Immunobiol., vol. 198, pp. 273-278 (1997)

Redox Regulation

Servicio de Inmunologia, Hospital de la Princesa y Centro de Biologia Molecular, and lInstitutode Investigaciones Biomedicas, CSIC-UAM, Madrid, Spain

Antioxidants and Ap· 1 Activation: A Brief Overview

PABLO G6MEZ DEL ARCO, SARA MARTINEZ-MARTINEZ, VICTOR CALVO!,ANGEL LUIS ARMESILLA, and JUAN MIGUEL REDONDO

Abstract

Activity of the transcription factor AP-l is controlled by different MAPK cascades that regulatethe different AP-l components at the transcriptional and posttranscriptional level. Recently,AP-l has been shown to behave as a redox-sensitive transcription factor that can be inducedunder both pro-oxidative and antioxidative conditions. In this overview we summarize the sig­naling pathways that converge on the activation of AP-l and the components of these pathwaysthat have been shown to be targets of antioxidants. The activation of AP-l by antioxidants mayaccount for the expression of a number of genes that mediate important functions under physio­logical conditions.

Signaling pathways involved in AP- 1 activation

AP-l is a transcription factor composed of homodimers or heterodimers ofmembers of Fos and Jun families which binds to the TRE (TPA responsive ele­ment) motif to mediate gene transcription. AP-l regulates the gene expressionprogram in response to signals generated by a wide array of extracellular stimuliincluding growth factors, tumor promoters, neurotransmitters, UV light, andcytokines (1, 2).

The activity of AP-l is regulated by the transcription factors involved in theactivation of the c-Ios and the c-jun promoter elements as well as by the post­translational modification of both the pre-formed and de novo synthesized AP-lmembers, which ensure the immediate induction of AP-l-dependent transcrip­tion. The transcriptional and posttranslational regulators of AP-l activity are inturn regulated through different signaling cascades, thereby explaining the versa­tility of AP-l to respond to such a spectrum of stimuli (1, 3, 4).

In mammalian cells, three different MAPK cascades have so far been involvedin the induction of AP-l activity; ERK 1 and 2, the SAPKsIJNKs and p38/RKIMpK2/CSBP kinase cascades (3,4).

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274 . P. G6MEZ DEL ARea et al.

The Ras/ERK pathway is triggered by growth factors and cytokines and itsactivation is related with the stimulation of tyrosine kinase receptors which elic­its a signaling cascade involving Ras activation, recruitment of Raf kinase to theplasma membrane, and sequential activation/phosphorylation of MEK 1,2, andERK 1, 2. The major regulatory element of the c-fos promoter, the serumresponse element (SRE), is recognized by the ternary complex factor (TCF)member Elk-1 and by the dimeric serum responsive factor (SRF). Elk-1 is phos-

EXTRACELLULAR STIMULI

c%~~s

~SRE

~Ras • Rac,Cdc42 Rac,Cdc42... ...... ...... ...

