Induction of beta3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEK-extracellular signal-regulated kinase signaling pathway.

Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf-MEK-extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of alpha6- and beta3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface alpha6- and beta3-integrin expression. Induced beta3-integrin was expressed on the cell surface as a heterodimer with alphav-integrin; however, the overall level of alphav-integrin expression was not altered by Ras or Raf. Raf-induced beta3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce beta3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated beta3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, beta3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on beta3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface alphavbeta3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

DAP10 and DAP12 form distinct, but functionally cooperative, receptor complexes in natural killer cells.

Many of the activating receptors on natural killer (NK) cells are multisubunit complexes composed of ligand-binding receptors that are noncovalently associated with membrane-bound signaling adaptor proteins, including CD3zeta, FcstraightepsilonRIgamma, DAP12, and DAP10. Because the DAP10 and DAP12 genes are closely linked, expressed in NK cells, and have remarkably similar transmembrane segments, it was of interest to determine the specificity of their interactions with ligand-binding receptors and to examine their signaling properties. Despite their similarities, DAP10, DAP12, FcstraightepsilonRIgamma, and CD3zeta form specific receptor complexes with their ligand-binding partners in NK cells and transfectants. The transmembrane regions of DAP10 and DAP12 are sufficient to confer specific association with their partners. Although cross-linking of either DAP10- or DAP12-associated receptors has been shown to be sufficient to trigger NK cell-mediated cytotoxicity against Fc receptor-bearing cells, substantial synergy was observed in the induction of cytokine production when both receptors were engaged. Activation of the Syk/ZAP70 tyrosine kinases by the immunoreceptor tyrosine-based activation motif-containing DAP12 adaptor and of the phosphatidylinositol 3-kinase pathway by the YxNM-containing DAP10 adaptor may play an important role in the stimulation of NK cells and T cells.

Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism.

In this study, the abilities of constitutive and conditional forms of the three Raf kinases to abrogate the cytokine dependency of FDC-P1 cells were examined. The constitutively active forms (delta) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous beta-estradiol. The vast majority of deltaRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of deltaRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from deltaA-Raf:ER than either deltaRaf-1:ER or deltaB-Raf:ER infected cells. To determine whether the regulatory phosphorylation sites in the Raf proteins were necessary for abrogation of cytokine dependency, they were changed by site-directed mutagenesis. Substitution with phenylalanine eliminated the transforming ability of the deltaB-Raf:ER and deltaRaf-1:ER kinases. However, a similar substitution in A-Raf did not extinguish its transforming activity. The activated Raf proteins induced essential downstream MEK1 activity as treatment with the MEK1 inhibitor, PD98059, suppressed Raf-mediated growth. Activated MAP kinases (ERK1 and ERK2) were detected in deltaRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the deltaRaf:ER proteins as removal of beta-estradiol caused the cells to stop growing and undergo apoptosis. The Raf-responsive cells were found to express autocrine growth factors, which promoted their growth. Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice. In summary, Raf kinases can abrogate cytokine dependency, prevent apoptosis and induce the tumorigenicity of a certain subpopulation of FDC-P1 cells by a MEK1-dependent mechanism.

A conditionally-active form of MEK1 results in autocrine tranformation of human and mouse hematopoietic cells.

The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using deltaMEK1:ER, a conditionally-active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of the human and murine hematopoietic cells lines TF-1, FDC-P1 and FL5.12. Cytokine-independent cells were obtained from TF-1, FDC-P1 and FL5.12 cells at frequencies of 2.5 x 10(-3), 5 x 10(-5) and 10(-7) respectively, indicating that not all cells expressing deltaMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to deltaMEK1:ER activation than those that remained cytokine-dependent. deltaME-K1:ER-responsive cells could be maintained long-term in the presence of beta-estradiol as well as the estrogen-receptor antagonist 4-Hydroxy-Tamoxifen and the anti-estrogen ICI 164383. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. Treatment of deltaMEKI:ER-responsive cells with a specific and selective inhibitor, PD98059, prevented growth in response to beta-estradiol. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that the activated deltaMEK1:ER may induce a pathway leading to autocrine proliferation. Treatment of MEK1-responsive cells with an anti-GM-CSF antibody, but not a control antibody, suppressed cell growth. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.

2B4-mediated activation of human natural killer cells.

