Synergistic action of protein kinase C theta and calcineurin is sufficient for Fas ligand expression and induction of a crmA-sensitive apoptosis pathway in Jurkat T cells.

Deletion of activated peripheral T cell clones by apoptosis requires the regulated expression of Fas ligand (FasL) and sensitization of these cells to CD95-mediated signaling. To investigate the signaling pathways responsible for FasL expression in T cells, we tested-besides subfamily-selective protein kinase C (PKC) inhibitors - the effect of constitutively active mutants of representatives of all PKC subfamilies, i.e. PKCalpha,epsilon,theta,iota, on FasL luciferase promoter reporter constructs. In synergy with a constitutively active form of protein phosphatase 2B calcineurin (CaN), only PKCtheta, but not PKCalpha,epsilon,iota, preferentially induced FasL promoter reporter activity and, consequently, FasL protein expression in Jurkat T cells. Activation of an inducible PKCtheta AE-estrogen receptor fusion mutant led to a CaN-dependent and rapid FasL reporter activity detected as early as 4 h after addition of 4-hydroxytamoxifen, incidating a direct effect of PKCtheta action on FasL expression. Consistently, in Jurkat T cells, expression of PKCtheta AE / CaN significantly enhanced FasL protein expression and apoptosis in a CD95-dependent manner since cell death was not observed in T cells co-expressing the caspase-8 inhibitor crmA. Taken together, our results support the notion that PKCtheta and CaN are sufficient to regulate apoptosis through FasL expression.

Villunger A ，Ghaffari-Tabrizi N ，Tinhofer I ，Krumböck N ，Bauer B ，Schneider T ，Kasibhatla S ，Greil R ，Baier-Bitterlich G ，Uberall F ，Green DR ，Baier G ... -  《EUROPEAN 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》

Differential implication of protein kinase C isoforms in cytotoxic T lymphocyte degranulation and TCR-induced Fas ligand expression.

CD8(+) cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKCtheta. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKCepsilon, and especially of PKCtheta, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKCtheta prevented the induction of the reporter. Finally, transfection with PKCalpha A/E, but not with PKCtheta A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKCtheta activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.

Pardo J ，Buferne M ，Martínez-Lorenzo MJ ，Naval J ，Schmitt-Verhulst AM ，Boyer C ，Anel A ... -  《INTERNATIONAL IMMUNOLOGY》

Protein kinase Ctheta, a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T cells.

Ghaffari-Tabrizi N ，Bauer B ，Villunger A ，Baier-Bitterlich G ，Altman A ，Utermann G ，Uberall F ，Baier G ... -  《EUROPEAN JOURNAL OF IMMUNOLOGY》

AMP-activated protein kinase mediates T cell activation-induced expression of FasL and COX-2 via protein kinase C theta-dependent pathway in human Jurkat T leukemia cells.

AMP-activated protein kinase (AMPK), an important regulator of energy homeostasis, is known to be activated during T cell activation. T cell activation by T cell receptor (TCR) engagement or its pharmacological mimics, PMA plus ionomycin (PMA/Io), induces immunomodulatory FasL and cyclooxygenase-2 (COX-2) expression. In this study, we examined the role and mechanisms of AMPK in PMA/Io-induced expression of FasL and COX-2 in Jurkat T human leukemic cells. Inhibition of AMPK by a pharmacological agent, compound C, or AMPKα1 siRNA suppressed expression of FasL and COX-2 mRNAs and proteins in PMA/Io-activated Jurkat cells. It also reduced secretion of FasL protein and prostaglandin E2, a main product of COX-2, in Jurkat cells and peripheral blood lymphocytes activated with PMA/Io or monoclonal anti-CD3 plus anti-CD28. Consistently, inhibition of AMPK blocked promoter activities of FasL and COX-2 in activated Jurkat cells. As protein kinase C theta (PKCθ) is a central molecule for TCR signaling, we examined any possible cross-talk between AMPK and PKCθ in activated T cells. Of particular importance, we found that inhibition of AMPK blocked phosphorylation and activation of PKCθ, suggesting that AMPK is an upstream kinase of PKCθ. Moreover, we showed that AMPK was directly associated with PKCθ and phosphorylated Thr538 of PKCθ in PMA/Io-stimulated Jurkat cells. We also showed that inhibition of PKCθ by rottlerin or dominant negative PKCθ reduced AMPK-mediated transcriptional activation of NF-AT and AP-1 in activated Jurkat cells. Taken together, these results suggest that AMPK regulates expression of FasL and COX-2 via the PKCθ and NF-AT and AP-1 pathways in activated Jurkat cells.

Lee JY ，Choi AY ，Oh YT ，Choe W ，Yeo EJ ，Ha J ，Kang I ... -  《-》