Regulation of T-cell death-associated gene 51 (TDAG51) expression in human T-cells

T-cell death-associated gene 51 (TDAG51) has been described to regulate T-cell receptor/CD3-dependent induction of CD95/Fas and subsequent activation-induced cell death (AICD) in a murine T-cell hybridoma. Using well-defined pharmacological inhibitors, we investigated the regulation of TDAG51 expression in human T-cells and the correlation with cell death. TDAG51 was induced in resting T-cells, lymphoid cell lines and AICD-susceptible as well as AICD-resistant T-cell clones, and induction was inhibited by MAP-kinase inhibitors and PKC inhibitor G?6983. No correlation between the effects of inhibitors on TDAG51 expression and cell death was observed. The constitutive TDAG51 expression in five pancreatic carcinoma cell lines was reduced by MAP-kinase inhibitors but not by G?6983. Furthermore, the inducible overexpression of TDAG51 in TetOn Jurkat cells did not modulate cellular proliferation, phorbolester/ionomycin-induced growth arrest, or the expression of various cell surface molecules. Our results indicate that the expression of TDAG51 in human T-cells does not correlate with AICD.     

Differential down-regulation of protein kinase C subspecies in KM3 cells

The down-regulation of protein kinase C (PKC) subspecies in KM3 cells (a pre-B, pre-T cell line) has been examined. The PKC from KM3 cells was resolved into two subspecies, type II (mainly beta II) and type III (alpha), upon hydroxyapatite column chromatography. Biochemical and immunocytochemical analysis revealed that, when these cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), the time course of down-regulation of the PKC subspecies was different; type II PKC was translocated and depleted from the cell more quickly than type III enzyme. The results suggest that each PKC subspecies plays a different role in the cellular response to TPA and probably to other external stimuli.     

Reduction of PKC alpha decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma

Protein kinase C (PKC) superfamily play key regulatory roles on the development of cancer. However, the exact role of these enzymes in human hepatocellular carcinoma (HCC) has not been well established. Using the RT-PCR and Western blotting to analyze the levels of PKC isoforms mRNA and protein in the five different differentiated hepatoma cell lines, we found that PKC alpha was highly expressed in the poor-differentiated HCC cell lines (SK-Hep-1 and HA22T/VGH) as compared with that in the well-differentiated HCC cell lines (PLC/PRF/5, Hep3B, and HepG2). When treated with PKC alpha antisense oligonucleotides (ODN), both HA22T/VGH and SK-Hep-1 cells lines showed the reduction of PKC alpha expression, as well as a deceleration in the growth rate and in the level of cyclin D1, but the increase in the levels of p53 and p21(WAF1/CIP1). Moreover, the reduction of PKC alpha expression also inhibited the migratory and invasive potential of both HA22T/VGH and SK-Hep-1 cells lines, and revealed a down-regulation of several migration/invasion-related genes (MMP-1, u-PA, u-PAR, and FAK). These phenomenon were also confirmed by DNA-based small interfering RNA (siRNA) PKC alpha and PKC alpha/beta specific inhibitor Go6976. Thus, the results indicated that PKC alpha may be associated with regulation of cell proliferation/migration/invasion in human poorly differentiated HCC cells, suggesting a role for the PKC alpha in the malignant progression of human HCC.     

T cell antigen receptor engagement stimulates c-raf phosphorylation and induces c-raf-associated kinase activity via a protein kinase C-dependent pathway

The c-raf kinase has been shown to be activated following stimulation of several tyrosine kinase growth factor receptors. We examined changes in c-raf following engagement of the T cell receptor for antigen (TCR), a stimulus which activates both a non-receptor tyrosine kinase and protein kinase C (PKC). We found that activation of the T-cell receptor on the T cell hybridoma 2B4 causes a rapid and stoichiometric hyperphosphorylation of c-raf and an increase in c-raf-associated kinase activity. Phosphoamino acid analysis showed that the phosphorylation was entirely on serine residues. High-resolution phosphopeptide mapping showed the appearance of a single major new phosphopeptide with TCR stimulation. That phosphopeptide was shown to comigrate with the major new phosphopeptide induced in response to phorbol ester. When cells were depleted of PKC by pretreatment with high concentrations of phorbol ester, TCR stimulation was no longer capable of inducing c-raf-associated kinase activity. To determine whether activation of the tyrosine kinase alone would activate c-raf, we examined the 2B4 variant cell line FL.8. In response to Thy-1 stimulation, these cells activate the tyrosine kinase but not protein kinase C due to a deficiency in TCR eta chain expression. We found that in contrast to Thy-1 stimulation of 2B4 cells, stimulation of FL.8 cells does not lead to the induction of c-raf-associated kinase activity, although phorbol ester activates the kinase to an equivalent degree in both cells. We conclude that T cell receptor activation of c-raf occurs via phosphorylation by the serine/threonine kinase PKC. Activation of c-raf through PKC represents a mechanism distinct from that reported for tyrosine kinase growth factor receptors.     