2B4 is a member of the CD2 subset of the immunoglobulin superfamily of cell surface receptors. Other members of this family include CD2, CD48, CD58, CD84, signaling lymphocytic activation molecule and Ly-9. Some of these molecules are activating structures expressed by natural killer cells and T cells. We have recently cloned and characterised the human homologue of 2B4 and found that the cytoplasmic domain of 2B4 can interact with SAP, a signaling adaptor protein that is mutated in the immunodeficiency X-linked lymphoproliferative disease (XLP). Additionally, the natural ligand of 2B4 has been identified as CD48. These findings have facilitated the investigation of the functional role of this receptor-ligand pair, and associated signal transduction pathways, on immune cells. In this study, it was found that the interaction between 2B4 on effector cells and CD48 on target cells induced NK-cell activation, as evidenced by increased cytotoxicity and secretion of IFN-gamma. The responses induced by ligation of 2B4 could be reduced by the co-ligation of inhibitory receptors expressed by NK cells, demonstrating that activation signals delivered via 2B4 can be regulated by the action of certain inhibitory receptors. Because the signalling pathway of 2B4 involves SAP, it is possible that 2B4-mediated NK-cell activation may be compromised in patients with XLP due to mutations in SAP. This may contribute to the phenotype and progression of this disease.

Complementation of defective colony-stimulating factor 1 receptor signaling and mitogenesis by Raf and v-Src.

Ras-activated signal transduction pathways are implicated in the control of cell proliferation, differentiation, apoptosis, and tumorigenesis, but the molecular mechanisms mediating these diverse functions have yet to be fully elucidated. Conditionally active forms of Raf, v-Src, and MEK1 were used to identify changes in gene expression that participate in oncogenic transformation, as well as in normal growth control. Activation of Raf, v-Src, and MEK1 led to induced expression of c-Myc and cyclin D1. Induction of c-Myc mRNA by Raf was an immediate-early response, whereas the induction of cyclin D1 mRNA was delayed and inhibited by cycloheximide. Raf activation also resulted in the induction of an established c-Myc target gene, ornithine decarboxylase (ODC). ODC induction by Raf was mediated, in part, by tandem E-boxes contained in the first intron of the gene. Activation of the human colony-stimulating factor 1 (CSF-1) receptor in NIH 3T3 cells leads to activation of the mitogen-activated protein (MAP) kinase pathway and induced expression of c-Fos, c-Myc, and cyclin D1, leading to a potent mitogenic response. By contrast, a mutated form of this receptor fails to activate the MAP kinases or induce c-Myc and cyclin D1 expression and fails to elicit a mitogenic response. The biological significance of c-Myc and cyclin D1 induction by Raf and v-Src was confirmed by the demonstration that both of these protein kinases complemented the signaling and mitogenic defects of cells expressing this mutated form of the human CSF-1 receptor. Furthermore, the induction of c-Myc and cyclin D1 by oncogenes and growth factors was inhibited by PD098059, a specific MAP kinase kinase (MEK) inhibitor. These data suggest that the Raf/MEK/MAP kinase pathway plays an important role in the regulation of c-Myc and cyclin D1 expression in NIH 3T3 cells. The ability of oncogenes such as Raf and v-Src to regulate the expression of these proteins reveals new lines of communication between cytosolic signal transducers and the cell cycle machinery.

Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells.

