TNF-alpha and IL-5 gene induction in IgE plus antigen-stimulated mast cells require common and distinct signaling pathways.

BACKGROUND: Mast cells produce a variety of cytokines and chemokines in a timely and tightly controlled fashion if stimulated via the FcepsilonRI. Evidence is accumulating that the transcriptional induction of the corresponding genes and the release of these mediators are dependent on common and mediator-specific components of the signal transduction and transcription factor machinery. METHODS: We addressed this issue by comparing the effects of mitogen activated protein (MAP) kinase pathway inhibitors and protein kinase C (PKC) inhibitors on the induction of TNF-alpha and IL-5 after IgE plus antigen (Ag) stimulation in CPII mouse mast cells using Western blot analyses and transient transfections of reporter gene plasmids. RESULTS: TNF-alpha shows a strict dependence on the MAP kinase pathway, while IL-5 is either activated by PMA-dependent PKCs or along the MAP kinase pathway. In addition, both mediators are sensitive to PKCmu inhibition, suggesting involvement of this atypical, non-PMA dependent PKC in the overall induction process. CONCLUSION: While the two cytokines were recently shown to be regulated by a member of the nuclear factor of activated T-cells (NF-AT) transcription factor family, activator protein 1 (AP1) was identified as a cofactor at the TNF-alpha promoter while a GATA family member comprised the cofactor at the IL-5 promoter. This suggests that the differences in requirement for signal transduction cascades are the result of a different usage of NF-AT cofactors for transcription of each cytokine in mast cells.

A novel splice variant of the transcription factor Nrf1 interacts with the TNFalpha promoter and stimulates transcription.

Common signaling chains of various receptor families, despite some similarities, are able to provoke quite different cellular responses. This suggests that they are linked to different cascades and transcription factors, dependent on the context of the ligand binding moiety and the cell type. The ITAM (immunoreceptor tyrosine-based activation motif) containing gamma chain of the FcepsilonRI, FcgammaRI, FcgammaRIII and the T-cell receptor is one of these shared signaling molecules. Here, we show that in the context of the FcgammaRIII, the gamma chain activates the transcription factor Nrf1 or a closely related protein that specifically interacts with the extended kappa3 site in the TNFalpha promoter. A novel splice variant of Nrf1 with a 411 bp deletion of the serine-rich region, resulting in an overall structure reminiscent of the BTB and CNC homology (Bach) proteins, was isolated from the corresponding DC18 cells. In a gel shift analysis, this bacterially expressed splice variant binds to the TNFalpha promoter site after in vitro phosphorylation by casein kinase II (CKII). In addition, cotransfection studies demonstrate that this splice variant mediates induced transcription at the TNFalpha promoter after stimulation/activation in a heterologous system.

Common and distinct signaling pathways mediate the induction of TNF-alpha and IL-5 in IgE plus antigen-stimulated mast cells.

A small number of signaling cascades represented by mitogen-activated protein kinases, phosphoinositol-3-kinase, protein kinase C, signal transducers and activators of transcription, Ca2+/calcineurin, and a few other molecules are linked to an incomparably large number of surface receptors. Parallel activation of several of these pathways and the existence of isozymes for a number of signal transmitting molecules generate the required complexity and specificity matching the receptor variety. Here we show that the proinflammatory mediator TNF-alpha and the growth factor IL-5 are activated along common and distinct signaling cascades in allergically stimulated murine mast cells. Both of them are dependent on Ca2+ influx, activation of calcineurin and nuclear factor of activated T cells as well as a member of the atypical PKC family, most likely PKCmu. Additionally, mitogen-activated protein kinases for TNF-alpha and members of the classical or nonclassical PKCs for IL-5, respectively, were identified as additional required pathways. Inhibition of the classical and nonclassical PKCs, however, does not abrogate IL-5 induction but instead leads to a switch to mitogen-activated protein kinases, which then become essential. The activated branches of this "salvage" signaling cascade are represented by extracellular signal-regulated kinase 1/2 and c-jun NH2 terminal kinase 1 in allergically stimulated mast cells.