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Thioredoxin Negatively Regulates p38 MAP Kinase Activation and IL-6 Production by Tumor Necrosis Factor-α
Biochemical and Biophysical Research Communications 258, 443– 447 (1999) Article ID bbrc.1999.0658, available online at http://www.idealibrary.com on
Thioredoxin Negatively Regulates p38 MAP Kinase Activation and IL-6 Production by Tumor Necrosis Factor-a Shu Hashimoto,* ,1 Ken Matsumoto,* Yasuhiro Gon,* Sachiko Furuichi,* Shuichiro Maruoka,* Ikuko Takeshita,* Kiichi Hirota,† Junji Yodoi,‡ and Takashi Horie* *First Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan; †Department of Anesthesia, Kyoto University Hospital, Kyto University, Kyto, Japan; and ‡Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan
Received April 5, 1999
We examined the regulatory role of a reduction/ oxidation (redox) control protein, thioredoxin (TRX), in tumor necrosis factor-a (TNF-a)-induced p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression utilizing TRX-transfected murine L929 cells (TRX14). The results showed that TNF-ainduced p38 MAP kinase activation and interleukin-6 (IL-6) production by TRX 14 were less than those by the parental L cells and the control transfected L cells (Neo-1). SB 203580 as the specific inhibitor for p38 MAP kinase activity inhibited TNF-a-induced IL-6 production by the parental L cells, indicating that TNF-a-activated p38 MAP kinase regulates IL-6 production by the cell lines used in this study. These results showed that overexpression of TRX negatively regulates p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production by TNF-a-stimulated cells, indicating that TRX is critical for p38 MAP kinase activation which regulates cytokine expression. © 1999 Academic Press
Many extracellular stimuli elicit the specific biological responses through the activation of mitogenactivated protein (MAP) kinase cascades (1). The activation of p38 MAP kinase which belongs to MAP kinase superfamily elicits a variety of biological responses including cytokine expression (2– 8). The mechanism of p38 MAP kinase activation has been extensively studied. We have independently shown p38 MAP kinase-mediated interleukin-8 (IL-8) expression (9) and thiol reducing agents including N-acetylcys1 Corresponding author: Shu Hashimoto, First Department of Internal Medicine, Nihon University School of Medicine, 30-1 Oyaguchikamimachi, Itabashi-ku, Tokyo 173-8610, Japan. Fax 1813-3972-2893. E-mail: [email protected].
teine (NAC)-mediated inhibition of IL-8 expression (10). These results indicated that cellular reduction/ oxidation (redox) state regulated by cellular thiols might be involved in p38 MAP kinase-mediated cytokine expression. However, a role of thioredoxin (TRX) which is a redox control protein in p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression has not been determined. TRX is a 12 kDa ubiquitous multifunctional protein having a redox-active disulfide/dithiol within its active site sequence, -Cys-Gly-Pro-Cys-, and operates together with NADPH and TRX reductase as a protein disulfide-reducing system (11–13). Human TRX was initially identified from the culture supernatants of a HTLV-1-infected cell line as an IL-2 receptor a-chain/ Tac inducer (14, 15). Cloning of the cDNA of this factor, ATL-derived factor (ADF), showed that it is a human homologue of a sulfhydryl reducing enzyme, TRX, first found in prokaryocytes (16, 17). TRX which is a redox control protein has a variety of biological activities (17). TRX has been reported to act as a antioxidant by reducing reactive oxygen species (ROS) (18), and protects cells against hydrogen peroxide- or tumor necrosis factor-a (TNF-a)-induced cytotoxicity, in which the involvement of ROS has been implicated (19, 20). Recently, it has been shown that TRX bound directly to the N-terminal portion of apoptosis signal-regulating kinase (ASK1) identified as a mitogen-activated protein (MAP) kinase kinase kinase which activates p38 MAP kinase and c-Jun-NH 2-terminal kinase (JNK) (21, 22). TRX has been reported to inhibit ASK-1 activity and reduce ASK1-dependent apoptosis through a direct inhibition of ASK-1 activity (22). Although the regulatory function of TRX in ASK-1-dependent apoptosis has been shown, a role of TRX in p38 MAP kinase-mediated cytokine expression has not been examined.