ERKI/2 JNKs/SAPKs p38/RK

t===:J,1c::,==::::J'1C::'==::::J'lt=,==::::JERKI/2 JNKs/SAPKs p38/RK

~~~.. \c-jnn::1ifP~ TRE

~

VAP-l

Figure 1. Regulation of AP-I by extracellular stimuli. Signals transmitted by membrane recep­tors are transduced into the nucleus through the ERK1I2, JNKs/SAPKs and p38/RK MAPKcascades. The transcription factors Elk-I, Sap-la, c-jun and ATF-2 couple the activation of dif­ferent MAPKs with the transcriptional activation of c-fos and c-jun promoters.

Antioxidant-induced AP-l activation . 275

phorylated by ERK-1 and ERK-2, thereby facilitating the SRE ternary complexformation and coupling the activation of the Ras/ERK pathway with the tran­scriptional induction of c-Ios (5).

The jNKs/SAPKs and p38/RK cascades are preferentially triggered by proin­flammatory cytokines and cellular stress. jNKs/SAPKs are phosphorylated bySEKl/MKK4 which is in turn phosphorylated and activated by MEKKl. Simi­larly, the activation/phosphorylation of MKK3/6 leads to the activation ofp38/RK (4, 6). These cascades can also be activated by members of the Rassuperfamily of small GTPases such as Rae and Cdc42, two Rho-like proteins,that in the case of the jNK/SAPK cascade have been reported to act synergisti­cally with Ras that appears to be an alternative upstream component of thispathway (4, 7). jNKs/SAPKs and p38/RK cascades are also connected with theactivation of AP-l. Thus, two members of the TCF family Elk-l and Sap-la aswell as ATF-2 and c-jun are substrates of jNKs/SAPKs in activated cells, andp38/RK has been reported to phosphorylate Elk-l and ATF-2 (4-8). Therefore,the activation of ERKs, JNKs/SAPKs and p38/RK cascades converge at theTCF level and the three pathways are potential regulators of the c-Ios genethrough the transcriptional activation of the SRE, whereas the jNKs/SAPKs andp38/RK pathways are also involved in the transcriptional and posttranslationalregulation of c-jun and/or ATF-2 (Fig. 1).

Antioxidants and AP- l-dependent gene expression

In different cell systems, AP-l behaves as a redox-sensitive transcription factorwhich is activated, to a different extent, under pro-oxidant conditions generatedby treatment with agents such as H 20 2, UV light, y radiation or differentcytokines among others (1, 2). Paradoxically, a number of antioxidants includingdithiocarbamates, the antioxidant enzyme thioredoxin and the glutathione pre­cursor N-acetylcysteine have been shown to stimulate the DNA binding andtranscriptional activity of AP-l (9-12). In addition, antioxidants activate thetranscription of the genes of the antioxidative enzymes NADP(H): quinonereductase (DT-diaphorase) and the glutathione S-transferase Ya subunit throughantioxidant responsive elements (AREs) (13), which in the case of DT­diaphorase include a critical AP-l site involved in the inducible transcription ofthe gene by phenolic antioxidants (14). Given the fact that antioxidants canmimic a hypoxic state in the cells, it is important to note that AP-1 is activatedunder hypoxic conditions (15, 16) as occurs with DT diaphorase (15).

We have recently shown that dithiocarbamates (DTCs) trigger the expres­sion of CDllc and the intercellular adhesion molecule-1 (ICAM-1) genes inmyeloid and endothelial cells, respectively. In U937 cells, DTCs induce theexpression of myeloid differentiation markers and promote other differentia­tion-associated changes. In these cells, the surface upregulation of the ~2 inte­grin CDllc by pyrrolidine dithiocarbamate (PDTC) correlates with the acti­vation of the CDllc gene promoter which contains a critical AP-1 site func­tionally involved in the response to PDTC and other differentiation agents

276 . P. G6MEZ DEL ARea et al.

(11, 17). Similarly, treatment with PDTC or N-Acetylcysteine (NAC) resultsin cell surface upregulation of the endothelial activation molecule ICAM-l inhuman umbilical vein cells (HUVEC). The activating effect of PDTC is alsoobserved at the mRNA and promoter levels and involves the activation of AP­1 (18, 19). Thus, it is possible that the presence of high levels of ICAM-lobserved at sites of endothelial injury in ischemia-reperfusion-situations maybe related not only with the pro-oxidative stress induced during the reperfu­sion step, but also with the anti-oxidative ischemic phase. Further analysis willbe required to clarify whether these adhesion molecules activated by antioxi­dants are actually involved in cellular-stress responses triggered under hypoxicconditions.