Molecular mechanism of the activation-induced cell death inhibition mediated by a p70 inhibitory killer cell Ig-like receptor in Jurkat T cells

In this study we investigated the molecular mechanism of the activation-induced cell death (AICD) inhibition mediated by a p70 inhibitory killer cell Ig-like receptor (KIR3DL1, also called NKB1) in Jurkat T cells. Using stable Jurkat transfectants that express KIR or CD8-KIR fusion proteins we have shown for the first time that KIR inhibits, in a ligation-independent manner, the AICD induced by PHA, PMA/ionomycin, or anti-CD3 Ab. The AICD inhibition mediated by KIR appears to result from the blockade of Fas ligand induction upon activation of the Jurkat transfectants. Moreover, the membrane-proximal 20 aa of the KIR cytoplasmic tail were determined to play a crucial role in this process. Since the membrane-proximal portion of the KIR cytoplasmic tail contains a putative protein kinase C (PKC) substrate site, we investigated the molecular interaction between KIR and PKC. Immunoprecipitation analysis demonstrated that KIR constitutively bound both to PKCalpha, a conventional Ca(2+)-dependent PKC, and to PKCtheta, a novel Ca(2+)-independent PKC. Furthermore, an in vitro kinase assay revealed that PKC activation was blocked after PHA stimulation in Jurkat transfectants expressing KIR. These observations were supported by the finding that a recombinant KIR cytoplasmic tail also appeared to inhibit PKCalpha activation in vitro. Taken together these data strongly suggest that KIR inhibits the AICD of T cells by blocking Fas ligand induction upon stimulation, in a process that seems to be accomplished by PKC recruitment to the membrane-proximal PKC binding site and subsequent inhibition of PKC activation against the activating stimuli.     

Down-regulation of protein kinase C in Swiss 3T3 fibroblasts is independent of its phosphorylating activity

The phorbol ester TPA induces down-regulation of protein kinase C (PKC) in Swiss-3T3 fibroblasts, as determined by the use of an alpha, beta, gamma PKC-specific antiserum. PKC is almost completely degraded 10 hours after TPA treatment of the cells and recovers within 72 hours. The staurosporine derivative K252a, known to inhibit PKC activity, causes strong suppression of TPA-induced (PKC-catalyzed) protein phosphorylation in Swiss-3T3 cells. Inhibition of protein phosphorylation by K252a is still effective when the process of down-regulation is completed. However, K252a does not influence TPA-induced down-regulation of PKC at all. Thus, down-regulation of PKC is not dependent on the enzyme's phosphorylating activity and, therefore, most likely not on its autophosphorylation as has been suggested by Ohno et al. [J. Biol. Chem. 265, 6296-6300 (1990)].     

Granzyme B, a new player in activation-induced cell death, is down-regulated by vasoactive intestinal peptide in Th2 but not Th1 effectors

Following antigenic stimulation and differentiation, Th1 and Th2 effector cells contribute differently to cellular and humoral immunity. Vasoactive intestinal peptide (VIP) induces Th2 responses by promoting Th2 differentiation and survival. In this study, we investigate the mechanisms for the protective effect of VIP against activation-induced cell death (AICD) of Th2 effectors. Surprisingly, microarray and protein data indicate that VIP prevents the up-regulation of granzyme B (GrB) in Th2 but not Th1 effectors. This is the first report of GrB expression in Th cells and of its involvement in activation-induced apoptosis. The enhanced responsiveness of Th2 cells to VIP is probably due to the higher expression of VIP receptors. The effect of VIP on Th2 survival and GrB expression is mediated through the VIP receptors 1 and 2 and cAMP signaling through exchange protein activated by cAMP and, to a lesser degree, protein kinase A. In addition to effects on GrB, VIP also down-regulates Fas ligand (FasL) and perforin (Pfr) expression. The extrinsic Fas/FasL pathway and the intrinsic GrB-dependent pathway act independently in inducing AICD. The mechanisms by which GrB induces cell death in Th1/Th2 effectors include both fratricide and suicide. Fratricide killing, prevalent in wild-type cells, is calcium and Pfr dependent, whereas the cell death of Pfr-deficient Th cells involves Fas and GrB but is calcium independent. This study identifies GrB as a new significant player in Th1/Th2 AICD and characterizes two mechanisms for the protective effect of VIP on Th2 survival, i.e., the down-regulation of GrB and FasL expression.     