Raf is a key serine-threonine protein kinase which participates in the transmission of growth, anti-apoptotic and differentiation messages. These signals can be initiated after receptor ligation and are transmitted to members of the MAP kinase cascade that subsequently activate transcription factors controlling gene expression. Raf is a member of a multigene family which includes: Raf-1, A-Raf and B-Raf. The roles that individual Raf kinases play in the regulation of normal and malignant hematopoietic cell growth are not clear. The following studies show that all three Raf kinases are functionally present in certain human hematopoietic cells, and their aberrant expression can result in abrogation of cytokine dependency. Cytokine-dependent TF-1 cells were infected with retroviruses encoding amino-terminal deleted (delta) A-Raf, B-Raf and Raf-1 proteins. These Raf proteins were conditionally inducible as they were fused to the hormone-binding domain of the estrogen receptor (ER). A hierarchy in the abilities of Raf-containing retroviruses to abrogate cytokine dependency was observed as deltaA-Raf:ER was 20- to 200-fold more efficient than either deltaRaf-1:ER or deltaB-Raf:ER, respectively. This result was unexpected as A-Raf is an intrinsically weaker kinase than either Raf-1 or B-Raf. The activated Raf proteins induced downstream MEK and MAP (ERK1 and ERK2) kinase activities in the cells which proliferated in response to Raf activation. Furthermore, a functional MEK signaling pathway was necessary as treatment of the cells with a MEK1-inhibitor suppressed Raf-mediated proliferation. To determine whether the regulatory phosphorylation residues contained in the modified Raf oncoproteins were necessary for transformation, they were altered by site-directed mutagenesis. Substitution of the regulatory phosphorylation tyrosine residues with phenylalanine in either A-Raf or Raf-1 reduced the capacity of these oncoproteins to abrogate cytokine dependency. In contrast, changing the critical aspartic acid residues of B-Raf to either tyrosine or phenylalanine increased the frequency of estradiol-responsive cells. Thus, the amino acids present in the regulatory residues modulated the capability of Raf proteins to abrogate the cytokine dependency of TF-1 cells. Differences in the levels of Raf and downstream kinase activities were observed between cytokine-dependent and estradiol-responsive deltaRaf:ER-infected cells as estradiol-responsive cells usually expressed more Raf and MEK activity than GM-CSF-dependent, deltaRaf:ER-infected cells. Abrogation of cytokine dependency by the activated deltaRaf:ER proteins was associated with autocrine growth factor synthesis which was sufficient to promote the growth of uninfected TF-1 cells. In summary, these observations indicate that the aberrant expression of certain activated deltaRaf:ER oncoproteins can alter the cytokine dependency of human hematopoietic TF-1 cells. These cells will be useful in evaluating the roles of the individual Raf oncoproteins in signal transduction, cell cycle progression, autocrine transformation, regulation of apoptosis and differentiation. Moreover, these Raf-infected cells may be important in evaluating the efficacy of novel anticancer drugs designed to inhibit Raf and downstream signal transduction molecules.

Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.

The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.

Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1.

The catalytic domains of the Raf family of protein kinases (deltaRaf) differ in their ability to activate MEK in vitro and in vivo and in their ability to oncogenically transform mammalian cells. The kinase domain of B-Raf is more active than the equivalent portion of Raf-1 which in turn is more active than A-Raf. In Raf-1 the phosphorylation or mutation to aspartic acid of two key tyrosine residues upstream of the ATP binding site has been demonstrated to significantly potentiate catalytic activity. In A-Raf the analogous amino acids are also tyrosine whereas in B-Raf they are aspartic acid. To determine if these differences in amino acid sequence influence the relative catalytic activity of the Raf kinase domains we constructed forms of deltaA-Raf, deltaB-Raf and deltaRaf-1 that encode either aspartic acid [DD], phenylalanine [FF] or tyrosine [YY] at these positions. These proteins were expressed both in mammalian cells as fusions with the hormone binding domain of the estrogen receptor and as epitope-tagged proteins in Sf9 insect cells to test their oncogenic and catalytic potentials. When expressed in Rat1 or 3T3 cells in the presence of hormone all of the deltaRaf-1:ER and deltaA-Raf:ER proteins were transforming with the exception of the [FF] form of deltaA-Raf. In general the [DD] forms of the deltaRaf-1:ER and deltaA-Raf:ER proteins were the most potently oncogenic which correlated with their ability to elicit activation of the MAP kinase pathway. Consistent with the transformation data, the catalytic activity of the [DD] forms of deltaA-Raf:ER and deltaRaf-1:ER was about ten times greater than the cognate [FF] and [YY] forms of the proteins. By contrast all of the deltaB-Raf:ER proteins were highly transforming and deltaB-Raf catalytic activity was largely unaffected by mutation of the aforementioned aspartic acids to either tyrosine or phenylalanine. Similar results were obtained with epitope-tagged forms of deltaA-Raf, deltaB-Raf and deltaRaf-1 expressed in Sf9 cells. These data provide support for the model that key tyrosine residues in the protein kinase domains of A-Raf and Raf-1 are important in the regulation of catalytic activity. In addition they demonstrate that the higher intrinsic activity of B-Raf cannot be explained simply by the presence of aspartic acids at the analogous positions.

Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1.

Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Ras and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of deltaRaf-1:ER, a conditionally active form of oncogenic human Raf-1. Promoter activation by deltaRaf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp -974 and -988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following deltaRaf-1:ER activation. Cotransfection of Ets-2 and p44 mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to deltaRaf-1:ER. Potentiated activation required both p44 MAP kinase catalytic activity and threonine 72 in the Pointed domain of Ets-2. Biochemical assays demonstrated the ability of the p42 and p44 MAP kinases to phosphorylate Ets-2 on threonine 72. Importantly, in intact cells, the kinetics of phosphorylation of Ets-2 on this residue closely mirror the activation of the p42 and p44 MAP kinases and the observed onset of HB-EGF gene transcription following deltaRaf-1:ER activation. These data firmly establish Ets-2 as a direct target of the Raf-MEK-MAP kinase signaling pathway and strongly implicate Ets-2 in the regulation of HB-EGF gene expression.