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In the present study, we examined the regulatory role of TRX in p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression. To this end, we examined p38 MAP kinase activation and interleukin 6 (IL-6) production with TNF-a stimulation utilizing TRX-transfected murine L929 cells. MATERIALS AND METHODS Reagents and cytokine. Murine recombinant TNF-a was obtained from CEDARLANE Laboratories Ltd. (Ontario, Canada). SB 203580 as a specific inhibitor for p38 MAP kinase activity (23), which was kindly provided by SmithKline Beecham and dissolved in dimethyl sulfoxide. Cell cultures. L929, a murine fibrosarcoma cell line (L cells), human TRX-transfected L929 cell clone, TRX14 (TRX14) and the control transfected clone, Neo-1 (Neo-1) used in this study were grown in culture medium which is Dulbecco’s modified Eagle’s medium (DMEM; Nissui Co. Ltd., Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (FCS; Mitsubishikasei Co. Ltd., Tokyo, Japan), streptomycin and penicillin (Meiji Pharmaceutical Co. Ltd., Tokyo, Japan). TRX14 and Neo-1 were continuously maintained in the presence of 200 mg/ml of G418. Characteristics of these cells were previously reported (24). The parental L929, Neo-1 and TRX14 (1 3 10 4 cells/ml) were placed onto tissue culture plate (Falcon 1007, Oxnard, CA) for western blot analysis of p38 MAP kinase and 24-well flat-bottomed tissue culture plate (Corning, Corning, NY) using culture medium and cultured at 37°C in humidified 5% CO 2 atmosphere. The cells were allowed to reach subconfluence, the culture medium was replaced with serum-free DMEM and the cells were cultured for 16 hours. For the western blot study, to examine the effect of TNF-a (10 ng/ml) on the threonine and tyrosine phosphorylation of p38 MAP kinase, serum-starved subconfluent cells were stimulated with TNF-a for the desired times. For the determination of IL-6 protein production and the effect of SB 203580 (10 mM) on it, serum-starved subconfluent cells that had been preincubated with serum-free DMEM containing or not SB 203580 for 1 hour were stimulated with TNF-a (10 ng/ml) and cultured for 24 hours, and the culture supernatants were collected, filtrated with a miliporefilter and stored at 280°C until assay. Measurement of IL-6. The concentrations of IL-6 in the culture supernatants were measured by commercially available ELISA kits (R & D Systems, Minneapolis, MN). ELISA was performed according to the manufacturer’s instruction. All samples were assayed in duplicate. Western blot analysis of the threonine and tyrosine phosphorylation of p38 MAP kinase. The threonine and tyrosine phosphorylation of p38 MAP kinase was analyzed by commercially available kits (PhosphoPlus p38 MAPK Antibody Kit, New England Biolabs, Inc., Beverly, MA). The kit employs anti-phospho-p38 MAP kinase which is specific for phosphorylated threonine and tyrosine kinase of p38 and dose not cross-react with phosphorylated threonine and tyrosine of ERK1/2 or JNK. Analysis of threonine and tyrosine phosphorylation of p38 MAP kinase was performed according to the manufacturer’s instructions. In order to show the amounts of p38 MAP kinase precipitated, blots were stripped and reprobed using phosphorylation-state independent p38 MAP kinase-specific antibody to determine total p38 MAP kinase levels (affinity purified rabbit polyclonal IgG). Statistical analysis. Statistical significance was analyzed using analysis of variance (ANOVA). P value less than 0.05 was considered significant.
RESULTS p38 MAP Kinase Activation in Parental L Cells, Neo-1 and TRX14 Activation of p38 MAP kinase is mediated by dual phosphorylation of the threonine and tyrosine residues of p38 MAP kinase. The levels of threonine and tyrosine phosphorylation reflect activation state of p38 MAP kinase (25). Therefore, we examined the threonine and tyrosine phosphorylation of p38 MAP kinase in TNF-a-stimulated parental L cells, the control transfected Neo-1 and the TRX-transfected TRX14. To this end, TNF-a-induced phosphorylation of p38 MAP kinase in TRX14 was compared with that in Neo-1 and the parental L cells. In preliminary experiments, serum-starved subconfluent cells were stimulated with TNF-a for 5, 10, 15, 30 and 60 minutes and the threonine and tyrosine phosphorylation of p38 MAP kinase was analyzed by western blotting. As the results, the amounts of phosphorylated threonine and tyrosine of p38 MAP kinase in each cell type was maximal at 5 minutes after stimulation (data not shown). Consequently, the threonine and tyrosine phosphorylation of p38 MAP kinase at 5 minutes after stimulation with TNF-a was examined in following experiments. The activation of p38 MAP kinase by TNF-a in TRX14 was less than that in Neo-1 and the parental L cells, whereas p38 MAP kinase activation in Neo-1 was comparable to that in the parental L cells (Fig. 1a, upper panel and Fig. 1b), indicating that overexpression of TRX negatively regulates TNF-a-induced activation of p38 MAP kinase. IL-6 Production by Parental L Cells, Neo-1 and TRX14 To clarify the regulatory role of TRX