A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation

A protein kinase C alpha (PKC alpha) cDNA confers increased phorbol ester binding activity to intact cells when transiently expressed in COS cells or expressed stably in transfected rat 3Y1 fibroblasts. A point mutant (PKC alpha K----R) of PKC alpha, where Lys368 at the putative ATP-binding site is replaced with Arg, confers enhanced phorbol ester binding activity to both transiently and stably expressed COS and 3Y1 cells, respectively. Like endogenous and exogenously expressed wild type PKC alpha, the mutant PKC alpha K----R is translocated from the cytosol to the particulate fraction when cells are treated with a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). On the other hand, the mutant PKC alpha K----R is not degraded when cells are treated with TPA, making a clear contrast to wild type PKC alpha; i.e. the mutant is resistant to TPA-mediated down-regulation. The mutant lacks kinase activity as expected, as judged by autophosphorylation and by a kinase assay using a peptide substrate, although the phorbol ester binding activity remains intact. These results suggest a link between the kinase activity of PKC alpha and the sensitivity to TPA-mediated proteolytic degradation. We propose that autophosphorylation of PKC alpha is a prerequisite for proteolytic cleavage associated with the down-regulation of PKC alpha.     

Distinct mechanisms of regulation of protein kinase C epsilon by hormones and phorbol diesters

In this study, we examined the effects of T cell activators on the regulation of protein kinase C (PKC) isozymes present in thymocytes. Using affinity-purified anti-PKC antisera, we determined that the major PKC isoforms in murine thymocytes are PKC beta and PKC epsilon. The CD4+/CD8+ thymocyte subset expressed high levels of both PKC beta and PKC epsilon, whereas the CD4-/CD8- subset expressed much less of both. PKC beta was down-regulated following treatment of thymocytes with phorbol 12-myristate acetate (PMA) (2 x 10(-8) M) or ionomycin (0.4 microM). In contrast, PMA did not induce the down-regulation of PKC epsilon. Ionomycin alone, however, induced PKC epsilon down-regulation, similar to its effect on PKC beta. Similar observations were made on a promonocytic cell line, U937, which expresses PKC alpha, PKC beta (Strulovici, B., Daniel-Issakani, S., Oto, E., Nestor, J., Jr., Chan, H., and Tsou, A.-P. (1989) Biochemistry 28, 3569-3576), and PKC epsilon. To facilitate the study of PKC beta and PKC epsilon, we established a Chinese hamster ovary cell line which expresses murine PKC epsilon in addition to endogenous PKC alpha and PKC beta. Both PKC isoforms (beta and epsilon) were mostly in particulate form. PMA treatment left the majority of immunoreactive PKC epsilon intact. By contrast, thrombin treatment caused the disappearance of particulate and cytosolic PKC epsilon (60% by 10 min and 80% by 1 h). PMA and thrombin promoted the down-regulation of PKC beta with similar kinetics (100% down-regulation by 3 h). These results indicate that: 1) thymocytes express PKC epsilon; and 2) this isozyme exhibits a novel form of regulation distinct from the other PKC isozymes.     

Different protein kinase C isozymes could modulate bradykinin-induced extracellular calcium-dependent and -independent increases in osteoblast-like MC3T3-E1 cells.

Effects of protein kinase C (PKC) on bradykinin (BK)-induced intracellular calcium mobilization, consisting of rapid Ca2+ release from internal stores and a subsequent sustained Ca2+ inflow, were examined in Fura-2-loaded osteoblast-like MC3T3-E1 cells. The sustained Ca2+ inflow as inferred with Mn2+ quench method was blocked by Ni2+ and a receptor-operated Ca2+ channel blocker SK&F 96365, but not by nifedipine. The short-term pretreatment with phorbol 12-myristate 13-acetate (PMA), inhibited BK-stimulated Ca2+ inflow, and the prior treatment with PKC inhibitors, H-7 or staurosporine, enhanced the initial internal release and reversed the PMA effect. Moreover, 6 h pretreatment with PMA caused similar effect on the BK-induced inflow to that obtained with PKC inhibitors, whereas 24 h pretreatment was necessary to affect the internal release. On the other hand, the translocation and down-regulation of PKC isozymes were examined after PMA treatment of MC3T3-E1 cells by immunoblot analyses of PKCs with the isozyme-specific antibodies. 6 h treatment with PMA induced down-regulation of PKC beta, whereas longer treatment was needed for down-regulation of PKC alpha. Taken together, it was suggested that the BK-induced initial Ca2+ peak and the sustained Ca2+ inflow through the activation of a receptor-operated Ca2+ channel, are differentially regulated by PKC isozymes alpha and beta, respectively, in osteoblast-like MC3T3-E1 cells.     

