Calcium-dependent activation of TNF family gene expression by Ca2+/calmodulin kinase type IV/Gr and calcineurin.

CD40 ligand (L), FasL, and TNF-alpha are members of the TNF family of cytokines. All are expressed by T lymphocytes shortly after activation but have distinct effector functions. Transcription of these genes can be induced by stimulation of T cells by calcium ionophore alone and requires the calcineurin-dependent transcription factor NF of activated T cells. We have examined a second calcium-dependent signaling pathway, mediated by calcium/calmodulin-dependent kinase IV (CaMKIV) in transcriptional activation of TNF family genes. In reporter gene assays using constructs driven by the promoters of human CD40L, FasL, or TNF-alpha along with vectors expressing constitutively active CaMKIV and calcineurin, we have demonstrated that each promoter is activated by calcineurin and CaMKIV in a synergistic fashion. Furthermore, specific inhibition of CaMKIV by chemical means and by a dominant negative mutant of CaMKIV impairs the ionomycin-induced activity of all three promoters as well as protein expression of CD40L and TNF-alpha. Our results indicate that activation of gene expression by calcineurin and CaMKIV is common to members of the TNF cytokine family.

Lobo FM ，Zanjani R ，Ho N ，Chatila TA ，Fuleihan RL ... -  《JOURNAL OF IMMUNOLOGY》

Protein kinase ctheta cooperates with calcineurin to induce Fas ligand expression during activation-induced T cell death.

Activation-induced cell death is mediated by the TCR-induced expression of the Fas ligand (FasL) on the surface of T cells, followed by binding to its receptor Fas. FasL expression is induced by stimulating T cells with a combination of phorbol ester and Ca2+ ionophore, implicating a role for protein kinase C (PKC) in this process. However, the precise mechanisms that regulate FasL expression, including the contribution of distinct T cell-expressed PKC isoforms, are poorly understood. Herein, we report that PKCtheta, a Ca2+-independent PKC isoform that we have previously isolated as a PKC enzyme selectively expressed in T cells, plays an important role in these processes. A constitutively active PKCtheta mutant preferentially induced FasL expression and activated the corresponding gene promoter; conversely, a dominant-negative PKCtheta mutant blocked FasL expression induced by anti-CD3 or PMA plus ionomycin stimulation. Furthermore, PKCtheta synergized with calcineurin to provide a potent stimulus for FasL promoter activation. Full activation of the promoter required its binding sites for the transcription factors NF-AT, AP-1, and NF-kappaB. The biological significance of these findings is implicated by the finding that rottlerin, a selective PKCtheta inhibitor, blocked FasL induction by anti-CD3 or PMA plus ionomycin stimulation and, consequently, protected human Jurkat T cells and the mouse T cell hybridoma A1.1 from activation-induced cell death.

Villalba M ，Kasibhatla S ，Genestier L ，Mahboubi A ，Green DR ，Altman A ... -  《JOURNAL OF IMMUNOLOGY》

Fas ligand induction in human NK cells is regulated by redox through a calcineurin-nuclear factors of activated T cell-dependent pathway.

Fas ligand (FasL) on cytotoxic lymphocytes is important for mediating apoptosis of activated lymphocytes and other target cells. We have reported that NK cell functions, such as proliferation, cell death, and killing activity, are subject to regulation by cellular redox status. Here, we report that expression of FasL protein and mRNA in activated NK cells is also regulated by redox. Ligation of CD16 on IL-2-preactivated NK cells resulted in reduction of intracellular peroxide level as well as induction of FasL expression. This CD16-induced FasL expression was suppressed by oxidative stress, including thiol deprivation or treatment with hydrogen peroxide (H2O2). Addition of thiol-reducing compounds, such as L-cystine, 2-ME, or N-acetyl cysteine, restored FasL expression. These data suggest that CD16 stimulation requires cellular reducing status for FasL induction in NK cells. Because FasL gene activation following CD16 cross-linking is regulated by the NF of activated T cells (NFAT), we examined the effect of oxidative stresses on NFAT activation. Electrophoretic mobility shift assays revealed that both thiol insufficiency and H2O2 treatment suppressed DNA-binding activity of NFAT and that addition of thiol-reducing compounds reversed or even enhanced it. Furthermore, these oxidative stresses inhibited activity of calcineurin, a serine/threonine phosphatase that regulates NFAT activation. These results suggest that suppression of calcineurin and NFAT activation is a mechanism by which oxidative stress inhibits FasL induction in activated NK cells and further support the hypothesis that thiol-reducing compounds might be required for maintenance of optimal NK functions under physiologic oxidative conditions.