in TNF-ainduced IL-6 production, we stimulated the parental L cells, Neo-1 and TRX14 with TNF-a and the concentrations of IL-6 in the culture supernatants were determined. The concentration of IL-6 in the culture supernatants from TRX14 were lower than those from the parental L cells and Neo-1, whereas those from Neo-1 were comparable to those from the parental L cells (Fig. 2), indicating that overexpression of TRX negatively regulates TNF-a-activated signal pathway which regulates IL-6 production. SB 203580 Inhibits IL-6 Production by Parental L Cell We examined the effect of SB 203580 as the specific inhibitor for p38 MAP kinase activity on IL-6 production by TNF-a-stimulated parental L cells in order to clarify the role of p38 MAP kinase in TNF-a-induced IL-6 production. Abrogation of p38 MAP kinase activity by SB 203580 repressed TNF-a-induced IL-6 pro-
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FIG. 1. p38 MAP kinase activation in parental L cells, Neo-1 and TRX14. The parental L cells, Neo-1 and TRX14 were stimulated with TNF-a (10 ng/ml) for 5 minutes. After separating proteins from cell lysate by 15% SDS-PAGE, the lysates containing 10 mg of protein were transferred to membranes, and blotted with a specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase (phospho-p38 MAP kinase; upper panel of Fig. 1a). Blots shown in the upper panel of Fig. 1a were stripped and reprobed using a p38 MAP kinase-specific antibody to show the amounts of p38 MAP kinase blotted (p38 MAP kinase; lower panel of Fig. 1a). Lane P, positive protein prepared from C-6 glioma cells stimulated with anisomycin for phosphorylated threonine and tyrosine of p38 MAP kinase; Lane N, negative protein prepared from C-6 glioma cells unstimulated with anisomycin. Three identical experiments independently performed gave similar results. The amounts of p38 MAP kinase phosphorylation were quantitated by NIH image analyzer and are presented as the amounts of p38 MAP kinase phosphorylation relative to control cells treated without agonist (1.0) (Fig. 1b). The results are expressed as mean 6 s.d. of relative amounts of p38 MAP kinase phosphorylation in three different experiments. *, p , 0.01 compared with relative amounts of p38 MAP kinase phosphorylation in TNF-a-stimulated parental L cells and Neo-1. Relative amounts of p38 MAP kinase phosphorylation in TNF-a-stimulated Neo-1 are not statistically different from TNF-a-stimulated parental L cell culture.
duction by the parental L cells (Fig. 3), indicating that TNF-a-activated p38 MAP kinase regulates IL-6 production by the cell lines used in this study. DISCUSSION The mechanism of activation and the function of MAP kinases have been extensively studied (1). Inflammatory cytokines activate p38 MAP kinase and JNK and elicit a variety of cellular functions including cytokine expression and chemical mediator release, and apoptosis (1, 4 –7, 26, 27). In the present study, we examined the regulatory role of TRX in p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression by TRX utilizing TRX-transfected murine L929 cells. The results showed that TNF-a caused p38 MAP kinase activation and abrogation of p38 MAP kinase activity by SB 203580 repressed IL-6 production by TNF-a-stimulated parental parental L cells. These results indicated that TNF-a-induced IL-6 production was mediated by p38 MAP kinase. In order to clarify the regulatory role of TRX in p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production, we utilized TRX-transfected L cells. As the results, overexpression of TRX negatively regulated p38 MAP kinase activation and IL-6 production.
FIG. 2. IL-6 production by parental L cells, Neo-1 and TRX14. Parental L cells, Neo-1 and TRX14 were cultured with either medium or TNF-a (10 ng/ml) and the concentrations of IL-6 in the culture supernatants were determined at 24 hours after cultivation as described in Materials and Methods. The results are expressed as the mean 6 s.d. of IL-6 concentrations (pg/ml/1 3 10 5 cells) in five different experiments. *, p , 0.01 compared with IL-6 concentrations in the culture supernatants from TNF-a-stimulated parental L cells and those from TNF-a-stimulated Neo-1. IL-6 concentrations in TNF-a-stimulated Neo-1 culture are not statistically different from TNF-a-stimulated parental L cell culture.
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FIG. 3. SB 203580 inhibits TNF-a-induced IL-6 production by parental L cells. The parental L cells that had been preincubated with medium or SB 203580 (10 mM) for 1 hour were cultured either with medium or TNF-a (10 ng/ml). The concentrations of IL-6 in the culture supernatants were determined at 24 hours after cultivation as described in Materials and Methods. The concentrations of dimethyl sulfoxide used in this study were 0.01%, which had no effect. The results are expressed as the mean 6 s.d. of IL-6 concentrations (pg/ml/1 3 10 5 cells) in three different experiments. *, p , 0.01 compared with IL-6 concentrations in the culture supernatants from TNF-a-stimulated parental L cells cultured without SB 203580.
