Immunosuppressant triptolide inhibits dendritic cell-mediated chemoattraction of neutrophils and T cells through inhibiting Stat3 phosphorylation and NF-kappaB activation

Triptolide, an active component purified from the medicinal plant Tripterygium wilfordii Hook F., is potent in anti-inflammation and immunosuppression. Dendritic cells (DC), one of important targets of immunosuppressants, play crucial roles in linking the innate immunity and adaptive immunity. However, the effects of triptolide on DC have not been fully elucidated. Chemoattraction of neutrophils and T cells by DC may favor their interactions and initiation of immune response. Here we demonstrate that triptolide significantly impairs DC-mediated chemoattraction of neutrophils and T cells both in vitro and in vivo by suppressing DC production of CC and CXC chemokines including MIP-1alpha, MIP-1beta, MCP-1, RANTES, TARC, and IP-10 in response to LPS. Furthermore, triptolide-mediated inhibition of NF-kappaB activation, Stat3 phosphorylation and increase of SOCS1 expression in DC may be involved in the inhibitory effect of triptolide. Our study provides a novel mechanistic explanation for the anti-inflammatory and immunosuppressive activities of triptolide.     

The essential role of MEKK3 signaling in angiotensin II-induced calcineurin/nuclear factor of activated T-cells activation

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes, such as T-cell activation, apoptosis, skeletal myocyte differentiation, and cardiac hypertrophy. We determined that active MEKK3 was capable of activating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes and reprogramming cardiac gene expression. In contrast, small interference RNA directed against MEKK3 and a dominant negative form of MEKK3 caused the reduction of NFAT activation in response to angiotensin II in cardiac myocytes. Genetic studies showed that MEKK3-deficient mouse embryo fibroblasts failed to activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring MEKK3 to the MEKK3-deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. We determined that angiotensin II induced MEKK3 phosphorylation. Thus, MEKK3 functions downstream of the AT1 receptor and is essential for calcineurin/NFAT activation. Finally, we determined that MEKK3-mediated activation of calcineurin/NFAT signaling was associated with the phosphorylation of modulatory calcineurin-interacting protein 1 at Ser(108) and Ser(112). Taken together, our studies reveal a previously unrecognized novel essential regulatory role of MEKK3 signaling in calcineurin/NFAT activation.     

CD147 inhibits the nuclear factor of activated T-cells by impairing Vav1 and Rac1 downstream signaling

CD147 is a transmembrane protein that plays crucial roles in the development and function of the reproductive, visual, and nervous systems. CD147 also exerts positive and negative actions in T-cells by still obscure mechanisms. In this study, we have analyzed the expression, localization, and function of CD147 during T-cell receptor signaling responses. We show here that CD147 is an integral component of the T-cell immune synapse and that its overexpression leads to the inhibition of NF-AT (nuclear factor of activated T-cells) activity induced by Vav1, a Rac1 exchange factor. This inhibitory activity is mediated by the CD147 intracellular tail and is totally independent of its extracellular or transmembrane regions. The molecular dissection of the influence of CD147 on the Vav1 pathway indicates that its inhibitory action takes place downstream of Vav1 and Rac1 but upstream of the serine/threonine kinases JNK and Pak1. The interference of CD147 with these pathways is highly specific because the overexpression of CD147 does not affect the activity of other GDP/GTP exchange factors or the stimulation of the ERK cascade. Finally, we show that the CD147 knockdown in Jurkat cells promotes higher levels of NF-AT stimulation and Pak1 phosphorylation upon T-cell receptor cross-linking. Instead, the lack of CD147 does not affect other signaling cascades that participate in the same cellular response. Taken together, these results indicate that CD147, via the selective inhibition of specific downstream elements of the Vav1/Rac1 route, contributes to the negative regulation of T-cell responses.     