Flow cytometric ratio analysis of the Hoechst 33342 emission spectrum: multiparametric characterization of apoptotic lymphocytes.

The apoptotic response to various stimuli is an important part of immune regulation, and the ability to identify apoptotic lymphocytes within a complex population is a prerequisite to a more detailed understanding of its role in vivo, We described a flow cytometric technique which utilizes viable cells and enables simultaneous identification of apoptotic cells and analyses of immunophenotype, cell cycle progression, membrane integrity and light scatter properties. It is based upon analysis of two regions of the emission spectrum of the DNA-binding vital dye hoechst 33342. We established a precise correlation between the ratio of red to blue fluorescence emission and apoptosis based upon nuclear morphology and the presence of characteristic DNA degradation patterns. In human peripheral blood lymphocytes (PBL) and mouse thymocytes we incorporated light scatter properties, cell cycle stage, relevant cell surface immunophenotypic markers (CD25 or CD4) and CD8) and a marker of plasma membrane integrity (merocyanine 540) to enable multiparametric phenotyping of apoptotic cells. We show that staurosporine-induced apoptosis of ConA-stimulated PBL is not correlated with cell cycle stage but is selective for activated cells since the frequency of large, CD25+ cells is decreased by staurosporine. Dexamethasone and ionomycin differ in their ability to induce apoptosis selectively in murine thymocyte subsets. Dexamethasone kills a broad spectrum of the CD4/8 immunophenotypes with no selectively for cell cycle stage. Ionomycin selectively deplete CD4+8+ cells, especially those in the Go/G1 region of the cell cycle, and spared CD4-8+ cells. This technique is broadly advantageous for in vitro and ex vivo models of apoptosis in that it interrogates individual viable cells and correlates membrane and nuclear apoptotic changes with standard flow cytometric immunophenotyping.

The immunosuppressant leflunomide inhibits lymphocyte proliferation by inhibiting pyrimidine biosynthesis.

Leflunomide is a novel immunosuppressive compound that is effective in the treatment of animal models of autoimmune disease and human rheumatoid arthritis. The mechanism of action is unknown. Here we show that leflunomide blocked 1) increases in nucleolar size and number, 2) upregulation of the nuclear protein antigens (PCNA and Ki-67), 3) increases in uridine incorporation and total RNA and DNA content, 4) cell cycle progression and 5) proliferation in mitogen-stimulated rat spleen mononuclear cells and human peripheral blood mononuclear cells (HPBMC). Exogenous uridine reversed the leflunomide-dependent inhibition of the normal increase in total RNA and DNA content in mitogen-stimulated HPBMC and rat spleen cells. Uridine reversed the leflunomide-dependent inhibition of cell cycle progression in stimulated rat cell cultures. Either uridine or cytidine, which can be converted to uridine by cytidine deaminase, reversed the antiproliferative effect of leflunomide in HPBMC. Dihydroorotate accumulated in leflunomide-treated human T-lymphoblastoid cells, suggesting that the compound inhibited the fourth enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase. The results support the hypothesis that the in vitro effects of leflunomide on T-lymphocytes are due to inhibition of de novo pyrimidine synthesis.

Leflunomide interferes with pyrimidine nucleotide biosynthesis.

Leflunomide is an anti-inflammatory and immunosuppressive agent which blocks proliferation of transformed cells and mitogen stimulated normal lymphocytes but does not block T cell signaling mechanisms at antiproliferative concentrations. These properties are consistent with a mechanism involving interference with nucleotide metabolism. Leflunomide had anti-proliferative activity against all cells tested here. The anti-proliferative activities could be reversed by addition of uridine or cytidine to the cultures although some species and cellular differences were observed. Purine nucleotides had no effect. Measurements of nucleotide pools in a human T cell line and mitogen stimulated rat spleen cells treated with leflunomide showed that leflunomide preferentially reduces pyrimidine nucleotide levels. These results indicate that inhibition of pyrimidine biosynthesis is responsible for the anti-proliferative effects of leflunomide.

The immunosuppressant leflunomide inhibits lymphocyte progression through cell cycle by a novel mechanism.