There are several possible mechanisms by which overexpression of TRX negatively regulates p38 MAP kinase activation. It has been shown that TRX bound directly to the N-terminal portion of ASK1 and overexpression of TRX inhibits ASK1 activity (21, 22). TRXmediated inhibition of ASK1 activity depends on their interaction and a reduced form of TRX is critical for the direct inhibition of ASK1 activity, but TRX-mediated inhibition of ASK1 activity is not due to scavenging effect of TRX on ROS (22). The TRX-transfected L cells (TRX14) used in this study was reported to show 2.9fold increase in TRX activity over the parental L cells. In contrast, the control transfected L cells, Neo-1, displayed no significant differences in TRX activity compared to the parental L cells (24). The present results with less activation of p38 MAP kinase in TRX14 with TNF-a stimulation might result from a direct inhibition of ASK1 activity by the interaction between a reduced form of overexpressed TRX and ASK1. Alternative mechanism of the negative regulation of p38 MAP kinase activation by TRX is considered. ROS generated by TNF-a have been proposed to act as signaling intermediates for TNF-a-induced cytokine expression and apoptosis, since ROS are generated by TNF-a stimulation (28, 29) and antioxidants inhibit TNF-a-induced cytokine expression and apoptosis (9, 22, 30, 31). Furthermore, ROS per se cause p38 MAP kinase activation (32–34). It might be possible that ROS generated by TNF-a act as signaling intermediates for p38 MAP kinase activation in this study. Since it has been shown that TNF-a causes ASK1 activation through ROS-mediated dimerization of ASK1 and NAC
reduces ASK1 activity (35), it might be possible that less activation of p38 MAP kinase in the TRX14 shown in this study resulted from scavenging ROS by thiol reducing properties of TRX. We have demonstrated that TRX reduces ROS generated by NADPH oxidase including Rac-1 resulting in the inhibition of p38 MAP kinase activation in response to epidermal growth factors (Hirota, K., et al., submitted for the publication). These results might support the hypothesis that the negative regulation by TRX of p38 MAP kinase activation in response to TNF-a resulted from scavenging ROS by TRX. Overexpression of TRX partially inhibited TNF-ainduced IL-6 production. Although a dose and redox state of TRX are critical for the inhibition of ASK1 activity (22) and JNK activation is also regulated by TRX through the interaction of TRX and ASK1 (21, 22), one possible intracellular signal is JNK-dependent pathway. JNK pathway has been reported to be involved in the cytokine expression (36, 37). Consequently, there might be parallel pathways to regulate IL-6 production with TNF-a stimulation in this study. From the data presented here, we conclude that TRX negatively regulates p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production by TNF-astimulated cells. These results indicate that TRX is critical for p38 MAP kinase activation which regulates cytokine expression as well as apoptosis. REFERENCES 1. Davis, R. J. (1994) Trends Biochem. Sci. 19, 470 – 473. 2. Han, J., Lee, J. D., Bibbs, L., and Ulevitch, R. L. (1994) Science 265, 808 – 811. 3. Foltz, I. N., Lee, J. C., Young, P. R., and Schrader, J. W. (1997) J. Biol. Chem. 272, 3296 –3301. 4. Sapiro, L., and Dinarello, C. A. (1995) Proc. Natl. Acad. Sci. USA. 92, 12230 –12234. 5. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Letter 364, 229 –233. 6. Beyaert, R., Cuenda, A., Berghe, W. V., Plaisance, S., Lee, J. C., Haegeman, G., Cohen, P., and Walter, F. (1996) EMBO J. 15, 1914 –1923. 7. Hahsimoto, S., Gon, Y., Matsumoto, K., Nakayama, T., Takeshita, I., and Horie, T. (1999) Am. J. Respir. Crit. Care Med. 159, 634 – 640. 8. Gon, Y., Hashimoto, S., Matsumoto, K., Nakayama, Y., Takeshita, I., and Horie, T. (1998) Biochem. Biophys. Res. Commun. 249, 156 –160. 9. Matsumoto, K., Hashimoto, S., Gon, Y., Nakayama, T., and Horie, T. (1998) J. Allergy Clin. Immunol. 101, 825– 831. 10. Matsumoto, K., Hashimoto, S., Gon, Y., Nakayama, T., Takizawa, H., and Horie, T. (1998) Respir. Med. 92, 512–519. 11. Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237–271. 12. Holmgren, A. (1989) J. Biol. Chem. 264, 13963–13966. 13. Holmgren, A. (1995) Methods Enzymol. 252, 199 –208. 14. Teshigwara, K., Maeda, K., Nishino, K., Nikaido, T., Uchiyama, T., Tsudo, M., Wano, Y., and Yodi, J. (1985) J. Mol. Cell. Immunol. 2, 17–26.