Mechanisms of AP-l activation by antioxidants

While many studies have addressed the involvement of different signal cascadesin the activation of AP-l by pro-oxidative signals, the mechanisms leading to

AP-l activation by antioxidants are poorly understood. In Hela cells, AP-lDNA binding and transactivation have been reported to be activated by differ­ent antioxidants. In these cells, AP-1 activation by PDTC involves the inductionof c-fos and c-jun mRNAs, protein synthesis and the transcriptional activation ofc-fos via the SRE (9).

We have analyzed the effect of several antioxidants on signal transductionpathways that converge on the SRE in T lymphocytes. Although PDTC alsoinduces c-fos mRNA in Jurkat T cells, this effect does not seem to be mediatedby the Ras/ERK pathway. In transfection experiments, the expression of domi­nant negative versions of Ras and Raf does not interfere with the transcriptionalactivation of AP-l by the dithiocarbamate, and the treatment with PDTC failsto phosphorylate/activate ERKs (12). However, exposure of Jurkat cells toPDTC prior to the addition of PMA, triggers a strong and sustained activationof ERK that is detected even after 24 hours of treatment, whereas that mediatedby the phorbol ester is rapid and transient. This effect of PDTC on the activityof ERK induced by PMA could be related to the synergism of PMA plus PDTCin activating AP-l, and suggests that a phosphatase(s) involved in the regulationof ERK might be inhibited by PDTC (12). Since the phosphatases MKP-l andPAC-l have been shown to regulate ERK activity during T cell activation, theyare potential targets of PDTC. Although PAC-l transcription is not significantlyaffected by the antioxidant (12), a direct analysis of the effect of PDTC on theactivity of both phosphatases will clarify this issue.

As occurs with a variety of stimuli that generate oxidative stress, we haveobserved that different antioxidant compounds including NAC, butylatedhydroxyanisole and PDTC activate JNKs/SAPKs in Jurkat cells (12). In the caseof PDTC, JNK activation is strong and sustained for at least 8 h after treatment,which correlates to the activation of c-jun mRNA transcripts that are also acti­vated in a prolonged fashion by this antioxidant (12, MARTINEZ-MARTINEZ, S.,and J. M. REDONDO submitted). This activation of JNK by PDTC is also likely

Antioxidant-induced AP-1 activation . 277

to account for increased the transactivation c-Jun that we have detected in cellstransfected with GaI4-c-jun chimeric constructs of c-jun (MARTINEZ-MARTINEZ,S., and J. M. REDONDO submitted).

The activation of JNKs/SAPKs by antioxidants may well explain the induc­tion of c-fos and AP-1 in the apparent absence of activation of the Ras/ERKpathway. Thus, Elk-1 could be activated by JNKs/SAPKs thereby triggering thetranscription of c-[os. In addition, JNKs/SAPKs by activation of c-jun and ATF­2 transcription factors could also mediate the transcription of c-jun (Fig. 1).Other pathways involving Rac and Cdc42 activation that may also be involvedin the activation of c-fos and c-jun by antioxidants remain to be explored. This isthe case of the p38/RK kinase cascade whose activation leads to the phosphory­lation of Elk-1, and ATF-2 transcription factors (6).

Although analysis of the role of MAPKs in the activation of AP-1 mediatedby antioxidants has shed some light on the mechanisms involved in this process,the pathways leading to MAPK activation by antioxidants remain to be ana­lyzed. At least in the case of T lymphocytes, blockade of PKC, tyrosine kinasesor calcineurin-dependent pathways with specific inhibitors, that results in totalor partial inhibition of JNK activation by PMA plus ionophore, fails to inhibitthe JNK activation by PDTC (12). Thus, the signaling component(s) of path­ways upstream of JNK (or other MAPKs) that are targetted by antioxidantsremain to be established. Future analysis will be necessary to elucidate the pre­cise signaling mechanisms by which antioxidants activate AP-1. The use of dom­inant negative versions of different components of the MAPK cascade may helpto localize putative redox-sensitive kinases or phosphatases that mediate theantioxidant signaling.