Transient down-regulation of PKC-zeta RNA following crosslinking of membrane IgM on WEHI-231 B lymphoma cells.

Regulation of protein kinase C (PKC) isoform mRNAs has been studied in the immature, murine B lymphoma WEHI-231 by the MAPPing protocol and by slot blot analysis of unamplified mRNA. This membrane IgM (mIgM)-positive cell line has been previously used as a model to study signal transduction by mIgM in immature B lymphocytes and the role of those signals in the induction of immune tolerance in the B cell compartment. Stimulation of the cells by anti-mu antibodies, phorbol ester, or Ca2+ ionophore caused growth arrest and death of the cells. IL 4 and IL 5 slowed the growth of the cells. Of these stimuli, only anti-mu stimulation affected PKC mRNA levels. Anti-mu treatment caused a transient decrease in the amount of PKC-zeta isoform mRNA within 3 hr. Within 24 hr levels returned toward normal. Anti-mu had little or no effect on the expression of mRNA for the alpha, beta, delta, or epsilon isoforms of PKC. WEHI-231 cells do not express PKC-gamma. Although anti-mu treatment blocked progression of the cells from the G0/G1 stage into the S phase of cell cycle, viable sort selected cells in either the G0/G1 or the S/G2/M phases showed no clear difference in the expression of PKC-zeta message. Thus, there is not preferential regulation of expression of PKC-zeta during stages of the cell cycle. The results show that mIgM on WEHI-231 cells can transduce a signal that is not mediated by PKC or Ca2+ mobilization alone. The signal causes transient, selective down-regulation of mRNA encoding the zeta PKC isoform.     

Selective response of gamma delta T-cell hybridomas to orthomyxovirus-infected cells.

A gamma delta T-cell hybridoma established from influenza virus-infected mice responded to a reproducible way when cultured with influenza virus-infected stimulators. Subclones of this line responded to cells infected with influenza viruses A/PR/8/34 (H1N1), X-31 (H3N2), and B/HK/8/73 but not to cells infected with vaccinia virus or Sendai virus. This spectrum of response to both type A and type B orthomyxoviruses has never been recognized for the alpha beta T-cell receptor-positive subsets. There was no response to cells infected with a panel of recombinant vaccinia viruses expressing all individual influenza virus proteins, and so it is unlikely that the stimulating antigen is of viral origin. The alternative is that the antigen is a cellular molecule induced in influenza virus-infected cells. Infectious virus was required for stimulation, and immunofluorescence studies showed increased expression of heat shock protein 60 (Hsp60) in influenza virus- but not Sendai virus- or vaccinia virus-infected cells. Both the hybridoma generated from influenza virus-infected mice and an established hybridoma which uses the same gamma delta T-cell receptor combination responded to recombinant Hsp60. Furthermore, the Hsp60-reactive hybridoma, which was obtained from an uninfected mouse, also responded to influenza virus-infected cells, indicating that Hsp60 may indeed be the target antigen.     

Role of protein kinase C in induction of gene expression and inhibition of cell proliferation by interferon alpha.

Recent studies have suggested that protein kinase C (PKC) may be involved in the mechanism of signal transduction by which members of the interferon (IFN) family regulate gene expression and cell phenotype. We have investigated the role of PKC in the control of cell growth and gene expression by IFN alpha in Daudi cells. Treatment of these cells with two analogues of staurosporine, which are potent inhibitors of PKC, completely blocked the induction by IFN alpha of the mRNA for 2',5'-oligoadenylate synthetase and the 6-16 gene. These compounds also inhibited cell proliferation and thymidine incorporation in this system. In contrast, the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) did not significantly inhibit the induction of these genes by IFN alpha and had no effect on Daudi cell growth or thymidine incorporation in the presence or absence of IFN alpha. No effect of IFN alpha on total PKC activity could be observed, and there were no significant changes in the overall levels of individual PKC isoforms or their mRNA following IFN alpha treatment. In contrast, treatment of Daudi cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, which also inhibits cell proliferation, strongly down-regulated PKC. These data suggest that the activity of a PKC species, or a closely related enzyme, may be required both for continued cell proliferation and the response to IFN alpha in Daudi cells, but that IFN-induced growth inhibition does not involve overall down-regulation or change in activity of PKC.     