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Thioredoxin Negatively Regulates p38 MAP Kinase Activation and IL-6 Production by Tumor Necrosis Factor-a Shu Hashimoto,* ,1 Ken Matsumoto,* Yasuhiro Gon,* Sachiko Furuichi,* Shuichiro Maruoka,* Ikuko Takeshita,* Kiichi Hirota,† Junji Yodoi,‡ and Takashi Horie* *First Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan; †Department of Anesthesia, Kyoto University Hospital, Kyto University, Kyto, Japan; and ‡Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japan
Received April 5, 1999
We examined the regulatory role of a reduction/ oxidation (redox) control protein, thioredoxin (TRX), in tumor necrosis factor-a (TNF-a)-induced p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression utilizing TRX-transfected murine L929 cells (TRX14). The results showed that TNF-ainduced p38 MAP kinase activation and interleukin-6 (IL-6) production by TRX 14 were less than those by the parental L cells and the control transfected L cells (Neo-1). SB 203580 as the specific inhibitor for p38 MAP kinase activity inhibited TNF-a-induced IL-6 production by the parental L cells, indicating that TNF-a-activated p38 MAP kinase regulates IL-6 production by the cell lines used in this study. These results showed that overexpression of TRX negatively regulates p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production by TNF-a-stimulated cells, indicating that TRX is critical for p38 MAP kinase activation which regulates cytokine expression. © 1999 Academic Press
Many extracellular stimuli elicit the specific biological responses through the activation of mitogenactivated protein (MAP) kinase cascades (1). The activation of p38 MAP kinase which belongs to MAP kinase superfamily elicits a variety of biological responses including cytokine expression (2– 8). The mechanism of p38 MAP kinase activation has been extensively studied. We have independently shown p38 MAP kinase-mediated interleukin-8 (IL-8) expression (9) and thiol reducing agents including N-acetylcys1 Corresponding author: Shu Hashimoto, First Department of Internal Medicine, Nihon University School of Medicine, 30-1 Oyaguchikamimachi, Itabashi-ku, Tokyo 173-8610, Japan. Fax 1813-3972-2893. E-mail: [email protected].
teine (NAC)-mediated inhibition of IL-8 expression (10). These results indicated that cellular reduction/ oxidation (redox) state regulated by cellular thiols might be involved in p38 MAP kinase-mediated cytokine expression. However, a role of thioredoxin (TRX) which is a redox control protein in p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression has not been determined. TRX is a 12 kDa ubiquitous multifunctional protein having a redox-active disulfide/dithiol within its active site sequence, -Cys-Gly-Pro-Cys-, and operates together with NADPH and TRX reductase as a protein disulfide-reducing system (11–13). Human TRX was initially identified from the culture supernatants of a HTLV-1-infected cell line as an IL-2 receptor a-chain/ Tac inducer (14, 15). Cloning of the cDNA of this factor, ATL-derived factor (ADF), showed that it is a human homologue of a sulfhydryl reducing enzyme, TRX, first found in prokaryocytes (16, 17). TRX which is a redox control protein has a variety of biological activities (17). TRX has been reported to act as a antioxidant by reducing reactive oxygen species (ROS) (18), and protects cells against hydrogen peroxide- or tumor necrosis factor-a (TNF-a)-induced cytotoxicity, in which the involvement of ROS has been implicated (19, 20). Recently, it has been shown that TRX bound directly to the N-terminal portion of apoptosis signal-regulating kinase (ASK1) identified as a mitogen-activated protein (MAP) kinase kinase kinase which activates p38 MAP kinase and c-Jun-NH 2-terminal kinase (JNK) (21, 22). TRX has been reported to inhibit ASK-1 activity and reduce ASK1-dependent apoptosis through a direct inhibition of ASK-1 activity (22). Although the regulatory function of TRX in ASK-1-dependent apoptosis has been shown, a role of TRX in p38 MAP kinase-mediated cytokine expression has not been examined.
