The mitogen-activated protein kinase pathway inhibits ceramide-induced terminal differentiation of a human monoblastic leukemia cell line, U937

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.     

Effects of pulsed ultrasound on embryonic development: an in vitro study

Precursor CD4+ T cells develop into effector Th1 and Th2 cells that play a central role in the immune response. We show that the JNK MAP kinase pathway is induced in Th1 but not in Th2 effector cells upon antigen stimulation. Further, the differentiation of precursor CD4+ T cells into effector Th1 but not Th2 cells is impaired in JNK2-deficient mice. The inability of IL-12 to differentiate JNK2-deficient CD4+ T cells fully into effector Th1 cells is caused by a defect in IFNgamma production during the early stages of differentiation. The addition of exogenous IFNgamma during differentiation restores IL-12-mediated Th1 polarization in the JNK2-deficient mice. The JNK MAP kinase signaling pathway, therefore, plays an important role in the balance of Th1 and Th2 immune responses.     

The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes

The extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and p38 MAP kinase pathways are triggered upon ligation of the antigen-specific T cell receptor (TCR). During the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4(-)CD8(-) into CD4(+)CD8(+) double positive (DP) thymocytes, positive selection of DP cells, and their maturation into CD4(+) cells. However, the ERK pathway is not required for negative selection. Here, we show that JNK is activated in DP thymocytes in vivo in response to signals that initiate negative selection. The activation of JNK in these cells appears to be mediated by the MAP kinase kinase MKK7 since high levels of MKK7 and low levels of Sek-1/MKK4 gene expression were detected in thymocytes. Using dominant negative JNK transgenic mice, we show that inhibition of the JNK pathway reduces the in vivo deletion of DP thymocytes. In addition, the increased resistance of DP thymocytes to cell death in these mice produces an accelerated reconstitution of normal thymic populations upon in vivo DP elimination. Together, these data indicate that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis in response to TCR-derived and other thymic environment- mediated signals.     

T cell antigen receptor-mediated activation of the Ras/mitogen-activated protein kinase pathway controls interleukin 4 receptor function and type-2 helper T cell differentiation

The central role of type-2 helper T (Th2) cells in the development of allergic responses and immune responses against helminthic parasites is well documented. The differentiation of Th2 cells from naive T cells requires both the recognition of antigen by T cell antigen receptors (TCR) and the activation of downstream signal-transduction molecules of the interleukin 4 receptor (IL-4R) pathway, including Jak1, Jak3, and STAT6. Little is known, however, about how these two distinct pathways cooperate with each other to induce Th2 cells. Here, we use a T cell-specific H-Ras-dominant-negative transgenic mouse to show that TCR-mediated activation of the Ras/mitogen-activated protein kinase pathway alters IL-4R function and is required for Th2 cell differentiation. The enhancement of IL-4R signaling seems to be a consequence of both direct "crosstalk" with the TCR signaling pathway and increased protein expression of downstream signaling molecules of the IL-4R pathway. Therefore, successful Th2 differentiation depends on the effectiveness of the TCR-mediated activation of the Ras/mitogen-activated protein kinase pathway in modifying the IL-4R-mediated signaling pathway.     

Stimulation of Jun N-terminal kinase (JNK) by gonadotropin-releasing hormone in pituitary alpha T3-1 cell line is mediated by protein kinase C, c-Src, and CDC42

The signaling of ligands operating via heterotrimeric G proteins is mediated by a complex network that involves sequential phosphorylation events. Signaling by the G protein-coupled receptor GnRH was shown to include elevation of Ca2+ and activation of phospholipases, protein kinase C (PKC) and extra-cellular signal-regulated kinase (ERK). In this study, GnRH was shown to activate Jun N-Terminal Kinase (JNK)/SAPK in alpha T3-1 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as well as tumor-promoting agent (TPA) also increased c-Src activity, which peaked at 2 min after GnRH stimulation and was sensitive both to PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which serves as a Src-dominant interfering kinase, and constitutively active forms of Src, together with JNK, confirmed the involvement of c-Src downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1, and MEK1 with JNK indicated that JNK activation by GnRH and TPA is mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a related mitogen-activated protein kinase (MAPK) pathway, did not affect JNK activation in alpha T3-1 cells. Taken together, our results suggest that GnRH stimulation of JNK activity is mediated by a unique pathway that includes sequential activation of PKC, c-Src, CDC42, and probably also MEKK1.     

Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation

The receptor tyrosine kinase Kit plays critical roles in hematopoiesis, gametogenesis and melanogenesis. In mast cells, Kit receptor activation mediates several cellular responses including cell proliferation and suppression of apoptosis induced by growth factor deprivation and gamma-irradiation. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules. We have investigated the role of phosphatidylinositol 3'-kinase (PI 3-kinase) and Src kinases in Kit-mediated cell proliferation and suppression of apoptosis induced both by factor deprivation and irradiation in bone marrow-derived mast cells (BMMC). Analysis of Kit-/- BMMC expressing mutant Kit receptors and the use of pharmacological inhibitors revealed that both signaling pathways contribute to these Kit-mediated responses and that elimination of both pathways abolishes them. We demonstrate that the PI 3-kinase and Src kinase signaling pathways converge to activate Rac1 and JNK. Analysis of BMMC expressing wild-type and dominant-negative mutant forms of Rac1 and JNK revealed that the Rac1/JNK pathway is critical for Kit ligand (KL)-induced proliferation of mast cells but not for suppression of apoptosis. In addition, KL was shown to inhibit sustained activation of JNK induced by gamma-irradiation and concomitant irradiation-induced apoptosis.     

Role of mitogen-activated protein kinases and c-Jun/AP-1 trans-activating activity in the regulation of protease mRNAs and the malignant phenotype in NIH 3T3 fibroblasts

Ras activates a multitude of downstream activities with roles in cellular proliferation, invasion and metastasis, differentiation, and programmed cell death. In this work we have evaluated the requirement of extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase kinase (JNKK), and c-Jun/AP-1 activities in transformation and extracellular matrix invasion of ras oncogene expressing NIH 3T3 fibroblasts by expressing stable mutant genes that constitutively inhibit these activities. Whereas the inhibition of ERK activity reverts the transformed and invasive phenotype, the inhibition of the JNK pathway and AP-1 trans-activating activities by JNKK[K129R] and c-Jun(TAM67) had no effect on the ability of the ras oncogene-expressing cells to grow in soft agar or invade Matrigel basement membrane. Thus an elevated JNK activity and/or c-Jun/AP-1 trans-activating activity are not absolute requirements for ras transformation or invasion through basement membrane, and the dependence on AP-1 activity for transformation is cell-specific. However, inhibition of JNK kinase (JNKK) in ras-transformed cells with normally elevated JNK activity switches the protease-dependent invasive phenotype from a urokinase plasminogen activator (uPA)-dependent to a cathepsin L (CL)-dependent invasive phenotype. Conversely, treatment of ras-transformed cells of low constitutive JNK activity with the JNK stimulator, anisomycin, converts the protease mRNA levels from those characteristic of a CL-dependent to a uPA-dependent phenotype. These protease phenotypes can be duplicated in untransformed NIH 3T3 cells that express platelet-derived growth factor receptors and m1 muscarinic receptors that selectively stimulate the ERK or JNK pathways, respectively. It is concluded that high ERK activity is required for both protease phenotypes, whereas the JNK pathway and c-Jun/AP-1 activity are not required for transformation but regulate a switch between uPA and CL protease phenotypes in both transformed and untransformed cells. In ras-transformed NIH 3T3 fibroblasts, the uPA- and CL-dependent protease phenotypes are redundant in their ability to invade through basement membrane.     

The A and the extended V N-terminal regions of streptococcal protein I/IIf mediate the production of tumour necrosis factor alpha in the monocyte cell line THP-1

Signal transduction is ubiquitously involved in the initiation of physiological signals that lead to growth and proliferation of cells. The signaling cascade mediated by the mitogen-activated protein kinase (MAPK) is considered essential for T cell growth and function. Therefore, it was of interest to determine the influence of age on the induction of MAPK in mitogen-activated T cells. T cells from young (4-6 months) and old (24-26 months) rats responded to concanavalin A (Con A) stimulation by increasing MAPK, c-jun amino terminal kinase (JNK), and p21ras activities. The time course of induction of MAPK/JNK and p21ras activities was similar in T cells isolated from young and old rats. The induction of JNK activity did not change significantly with age; however, the induction of MAPK and p21ras activities was significantly less (50 to 65%) in T cells from old rats than in T cells from young rats. Although the relative protein levels of p42 and p44 MAPK did not change with age, the proportion of the phosphorylated p44 MAPK decreased with age. In addition, it was found that the in vitro kinase activities of the T cell receptor-associated protein tyrosine kinase Lck (p56Lck) and ZAP-70 but not Fyn (p59Fyn) were lower in T cells from old rats than in T cells from young rats. The decline in activities of these signaling molecules with age was not associated with changes in their corresponding protein levels. Thus, our results demonstrate that aging alters the activation of the signal transduction cascade that leads to T cell activation.     

Sucrase-isomaltase and other brush border glycosidases in colorectal tumors

CD28 ligation delivers a costimulatory signal important in T cell activation. This study demonstrates that the disruption of the CD28/B7 pathway early in the nonobese diabetic mouse strain, using CD28-/- and CTLA41g transgenic mice, promoted the development and progression of spontaneous autoimmune diabetes. Functional analyses of T cells isolated from CD28-deficient mice demonstrated that the GAD-specific T cells produced enhanced Th1-type cytokines (IL-2 and IFN gamma) and diminished Th2-type cytokine, IL-4. Moreover, there was a significant decrease in serum levels of anti-GAD antibodies of the IgG1 isotype consistent with a profound suppression of Th2-type responses in these animals. Thus, the early differentiation of naive diabetogenic T cells into the Th2 subset is dependent upon CD28 signaling and extends our understanding of the importance of Th1/Th2 balance in the regulation of this spontaneous autoimmune disease.     