Leflunomide is a novel and effective immunosuppressant that holds promise as a therapeutic agent, but the mechanism of action is unknown. Here we provide evidence that leflunomide is a general cytostatic agent for a wide range of cells. The IC50 for proliferation in transformed cell lines ranged from 2 to 16 microM. The mean IC50 for proliferation of mitogen-stimulated rat lymphocytes (86 nM) was much lower than for mouse (3.5 microM) or human (12.5 microM) lymphocytes. Initial signal transduction events (epidermal growth factor receptor-stimulated phosphotyrosine formation and phytohemagglutinin-stimulated Ca++ mobilization) were unaffected by antiproliferative concentrations of leflunomide. Leflunomide was equally as effective against mitogenic stimuli that bypass initial signaling events as it was against surface receptor-mediated mitogens. Leflunomide was fully potent when added 8 hr after the mitogenic stimulus. Cytokine dependent T-cell growth also was blocked by leflunomide. Leflunomide caused only partial reductions of autocrine cytokine production or cytokine receptor expression. Leflunomide blocked completely the progression of rat lymphocytes beyond early S-phase of cell cycle and inhibited entry of human T-cells into the G2 and M-phases without causing cell death. Inhibition of proliferation could not be reversed by purine nucleosides. The results suggest that leflunomide's mechanism of action differs from that of other immunosuppressive agents such as corticosteroids, Cyclosporine A, rapamycin or mycophenolic acid.

Evidence for clonal heterogeneity of the expression of six protein kinase C isoforms in murine B and T lymphocytes.

Protein kinase C (PKC), which plays a pivotal role in lymphocyte activation, represents a homologous family of at least nine proteins. Seven genes that encode PKC proteins have been identified. Since the regulatory properties and substrate specificities of the isoforms are not identical in vitro, it is possible that each isoform plays a unique role in cell activation. Toward an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of mRNA encoding six of the isoforms (alpha, beta, gamma, delta, epsilon, and zeta) in T cell clones and B cell lines. PKC isoform phenotyping was done by MAPPing using isoform-specific primers and slot-blot analyses of mRNA were performed using specific probes. T cell clones and B cell lines were determined to express levels of the delta, epsilon, and zeta isoforms of PKC that were detectable by MAPPing. Plasmacytomas did not express PKC-beta message detectable by MAPPing. Slot blot analyses and Western blot analyses with peptide-specific antibody confirmed that B cell plasmacytomas did not express PKC-beta mRNA or protein. T cell clones and B cell lines were similar in that none expressed PKC-gamma. In cells that expressed PKC isoforms that were detectable by the MAPPing protocol, there was heterogeneity in the relative abundance of isoform mRNA (PKC-delta and -beta) and protein (PKC-beta and -epsilon). Such diversity of isoform expression could be responsible for the differential responsiveness of lymphocyte clones to activating stimuli.

Stimulation of a T helper cell class 2 clone with immobilized anti-T cell receptor antibody activates a Ca2+ and protein kinase C-independent lethal signaling pathway.

Stimulation of an IL-2-dependent variant of the Th2 clone D10.G4.1 with antibodies (Ab) specific for CD3 epsilon or the TCR-alpha beta caused either activation of the clone to secrete the autocrine lymphokine IL-4, or lethal activation in which the cells secreted high quantities of IL-4 but then died within 2 days. High densities of immobilized Ab delivered a lethal signal, whereas soluble forms of Ab and low densities of immobilized Ab caused productive activation in which cell viability was maintained. Lethal activation was not prevented by accessory cells, IL-1, or IL-2, or by co-cross-linkage of CD4 and TCR. The lethal signal was not mediated via a soluble effector from the activated cells. Lethal signaling was insensitive to cyclosporin A or dexamethasone. Studies with activators of protein kinase C (PKC), and PKC inhibitors, indicated that direct activation of PKC was not sufficient for lethal signaling. Nor could direct activation of PKC prevent the lethal signal. The lethal signal was not caused by Ca2+ mobilization mediated by Ca2+ ionophore and there was no evidence of apoptosis. The combination of a PKC activator and Ca2+ ionophore was not lethal, thereby showing that together these events are not sufficient. That these signal pathways were not necessary for lethal activation was evidenced by their inability to lower the density of immobilized anti-CD3 required to cause cell death. In this model, ligation of the TCR specifically activates a Ca2+/PKC-independent lethal signal transduction pathway.

Expression of protein kinase C isoenzymes in thymocyte subpopulations and their differential regulation.