Furuke K ，Shiraishi M ，Mostowski HS ，Bloom ET ... -  《JOURNAL OF IMMUNOLOGY》

Differential induction of cytokine genes and activation of mitogen-activated protein kinase family by soluble CD40 ligand and TNF in a human follicular dendritic cell line.

Follicular dendritic cells (FDC)3 play crucial roles in germinal center (GC) formation and differentiation of GC B cells. Many aspects of FDC function are influenced by contact with B or T cells, and by cytokines produced in the GC, which involve stimulation of CD40 and TNF-alpha receptors on FDC. In this study, using an established FDC line, HK cells, we compared the effects of CD40 and TNF receptor triggering on cytokine induction and activation of mitogen-activated protein kinase family. We show that HK cells spontaneously produced IL-6, M-CSF, and G-CSF mRNA. Both the soluble form of CD40 ligand (sCD40L) and TNF increased the level of M-CSF and G-CSF mRNA. While TNF strongly induced IL-6 mRNA, its expression was not affected by sCD40L treatment, differing from the strong IL-6 induction in other cell types upon CD40 stimulation. In addition, sCD40L treatment resulted in activation of extracellular signal-related kinase 1 and 2 (ERK1/2) and p38 without significant increase in c-Jun N-terminal kinase (JNK) activity. Lack of JNK activation differs in that most B cells respond to CD40 stimulation by inducing JNK activity strongly, suggesting distinct characteristics of CD40 signaling in FDC. Compared with the effects of sCD40L, TNF was capable of inducing JNK activity in addition to the activation of ERK1/2 and p38. Furthermore, the proximal signaling elements activated by TNF differed from those activated by sCD40L, in that TNF did not require PMA-sensitive protein kinase C isoforms in the activation of ERK and p38, whereas sCD40L did. However, signals activated by these stimuli converged on cytokine gene expression in a synergistic manner, which may have implication in augmenting FDC function during GC reaction.

Park SM ，Kim HS ，Choe J ，Lee TH ... -  《JOURNAL OF IMMUNOLOGY》

Regulation and properties of the rat Ca2+/calmodulin-dependent protein kinase IV gene and its protein products.

Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric multifunctional enzyme that is expressed only in subanatomical portions of the brain, T lymphocytes, and postmeiotic male germ cells. It is present in the nucleus of the cells in which it is expressed and can phosphorylate and activate the cyclic AMP response element binding proteins CREB and CREM tau in a manner analogous to protein kinase A. In the absence of Ca2+/calmodulin, CaMKIV is inactive. Activation requires three events: 1) binding of Ca2+/calmodulin; 2) phosphorylation of a single threonine residue present in the activation loop by a separate protein kinase that is also Ca2+/calmodulin-dependent; and 3) autophosphorylation of serine residues present in the extreme N-terminus that is required to relieve a novel form of autoinhibition. The gene for rat CaMKIV has been cloned and found to span 42 kb of DNA. The gene encodes three proteins: namely, the alpha and beta forms of CaMKIV that differ only in that the beta form contains a 28 amino acid N-terminal extension as well as calspermin. Calspermin is the C-terminal 169 amino acids of CaMKIV that binds Ca2+/calmodulin and is expressed only in postmeiotic male germ cells. The promoter for calspermin resides in the penultimate intron of the CaMKIV gene and is regulated by two CREs. This promoter is sufficient to faithfully target expression of a reporter gene to the postmeiotic male germ cells of transgenic mice. Transgene expression can be induced in cells from the transgenic mice that do not normally express it by transfection of CREM tau and CaMKIV. These data suggest that rearrangement of chromatin during meiosis together with the expression of CREM tau at high levels are sufficient to control expression of the calspermin promoter during spermatogenesis. On the other hand, the developmental expression of CaMKIV in brain and thymus appears to be controlled by thyroid hormone mediated via the thyroid hormone receptor alpha. In T lymphocytes, CaMKIV will phosphorylate CREB in response to signals that result in T cell activation. Transgenic mice that express a kinase minus mutant of CaMKIV specifically in thymic T cells show a marked reduction of total thymic cellularity. The remaining T cells undergo a much greater than normal rate of spontaneous apoptosis when placed in culture. These cells fail to generate the signals to phosphorylate CREB and produce significantly less of the cytokine Interleukin-2 (IL-2) in response to agents that either increase intracellular Ca2+ and/or activate protein kinase C. Collectively, the data suggest that CaMKIV may be involved both in preventing apoptosis during T cell development and also in the early cascade of events that is required to activate the mature T cells in response to a mitogenic stimulus.

Means AR ，Ribar TJ ，Kane CD ，Hook SS ，Anderson KA ... -  《-》