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In the present study, we examined the regulatory role of TRX in p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression. To this end, we examined p38 MAP kinase activation and interleukin 6 (IL-6) production with TNF-a stimulation utilizing TRX-transfected murine L929 cells. MATERIALS AND METHODS Reagents and cytokine. Murine recombinant TNF-a was obtained from CEDARLANE Laboratories Ltd. (Ontario, Canada). SB 203580 as a specific inhibitor for p38 MAP kinase activity (23), which was kindly provided by SmithKline Beecham and dissolved in dimethyl sulfoxide. Cell cultures. L929, a murine fibrosarcoma cell line (L cells), human TRX-transfected L929 cell clone, TRX14 (TRX14) and the control transfected clone, Neo-1 (Neo-1) used in this study were grown in culture medium which is Dulbecco’s modified Eagle’s medium (DMEM; Nissui Co. Ltd., Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (FCS; Mitsubishikasei Co. Ltd., Tokyo, Japan), streptomycin and penicillin (Meiji Pharmaceutical Co. Ltd., Tokyo, Japan). TRX14 and Neo-1 were continuously maintained in the presence of 200 mg/ml of G418. Characteristics of these cells were previously reported (24). The parental L929, Neo-1 and TRX14 (1 3 10 4 cells/ml) were placed onto tissue culture plate (Falcon 1007, Oxnard, CA) for western blot analysis of p38 MAP kinase and 24-well flat-bottomed tissue culture plate (Corning, Corning, NY) using culture medium and cultured at 37°C in humidified 5% CO 2 atmosphere. The cells were allowed to reach subconfluence, the culture medium was replaced with serum-free DMEM and the cells were cultured for 16 hours. For the western blot study, to examine the effect of TNF-a (10 ng/ml) on the threonine and tyrosine phosphorylation of p38 MAP kinase, serum-starved subconfluent cells were stimulated with TNF-a for the desired times. For the determination of IL-6 protein production and the effect of SB 203580 (10 mM) on it, serum-starved subconfluent cells that had been preincubated with serum-free DMEM containing or not SB 203580 for 1 hour were stimulated with TNF-a (10 ng/ml) and cultured for 24 hours, and the culture supernatants were collected, filtrated with a miliporefilter and stored at 280°C until assay. Measurement of IL-6. The concentrations of IL-6 in the culture supernatants were measured by commercially available ELISA kits (R & D Systems, Minneapolis, MN). ELISA was performed according to the manufacturer’s instruction. All samples were assayed in duplicate. Western blot analysis of the threonine and tyrosine phosphorylation of p38 MAP kinase. The threonine and tyrosine phosphorylation of p38 MAP kinase was analyzed by commercially available kits (PhosphoPlus p38 MAPK Antibody Kit, New England Biolabs, Inc., Beverly, MA). The kit employs anti-phospho-p38 MAP kinase which is specific for phosphorylated threonine and tyrosine kinase of p38 and dose not cross-react with phosphorylated threonine and tyrosine of ERK1/2 or JNK. Analysis of threonine and tyrosine phosphorylation of p38 MAP kinase was performed according to the manufacturer’s instructions. In order to show the amounts of p38 MAP kinase precipitated, blots were stripped and reprobed using phosphorylation-state independent p38 MAP kinase-specific antibody to determine total p38 MAP kinase levels (affinity purified rabbit polyclonal IgG). Statistical analysis. Statistical significance was analyzed using analysis of variance (ANOVA). P value less than 0.05 was considered significant.
RESULTS p38 MAP Kinase Activation in Parental L Cells, Neo-1 and TRX14 Activation of p38 MAP kinase is mediated by dual phosphorylation of the threonine and tyrosine residues of p38 MAP kinase. The levels of threonine and tyrosine phosphorylation reflect activation state of p38 MAP kinase (25). Therefore, we examined the threonine and tyrosine phosphorylation of p38 MAP kinase in TNF-a-stimulated parental L cells, the control transfected Neo-1 and the TRX-transfected TRX14. To this end, TNF-a-induced phosphorylation of p38 MAP kinase in TRX14 was compared with that in Neo-1 and the parental L cells. In preliminary experiments, serum-starved subconfluent cells were stimulated with TNF-a for 5, 10, 15, 30 and 60 minutes and the threonine and tyrosine phosphorylation of p38 MAP kinase was analyzed by western blotting. As the results, the amounts of phosphorylated threonine and tyrosine of p38 MAP kinase in each cell type was maximal at 5 minutes after stimulation (data not shown). Consequently, the threonine and tyrosine phosphorylation of p38 MAP kinase at 5 minutes after stimulation with TNF-a was examined in following experiments. The activation of p38 MAP kinase by TNF-a in TRX14 was less than that in Neo-1 and the parental L cells, whereas p38 MAP kinase activation in Neo-1 was comparable to that in the parental L cells (Fig. 1a, upper panel and Fig. 1b), indicating that overexpression of TRX negatively regulates TNF-a-induced activation of p38 MAP kinase. IL-6 Production by Parental L Cells, Neo-1 and TRX14 To clarify the regulatory role of TRX in