The expression of the different protein kinase C (PKC) isozymes in mouse thymocytes was studied to determine if there is a correlation between isozyme expression and thymocyte phenotype. Expression of PKC isozymes in thymocyte subsets (distinguished by the CD4 or CD8 Ag) was determined by message amplification phenotyping. The expression of mRNA for PKC-alpha, -beta, -epsilon, and -zeta, but not -gamma or -delta isozymes, was detected in all of the unstimulated thymocyte subpopulations analyzed. Thus no differences in the pattern of PKC isozyme expression were found that could be correlated with thymocyte phenotype. However, it was noted that the levels of PKC mRNA expression were affected by different stimuli in unfractionated thymocytes. Whereas mRNA levels of PKC-alpha and -beta were down-regulated by PMA and ionomycin treatment, no significant changes were seen in the levels of PKC-epsilon mRNA with these agents. PKC-epsilon mRNA decreased in thymocytes exposed to Con A similar to what has been reported for PKC-epsilon protein. PKC-zeta mRNA was also down-regulated by PMA or ionomycin, and the combination of both compounds caused a more rapid and drastic effect. Finally, PKC-delta mRNA expression was induced transiently in thymocytes only after exposure to PMA or Con A, and this induction was inhibited by ionomycin treatment. These results indicate that message levels of specific isoforms of PKC are uniquely regulated and suggest an additional level of control of PKC activity in activated lymphocytes.

Characterization of the m4 muscarinic receptor Ca2+ response in a subclone of PC-12 cells by single cell flow cytometry. Inhibition of the response by bradykinin.

We have studied Ca2+ mobilization mediated by the constitutively expressed muscarinic receptor on a subclone of PC-12 cells. The subclone, ACH2, was isolated with a flow cytometer by selection of single cells that exhibited a strong intracellular Ca2+ response to acetylcholine (ACh). Cell to cell heterogeneity of resting Ca2+ levels was markedly reduced in the subclone and homogeneity of the population response was also dramatically improved. ACH2 cells were highly sensitive to ACh and the Ca2+ response in all cells was blocked by muscarinic antagonists. Membranes from ACH2 exhibited muscarinic binding affinities which were not typical of M1, M2, or M3 receptors but were consistent with the profile of the putative m4 receptor. The same percentage of cells responded to ACh whether or not extracellular Ca2+ was reduced with EGTA, but the response was eliminated in all cells by preincubation with pertussis toxin. Thus, the constitutive m4 receptor on ACH2 cells is efficiently coupled to intracellular Ca2+ release by a pertussis toxin-sensitive mechanism. Stimulation of the ACH2 cells by bradykinin (BK) evoked a Ca2+ response in 90% of the cells. Prestimulation with BK diminished the magnitude of the muscarinic Ca2+ response but did not reduce the number of cells which responded to ACh. Inhibition was partially attributed to inhibition of a Ca2+ influx pathway in resting cells. Thus, the signaling mechanism coupled to the m4 muscarinic receptor can be inhibited by signals initiated by the BK receptor.

Flow cytometric analysis of internal calcium mobilization via a B2-bradykinin receptor on a subclone of PC-12 cells.

Single cell Ca2+ mobilization was studied by nonparametric, quantitative flow cytometry using a sort-selected subclone of PC-12 cells. The response of the parent PC-12 population to bradykinin (BK) was very heterogeneous and of a relatively low magnitude. Cells that exhibited maximal Ca2+ mobilization were singly sorted by flow cytometry, cultured, and reanalyzed. In one subclone, referred to as BK1, BK or the B2-BK receptor agonists Lys-BK and Met-Lys-BK (10 pM-1 microM) induced robust Ca2+ transients in 80% of the cells. All three peptides produced the same maximal responses. The B1-BK receptor agonist Des-Arg9-BK (1 nM-1 microM) failed to elicit Ca2+ mobilization in these cells. The responses to BK (10 and 100 nM) were inhibited by preincubation with the B2-receptor antagonists D-Arg0-Hyp3-thienyl5,8-D-Phe7-BK and D-Arg0-Hyp3-D-Phe7 (0.1 nM-10 microM) in a concentration-dependent manner. Des-Arg9-Leu8-BK, a B1-receptor antagonist, failed to block the BK responses at 0.1-10 microM. The agonist/antagonist profile of the BK responses indicated that the B2-BK receptor mediated the Ca2+ response in the BK1 subclone. Thus, flow cytometric analysis of a receptor-mediated Ca2+ response can be employed to select a homogeneously responsive subclone from a heterogeneous, clonal population that can improve the resolution of receptor-mediated second messenger generation at the single cell level.

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.