Acknowledgements

This work was supported by grants from Ministerio de Educacion y Ciencia of Spain SAF94.0187,and Comunidad Autonoma de Madrid (CAM.) AE281195 and AC046/96 to]' M. R.

References

1. KARIN, M. 1995. The regulation of AP-1 activity by mitogen-activated protein kinases.]. BioI. Chern. 270: 16483.

2. ANGEL, P., and M. KARIN. 1991. The role of Jun, Fos and the AP-1 complex in cell prolifera­tion and transformation. Biochem. Biophys. acta 1072: 129.

3. TREISMAN, R. 1996. Regulation of transcription by MAP kinase cascades. Curf. Opin. Cell.BioI. 8: 205.

4. Su, B., and M. KARIN. 1996. Mitogen-activated protein kinase cascades and regulation ofgene expression. Curf. Opin. Immunol. 8: 402.

5. HILL, C. S., and R. TREISMAN. 1995. Transcriptional regulation by extracellular signals:mechanisms and specificity. Cell. 80: 199.

6. KYRIAKIS,.r. M., and.r. AVRucH. 1996. Sounding the alarm: protein kinase cascades activatedby stress and inflammation. J. BioI. Chern. 271: 24313.

7. CAHILL, M. A., R. JANKNECHT, and A. NORDHEIM. 1996. Signalling pathways: Jack of allcascades. Curf. BioI. 6: 16.

278 . P. GbMEZ DEL ARCO et al.

8. JANKNECHT, R., and T. HUNTER. 1997. Activation of the Sap-1a transcription factor by thec-Jun N-terminal kinase ONK) mitogen-activated protein kinase. J. BioI. Chern. 272: 4219.

9. MEYER, M., R. SCHRECK, and P. A. BAEUERLE. 1993. H 20 2 and antioxidants have oppositeeffects on activation of NF-KB and AP-1 in intact cells: AP-1 as secondary antioxidant­responsive factor. EMBO J. 12: 2005.

10. SCHENK, H., M. KLEIN, W. ERDBROGGER, W. DROGE, and K. SCHULZE-OSTHOFF. 1994. Dis­tinct effects of thioredoxin and other antioxidants on the activation of NF-KB and AP-1.Proc. Natl. Acad. Sci. 91: 1672.

11. ARAGONES, J., C. LbPEZ-RODRIGUEZ, A. CORBI, P. GbMEZ DEL ARCO, M. LbPEZ-CABRERA,M. 0. DE LANDAzuRI, and J. M. REDONDO. 1996. Dithiocarbamates trigger differentiation ainduction of CDllc gene through AP-1 in the myeloid lineage. J. BioI. Chern. 271: 10924.

12. GbMEZ DEL ARCO, P., S. MARTiNEZ-MARTINEZ, V. CALVO, A. L. ARMESILLA, and J. M.REDONDO. 1996. JNK (c-Jun NH2-terminal kianse) is a target for antioxidants in T lympho­cytes. J. BioI. Chern. 271: 26335.

13. RUSHMORE, T. H., M. R. MORTON, and C. B. PICKETT. 1991. The antioxidant responsive ele­ment. J. BioI. Chern. 266: 11632.

14. Lr, Y, and K. JAISWAL. 1992. Regulation of human NAD(P)H-quinone oxidoreductase gene.J. BioI. Chern. 267: 15097.

15. YAO, K.-S., S. XANTHOUDAKIS, T. CURRAN, and P. J. O'DWYER. 1994. Activation of AP-1and of nuclear redox factor, Ref-I, in the response of HT29 colon cancer cells to hypoxia.Mol. Cell. BioI. 14: 5997.

16. RUPEC, R. A., and P. A. BAEUERLE. 1995. The genomic response of tumor cells to hypoxiaand reoxygenation. Differential activation of transcription factors AP-1 and NF-KB. Eur. J.Biochem. 234: 632.

17. LbpEZ-RODRiGUEZ, C. H. C. KLUIN-NELEMANS, and A. CORBI. 1996. AP-1 regulates thebasal and developmentally induced transcription of the CD11c leukocyte integrin gene.J. Immunoi. 156: 3780.

18. MuNOZ, c., M. C. CASTELLANOS, A. ALFRANCA, A. VARA, M. A. ESTEBAN, J. M.REDONDO, and M. 0. DE LANDAZURI. 1996. Transcriptional up-regulation of intracellularadhesion molecule-1 in human endothelial cells by the antioxidant pyrrolidine dithiocarba­mate involves the activation of activating protein-I. J. Immunoi. 157: 3587.

19. Mu - OZ, c., D. PASCUAL-SALCEDO, M. C. CASTELLANOS, A. ALFRANCA, J. ARAGONES, A.VARA, J. M. REDONDO, and M. 0. DE LANDAZURI. 1996. Pyrrolidine dithiocarbamateinhibits the production of interieukin-6, interieukin-8, and granulocyte-macrophagecolony-stimulating factor by human endothelial cells in response to inflammatory media­tors: modulation of NF-KB and AP-1 transcription factors activity. Blood 88: 3482.

Dr. J. M. REDONDO, CSIC-UAM Facultad de Ciencias, Centro de Biologia Molecular, Canto­blanco, 28049 Madrid, Spain. Phone: 34-1-3978426; Fax: 34-1-3974799.