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Calcium signals lead to the translocation of nuclear factor of activated T cells (NFAT) from the cytoplasm to the nucleus. This process is regulated by the calciumactivated phosphatase calcineurin, which can be cotransported with NFAT to the nucleus to maintain it transcriptionally active for the duration of calcium signaling. When the calcium signal ceases, NFAT is exported to the cytoplasm, and different NFAT kinases have been reported to oppose calcineurin activities and regulate the nuclear export of NFAT. Here we show that p38 MAPK phosphorylates in vitro and interacts in vivo with NFATp. Furthermore, the activation of this pathway in HeLa cells by cotransfection with activated MKK6 and p38 counteracts the calcium-induced nuclear accumulation of NFATp but not that of NFATc. By contrast, activation of JNK or ERK pathways failed to modify the nuclear shuttling of NFATp. Consistently, activation of p38, but not the JNK MAPK pathway, results in the inhibition of NFATp-driven transcription. In addition, the inhibition of the nuclear accumulation of NFATp by p38 appears to be mediated through the activation of NFATp nuclear export and takes place in a Leptomycin B-sensitive fashion, suggesting the involvement of the exportin CRM1 in this process. Thus, the p38 signal transduction pathway appears to play an important role in the regulation of the nuclear shuttling of NFATp and in cellular homeostasis.

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Although important advances have been made in the development of antibiotics and medical intensive care technology in recent years, systemic response to infection remains a major health problem, with growing incidence and high mortality rates. Here we demonstrate the ability of the antioxidant agent pyrrolidine dithiocarbamate (PDTC) to inhibit the in vivo activation of NF-O B in lung and liver tissues, as well as the systemic release of TNF- § in lipopolysaccharide (LPS)-treated mice. The in vivo effect of PDTC on NF-O B activation in liver tissues involved the inhibition of both LPS-induced I O B- § degradation and the translocation of the p50 and p65 NF-O B subunits to the nucleus. In addition to protecting mice against lethal LPS doses, PDTC curtailed TNF- § -induced lethal shock. This effect was observed even after LPS injection, and when PDTC was administered at a time when TNF- § was already at maximum levels in serum. PDTC-treated mice survived despite high IL-1 g and IL-6 levels, induction of VCAM-1 and ICAM-1 expression or leukocyte infiltration in tissues known to be associated with LPS-induced shock, indicating that PDTC does not act by modifying these responses. Taken together, these results indicate that PDTC interferes with the production as well as the action of TNF- § , and points to a possible approach toward the treatment of septic shock.

It has recently been shown that the alteration of the cell-redox status affects the transcription factor expression and activity. Dithiocarbamates (DTCs) are potent antioxidant agents that can switch the expression of genes dependent on the activation of the transcription factors AP-1 and NF kappa B. In this study, we show that these agents triggered the expression of genes involved in myeloid differentiation of the promonocytic U-937 cell line. DTCs promoted differentiation-associated changes that included the surface up-regulation of beta 2-integrins (CD11a-c/CD18), cell growth arrest concomitant with transferrin receptor (CD71) down-modulation, induction of the nonspecific esterase enzyme, and a rapid drop in the mRNA levels of c-myc. A further analysis, focused on the molecular mechanisms leading to the activation of CD11c expression, revealed that the pyrrolidine derivative of DTC (PDTC) increased CD11c mRNA levels and augmented its gene promoter activity. Transfection experiments with reporter constructs harboring different promoter regions of CD11c gene, indicated the presence of a functional DTC-responsive region located between positions -160 and +40 of the promoter. Gel retardation assays revealed that the PDTC-induced DNA-protein complexes were restricted to members of the Fos and Jun families that bound to an AP-1 site located at position -60 from the transcription start site. A role for this site was confirmed by in vitro mutagenesis experiments that indicated the functional importance of this site for the CD11c gene transcriptional activation in response to PDTC. The effect of DTCs on myeloid cell differentiation supports a possible role for these agents in the therapy of some bone marrow-derived malignancies.

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AP-1 has been shown to behave as a redox-sensitive transcription factor that can be activated by both oxidant and antioxidant stimuli. However, the mechanisms involved in the activation of AP-1 by antioxidants are largely unknown. In this study we show that the structurally unrelated antioxidant agents pyrrolidine dithiocarbamate (PDTC), butylated hydroxyanisole, and Nacetylcysteine activated JNK (c-Jun NH 2 -terminal kinase) in Jurkat T cells. This activation differed substantially from that mediated by phorbol 12-myristate 13-acetate (PMA) and Ca 2؉ ionophore or produced by costimulation with antibodies against the T cell receptor-CD3 complex and to CD28. The activation of JNK by classical T cell stimuli was transient, whereas that mediated by PDTC and butylated hydroxyanisole (but not N-acetylcysteine) was sustained. The kinetics of JNK activation correlated with the expression of c-jun which was transient after stimulation with PMA plus ionophore and prolonged in response to PDTC, which also transiently induced c-fos. In addition, JNK activation by PMA plus ionophore was sensitive to inhibitors of signaling pathways involving Ca 2؉, protein kinase C, and tyrosine phosphorylation, which failed to inhibit the activation mediated by PDTC. Transfection of transdominant negative expression vectors of ras and raf, together with AP-1-dependent reporter constructs, as well as Western blot analysis using anti-ERK (extracellular signal-regulated kinase) antibodies, indicated that the Ras/Raf/ERK pathway did not appear to mediate the effect of the antioxidant. However, the combined treatment with PDTC and PMA, two agents that synergize on AP-1 activation, resulted in the persistent phosphorylation of ERK-2. In conclusion, our results identify JNK as a target of antioxidant agents which can be regulated differentially under oxidant and antioxidant conditions.