes encoding the catalytic and regulatory subunits of the enzyme gamma-glutamylcysteine synthetase (GCS) were observed in HepG2 human hepatocarcinorna cells after exposure to pyrrolidine dithiocarbamate (PDTC). PDTC was demonstrated to manifest both antioxidant and pro-oxidant properties in HepGP cells, as assessed by the fluorescence of the redox-sensitive dye dihvdrorhodamine 123 and by the oxidation of glutathione,resPe&ely. Attempts to characterize the sienalhne nathwav from PDTC exnosure to the increases in the exupresSio~of the &S catalytic and regulatory subunit genes demonstrated that induction by PDTC could be partiaEy~blocked by treatment with the thiol agent N-acetylcysteine and by the copper chelator bathocuproine dlulfonic add. These flncimgs suggested that the upregulation of the two genes resulted from a PDTC-induced pro-oxidant signal which was partially copper-dependent. Preliminary evaluation of cis elementa within the genes’promoters conferring PDTC responsiveness in HepG2 cells indicated a role for a ureviouslv characterized Antioxidant Resnonse Element (ARE) hthin the 5’-flanking region of the GCS &tlytlc subunit gene and an ARE/AP-1 binding site pair within the promoter of the GCS reeulatorv subunit eene. In summarv, theaestudies demon&ated~that PDTC exposure eli& a cellular response In HepG2 cells characterized by the induction of the genes encoding the two subunits of the enzyme GCS and increased de nova synthesis of the cellular protectant glutathione.
OXYGEN
’ 9
8
s53