TNF-ainduced IL-6 production, we stimulated the parental L cells, Neo-1 and TRX14 with TNF-a and the concentrations of IL-6 in the culture supernatants were determined. The concentration of IL-6 in the culture supernatants from TRX14 were lower than those from the parental L cells and Neo-1, whereas those from Neo-1 were comparable to those from the parental L cells (Fig. 2), indicating that overexpression of TRX negatively regulates TNF-a-activated signal pathway which regulates IL-6 production. SB 203580 Inhibits IL-6 Production by Parental L Cell We examined the effect of SB 203580 as the specific inhibitor for p38 MAP kinase activity on IL-6 production by TNF-a-stimulated parental L cells in order to clarify the role of p38 MAP kinase in TNF-a-induced IL-6 production. Abrogation of p38 MAP kinase activity by SB 203580 repressed TNF-a-induced IL-6 pro-
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FIG. 1. p38 MAP kinase activation in parental L cells, Neo-1 and TRX14. The parental L cells, Neo-1 and TRX14 were stimulated with TNF-a (10 ng/ml) for 5 minutes. After separating proteins from cell lysate by 15% SDS-PAGE, the lysates containing 10 mg of protein were transferred to membranes, and blotted with a specific antibody to phosphorylated threonine and tyrosine of p38 MAP kinase (phospho-p38 MAP kinase; upper panel of Fig. 1a). Blots shown in the upper panel of Fig. 1a were stripped and reprobed using a p38 MAP kinase-specific antibody to show the amounts of p38 MAP kinase blotted (p38 MAP kinase; lower panel of Fig. 1a). Lane P, positive protein prepared from C-6 glioma cells stimulated with anisomycin for phosphorylated threonine and tyrosine of p38 MAP kinase; Lane N, negative protein prepared from C-6 glioma cells unstimulated with anisomycin. Three identical experiments independently performed gave similar results. The amounts of p38 MAP kinase phosphorylation were quantitated by NIH image analyzer and are presented as the amounts of p38 MAP kinase phosphorylation relative to control cells treated without agonist (1.0) (Fig. 1b). The results are expressed as mean 6 s.d. of relative amounts of p38 MAP kinase phosphorylation in three different experiments. *, p , 0.01 compared with relative amounts of p38 MAP kinase phosphorylation in TNF-a-stimulated parental L cells and Neo-1. Relative amounts of p38 MAP kinase phosphorylation in TNF-a-stimulated Neo-1 are not statistically different from TNF-a-stimulated parental L cell culture.
duction by the parental L cells (Fig. 3), indicating that TNF-a-activated p38 MAP kinase regulates IL-6 production by the cell lines used in this study. DISCUSSION The mechanism of activation and the function of MAP kinases have been extensively studied (1). Inflammatory cytokines activate p38 MAP kinase and JNK and elicit a variety of cellular functions including cytokine expression and chemical mediator release, and apoptosis (1, 4 –7, 26, 27). In the present study, we examined the regulatory role of TRX in p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression by TRX utilizing TRX-transfected murine L929 cells. The results showed that TNF-a caused p38 MAP kinase activation and abrogation of p38 MAP kinase activity by SB 203580 repressed IL-6 production by TNF-a-stimulated parental parental L cells. These results indicated that TNF-a-induced IL-6 production was mediated by p38 MAP kinase. In order to clarify the regulatory role of TRX in p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production, we utilized TRX-transfected L cells. As the results, overexpression of TRX negatively regulated p38 MAP kinase activation and IL-6 production.
FIG. 2. IL-6 production by parental L cells, Neo-1 and TRX14. Parental L cells, Neo-1 and TRX14 were cultured with either medium or TNF-a (10 ng/ml) and the concentrations of IL-6 in the culture supernatants were determined at 24 hours after cultivation as described in Materials and Methods. The results are expressed as the mean 6 s.d. of IL-6 concentrations (pg/ml/1 3 10 5 cells) in five different experiments. *, p , 0.01 compared with IL-6 concentrations in the culture supernatants from TNF-a-stimulated parental L cells and those from TNF-a-stimulated Neo-1. IL-6 concentrations in TNF-a-stimulated Neo-1 culture are not statistically different from TNF-a-stimulated parental L cell culture.
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FIG. 3. SB 203580 inhibits TNF-a-induced IL-6 production by parental L cells. The parental L cells that had been preincubated with medium or SB 203580 (10 mM) for 1 hour were cultured either with medium or TNF-a (10 ng/ml). The concentrations of IL-6 in the culture supernatants were determined at 24 hours after cultivation as described in Materials and Methods. The concentrations of dimethyl sulfoxide used in this study were 0.01%, which had no effect. The results are expressed as the mean 6 s.d. of IL-6 concentrations (pg/ml/1 3 10 5 cells) in three different experiments. *, p , 0.01 compared with IL-6 concentrations in the culture supernatants from TNF-a-stimulated parental L cells cultured without SB 203580.