Downstream of Crk adaptor signaling pathway: activation of Jun kinase by v-Crk through the guanine nucleotide exchange protein C3G

Crk, which belongs to the adaptor family of proteins composed of Src homology 2 (SH2) and SH3 domains, has a putative role in signaling. However, the downstream events of Crk signaling remain unclear. In this study, we found that Jun kinase (JNK) is moderately activated by v-Crk in both NIH 3T3 cells and chicken embryo fibroblasts. Transient expression of v-Crk, c-Crk-I, or c-Crk-II activated JNK1 in human embryo kidney cells, 293T. Coexpression of a guanine nucleotide exchange protein C3G, which specifically binds to Crk's SH3 domain, further enhanced the JNK activity as well as growth rate and anchorage-independent growth of v-Crk NIH 3T3 cells. Furthermore, overexpression of a dominant-negative form of C3G lacking the guanine nucleotide exchange domain abolished both the JNK activity and the colony forming potential of v-Crk NIH 3T3 cells. The requirement for JNK activation in v-Crk induced transformation was demonstrated by the suppression of colony forming activity of v-Crk NIH 3T3 cells when a dominant-negative form of JNK kinase, Sek1/MKK4 is expressed in these cells. These data strongly suggest the existence of a novel signaling cascade involving an adaptor protein v-Crk, which transmits signals through C3G toward JNK activation.     

Early activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase in response to binding of simian immunodeficiency virus to Jurkat T cells expressing CCR5 receptor

We have shown that the binding of simian immunodeficiency virus (SIV) to Jurkat T cells expressing CD4 receptor strongly induces mitogen-activated protein (MAP) kinase kinase (MEK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) and only weakly induces p38 MAP kinase and c-Jun N-terminal kinase (JNK). Similarly, T-tropic NL4-3 virus, which uses both CD4 and CXCR4 receptors for entry, stimulated in these cells the MEK/ERK MAP kinase (MAPK) pathway in a CD4 receptor-dependent manner (Popik and Pitha, 1998). In contrast, both macrophage-tropic SIVmac316 and T cell-tropic SIVmac239, which in addition to CD4 require CCR5 coreceptor for entry, significantly enhanced early MEK/ERK, p38 MAPK, and JNK signaling in Jurkat cells expressing constitutively or transiently the CCR5 receptor. Together, this study provides the evidence that viruses using CXCR4 or CCR5 receptors for entry may differentially use signaling properties of their specific coreceptors to stimulate MAP kinase cascades. In addition, although SIVmac239 and SIVmac316 use different structural domains of the CCR5 receptor for entry, both viruses stimulate early phosphorylation of MEK, ERK, p38, and JNK independently of their tropism and replication.     

Enhancement of fibroblast collagenase (matrix metalloproteinase-1) gene expression by ceramide is mediated by extracellular signal-regulated and stress-activated protein kinase pathways

Inflammatory cytokines tumor necrosis factor-alpha and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKbeta. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders.     

Role of NK1.1+ T cells in a TH2 response and in immunoglobulin E production

Immune responses dominated by interleukin-4 (IL-4)-producing T helper type 2 (TH2) cells or by interferon gamma (IFN-gamma)-producing T helper type 1 (TH1) cells express distinctive protection against infection with different pathogens. Interleukin-4 promotes the differentiation of na?ve CD4+ T cells into IL-4 producers and suppresses their development into IFN-gamma producers. CD1-specific splenic CD4+NK1.1+ T cells, a numerically minor population, produced IL-4 promptly on in vivo stimulation. This T cell population was essential for the induction of IL-4-producing cells and for switching to immunoglobulin E, an IL-4-dependent event, in response to injection of antibodies to immunoglobulin D.     

T cells reactive to a single immunodominant self-restricted allopeptide induce skin graft rejection in mice

Alloreactive T lymphocytes can respond to foreign MHC complexed with foreign peptides through the direct pathway of allorecognition and can additionally recognize allopeptides expressed in the context of recipient (self) MHC through the indirect pathway. To better elucidate how indirect pathway-responsive CD4(+) T cells mediate allograft rejection, we isolated and characterized a TH1 T cell line from BALB/c recipients of B10.A skin that responds to a defined immunodominant, self-restricted allopeptide, I-Abetak58-71. When transferred into BALB/c severe combined immunodeficiency recipients of B10.A skin allografts, this cell line specifically induced a form of skin graft rejection characterized by the presence of TH1 cytokines, macrophage infiltration, and extensive fibrosis. Recall immune responses and immunofluorescence of the rejecting skin revealed only the presence of the peptide-specific T cells within the recipient animals, with no evidence of a direct pathway alloresponse. These studies demonstrate that T cells reactive to a single self-restricted allopeptide can mediate a form of allogeneic skin graft rejection that exhibits characteristics of a chronic, fibrosing process.