Elevated levels of protein kinase C activity and alpha-isoenzyme expression in murine peritoneal B cells

Conventional murine splenic B cells are stimulated to initiate DNA synthesis by the combination of a phorbol ester protein kinase C (PKC) agonist, and a calcium ionophore; in contrast, recent work from this laboratory has shown that peritoneal B cells, enriched for the Ly-1+ B cell subset, differ in that they proliferate in response to the single signal provided by phorbol ester, acting alone. To elucidate the mechanism responsible for the abbreviated signaling requirement of peritoneal B cells, studies of intracellular Ca2+ and PKC were carried out. Measurements using the calcium sensitive dye, Indo-1, showed that base line levels of intracellular Ca2+ in peritoneal B cells were similar to those of splenic B cells, and that there was no change as a result of phorbol ester treatment. However, measurements of PKC based on the phosphorylation of histone showed enzymatic activity in peritoneal B cells to be about 60% greater than that of splenic B cells on a per microgram protein basis. Furthermore, this difference was accentuated by phorbol ester treatment, so that after 4 h, membrane and cytosol fractions from peritoneal B cells contained more than 5 times the PKC activity of the corresponding splenic B cell fractions because the down-regulation of PKC was relatively delayed in peritoneal B cells. This could not be accounted for by the onset of new PKC synthesis, but may relate to the finding that peritoneal B cells express more of the alpha-isoenzyme of PKC than splenic B cells, as shown by immunoblot analysis. Together with data from experiments using the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride(H7), these results suggest that PKC activity remaining hours after phorbol ester treatment may contribute to the unusual phorbol ester responsiveness of peritoneal B cells, and indicate that B cells from separate anatomic locations differ in terms of several parameters relating to the activity and behavior of PKC.     

Regulation of the maintenance of peripheral T-cell anergy by TAB1-mediated p38 alpha activation

In anergic T cells, T-cell receptor (TCR)-mediated responses are functionally inactivated by negative regulatory signals whose mechanisms are poorly understood. Here, we show that CD4(+) T cells anergized in vivo by superantigen Mls-1(a) express a scaffolding protein, transforming growth factor beta-activated protein kinase 1-binding protein 1 (TAB1), that negatively regulates TCR signaling through the activation of mitogen-activated protein kinase p38 alpha. TAB1 was not expressed in naive and activated CD4(+) T cells. Inhibition of p38 activity in anergic T cells by a chemical inhibitor resulted in the recovery of interleukin 2 (IL-2) and the inhibition of IL-10 secretion. T-cell hybridoma 2B4 cells transduced with TAB1-containing retrovirus (TAB1-2B4 cells) showed activated p38 alpha, inhibited extracellular signal-regulated kinase (ERK) activity, culminating in reduced IL-2 levels and increased IL-10 production. The use of a p38 inhibitor or cotransfection of a dominant-negative form of p38 in TAB1-2B4 cells resulted in the recovery of ERK activity and IL-2 production. These results imply that TAB1-mediated activation of p38 alpha in anergic T cells regulates the maintenance of T-cell unresponsiveness both by inhibiting IL-2 production and by promoting IL-10 production.     

TLR4/MYD88-dependent, LPS-induced synthesis of PGE2 by macrophages or dendritic cells prevents anti-CD3-mediated CD95L upregulation in T cells

Antigen-presenting cells (APCs) control T-cell responses by multiple mechanisms, including the expression of co-stimulatory molecules and the production of cytokines and other mediators that control T-cell proliferation, survival and differentiation. Here, we demonstrate that soluble factor(s) produced by Toll-like receptor (TLR)-activated APCs suppress activation-induced cell death (AICD). This effect was observed in non-stimulated APCs, but it was significantly increased after lipopolysaccharide (LPS) treatment. Using different KO mice, we found that the LPS-induced protective factor is dependent on TLR4/MyD88. We identified the protective factor as prostaglandin E(2) (PGE(2)) and showed that both APC-derived supernatants and PGE(2) prevented CD95L upregulation in T cells in response to TCR/CD3 stimulation, thereby avoiding both AICD and activated T cell killing of target macrophages. The PGE(2) receptors, EP2 and EP4, appear to be involved since pharmacological stimulation of these receptors mimics the protective effect on T cells and their respective antagonists interfere with the protection induced by either APCs derived or synthetic PGE(2). Finally, the engagement of EP2 and EP4 synergistically activates protein kinase A (PKA) and exchange protein directly activated by cAMP pathways to prevent AICD. Taken together, these results indicate that APCs can regulate T-cell levels of CD95L by releasing PGE(2) in response to LPS through a TLR4/MyD88-dependent pathway, with consequences for both T cell and their own survival.