There are several possible mechanisms by which overexpression of TRX negatively regulates p38 MAP kinase activation. It has been shown that TRX bound directly to the N-terminal portion of ASK1 and overexpression of TRX inhibits ASK1 activity (21, 22). TRXmediated inhibition of ASK1 activity depends on their interaction and a reduced form of TRX is critical for the direct inhibition of ASK1 activity, but TRX-mediated inhibition of ASK1 activity is not due to scavenging effect of TRX on ROS (22). The TRX-transfected L cells (TRX14) used in this study was reported to show 2.9fold increase in TRX activity over the parental L cells. In contrast, the control transfected L cells, Neo-1, displayed no significant differences in TRX activity compared to the parental L cells (24). The present results with less activation of p38 MAP kinase in TRX14 with TNF-a stimulation might result from a direct inhibition of ASK1 activity by the interaction between a reduced form of overexpressed TRX and ASK1. Alternative mechanism of the negative regulation of p38 MAP kinase activation by TRX is considered. ROS generated by TNF-a have been proposed to act as signaling intermediates for TNF-a-induced cytokine expression and apoptosis, since ROS are generated by TNF-a stimulation (28, 29) and antioxidants inhibit TNF-a-induced cytokine expression and apoptosis (9, 22, 30, 31). Furthermore, ROS per se cause p38 MAP kinase activation (32–34). It might be possible that ROS generated by TNF-a act as signaling intermediates for p38 MAP kinase activation in this study. Since it has been shown that TNF-a causes ASK1 activation through ROS-mediated dimerization of ASK1 and NAC
reduces ASK1 activity (35), it might be possible that less activation of p38 MAP kinase in the TRX14 shown in this study resulted from scavenging ROS by thiol reducing properties of TRX. We have demonstrated that TRX reduces ROS generated by NADPH oxidase including Rac-1 resulting in the inhibition of p38 MAP kinase activation in response to epidermal growth factors (Hirota, K., et al., submitted for the publication). These results might support the hypothesis that the negative regulation by TRX of p38 MAP kinase activation in response to TNF-a resulted from scavenging ROS by TRX. Overexpression of TRX partially inhibited TNF-ainduced IL-6 production. Although a dose and redox state of TRX are critical for the inhibition of ASK1 activity (22) and JNK activation is also regulated by TRX through the interaction of TRX and ASK1 (21, 22), one possible intracellular signal is JNK-dependent pathway. JNK pathway has been reported to be involved in the cytokine expression (36, 37). Consequently, there might be parallel pathways to regulate IL-6 production with TNF-a stimulation in this study. From the data presented here, we conclude that TRX negatively regulates p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production by TNF-astimulated cells. These results indicate that TRX is critical for p38 MAP kinase activation which regulates cytokine expression as well as apoptosis. REFERENCES 1. Davis, R. J. (1994) Trends Biochem. Sci. 19, 470 – 473. 2. Han, J., Lee, J. D., Bibbs, L., and Ulevitch, R. L. (1994) Science 265, 808 – 811. 3. Foltz, I. N., Lee, J. C., Young, P. R., and Schrader, J. W. (1997) J. Biol. Chem. 272, 3296 –3301. 4. Sapiro, L., and Dinarello, C. A. (1995) Proc. Natl. Acad. Sci. USA. 92, 12230 –12234. 5. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., and Lee, J. C. (1995) FEBS Letter 364, 229 –233. 6. Beyaert, R., Cuenda, A., Berghe, W. V., Plaisance, S., Lee, J. C., Haegeman, G., Cohen, P., and Walter, F. (1996) EMBO J. 15, 1914 –1923. 7. Hahsimoto, S., Gon, Y., Matsumoto, K., Nakayama, T., Takeshita, I., and Horie, T. (1999) Am. J. Respir. Crit. Care Med. 159, 634 – 640. 8. Gon, Y., Hashimoto, S., Matsumoto, K., Nakayama, Y., Takeshita, I., and Horie, T. (1998) Biochem. Biophys. Res. Commun. 249, 156 –160. 9. Matsumoto, K., Hashimoto, S., Gon, Y., Nakayama, T., and Horie, T. (1998) J. Allergy Clin. Immunol. 101, 825– 831. 10. Matsumoto, K., Hashimoto, S., Gon, Y., Nakayama, T., Takizawa, H., and Horie, T. (1998) Respir. Med. 92, 512–519. 11. Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237–271. 12. Holmgren, A. (1989) J. Biol. Chem. 264, 13963–13966. 13. Holmgren, A. (1995) Methods Enzymol. 252, 199 –208. 14. Teshigwara, K., Maeda, K., Nishino, K., Nikaido, T., Uchiyama, T., Tsudo, M., Wano, Y., and Yodi, J. (1985) J. Mol. Cell. Immunol. 2, 17–26.
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