Wortmannin inhibits activation of nuclear transcription factors NF-kappaB and activated protein-1 induced by lipopolysaccharide and phorbol ester

The effect of the expression of murine Bax protein on growth and vitality was examined in Saccharomyces cerevisiae and compared with the effect of Bax in mutant cells lacking functional mitochondria. The cytotoxic effect of Bax on yeast does not require functional oxidative phosphorylation, respiration, or mitochondrial proteins (ADP/ATP carriers) implicated in the formation of the permeability transition pore in mammalian mitochondria. In the wild type S. cerevisiae the expression of Bax does not result in a severe effect on mitochondrial membrane potential and respiration. On the basis of Bax induced differences in the fluorescence of green fluorescent protein fused to mitochondrial proteins, it is proposed that Bax may interfere with one essential cellular process in yeast: the mitochondrial protein import pathway that is specific for the proteins of the mitochondrial carrier family.     

Activator protein 1 (AP-1)- and nuclear factor kappaB (NF-kappaB)-dependent transcriptional events in carcinogenesis

Generation of reactive oxygen species (ROS) during metabolic conversion of molecular oxygen imposes a constant threat to aerobic organisms. Other than the cytotoxic effects, many ROS and oxidants are also potent tumor promoters linking oxidative stress to carcinogenesis. Clonal variants of mouse epidermal JB6 cells originally identified for their differential susceptibility to tumor promoters also show differential reduction-oxidation (redox) responses providing a unique model to study oxidative events in tumor promotion. AP-1 and NF-kappaB, inducible by tumor promoters or oxidative stimuli, show differential protein levels or activation in response to tumor promoters in JB6 cells. We further demonstrated that AP-1 and NF-kappaB are both required for maintaining the transformed phenotypes where inhibition of either activity suppresses transformation response in JB6 cells as well as human keratinocytes and transgenic mouse. NF-kappaB proteins or extracellular signal-regulated kinase (ERK) but not AP-1 proteins are shown to be sufficient for conversion from transformation-resistant to transformation-susceptible phenotype. Insofar as oxidative events regulate AP-1 and NF-kappaB transactivation, these oxidative events can be important molecular targets for cancer prevention.     

Estrogen controls embryonic stem cell proliferation via store-operated calcium entry and the nuclear factor of activated T-cells (NFAT)

Embryonic stem cells (ESCs) can self-renew indefinitely and differentiate into all cell lineages. Calcium is a universal second messenger which regulates a number of cellular pathways. Previous studies showed that store-operated calcium channels (SOCCs) but not voltage-operated calcium channels are present in mouse ESCs (mESCs). In this study, store-operated calcium entry (SOCE) was found to exist in mESCs using confocal microscopy. SOCC blockers lanthanum, 2-aminoethoxydiphenyl borate (2-APB) and SKF-96365 reduced mESC proliferation in a concentration-dependent manner, suggesting that SOCE is important for ESC proliferation. Pluripotent markers, Sox-2, Klf-4, and Nanog, were down-regulated by 2-APB, suggesting that self-renewal property of mESCs relies on SOCE. 17β-estradiol (E2) enhanced mESC proliferation. This enhanced proliferation was associated with an increment of SOCE. Both stimulated proliferation and increased SOCE could be reversed by SOCC blockers suggesting that E2 mediates its stimulatory effect on proliferation via enhancing SOCE. Also, cyclosporin A and INCA-6, inhibitors of calcineurin [phosphatase that de-phosphorylates and activates nuclear factor of activated T-cells (NFAT)], reversed the proliferative effect of E2, indicating that NFAT is involved in E2-stimulated proliferation. Interestingly, E2 caused the nuclear translocation of NFATc4, and this could be reversed by 2-APB. These results suggested that NFATc4 is the downstream target of E2-induced SOCE. The present investigation provides the first line of evidence that SOCE and NFAT are crucial for ESCs to maintain their unique characteristics. In addition, the present investigation also provides novel information on the mechanisms of how E2, an important female sex hormone, affects ESC proliferation.     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.