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Distinct Ras effector pathways are involved in Fc epsilon R1 regulation of the transcriptional activity of Elk-1 and NFAT in mast cells.

Turner H, Cantrell DA - J. Exp. Med. (1997)

Bottom Line: Ras mediates diverse effects via its diverse effector pathways, which may include other members of the Ras GTPase family such as RhoA and Rac-1.The effector pathway for Ras activation of NFAT is not Raf-1/MEK.We identify that the Rac-1 GTPase is critical in Fc epsilon R1 regulation of NFAT, acting either in parallel with or as an effector of Ras.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London, United Kingdom.

ABSTRACT
Activation of Ras GTPases is a conserved feature of antigen receptor signaling, including Fc epsilon R1 activation of mast cells. Antigenic cross-linking of the Fc epsilon R1 on mast cells results in secretion of allergic mediators and induction of immediate early and cytokine genes. Here we examine the role of Ras in coupling the Fc epsilon R1 to transcriptional regulation. The transcription factors Elk-1, an immediate early gene regulator and the nuclear factor of activated T cells (NFAT), in the context of the IL-4 gene, are identified as Ras targets in mast cells. Ras mediates diverse effects via its diverse effector pathways, which may include other members of the Ras GTPase family such as RhoA and Rac-1. We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells. Activation of the "classical" Ras/Raf-1/MEK/ ERK cascade is necessary and sufficient for Fc epsilon R1 induction of Elk-1. Ras function is required, but not sufficient for Fc epsilon R1 induction of NFAT. However, activation or inhibition of Ras markedly shifts the antigen dose-response for Fc epsilon R1 induction of NFAT. The effector pathway for Ras activation of NFAT is not Raf-1/MEK. We identify that the Rac-1 GTPase is critical in Fc epsilon R1 regulation of NFAT, acting either in parallel with or as an effector of Ras. These data place Ras in a crucial position in mast cells, regulating disparate nuclear targets. Moreover, we identify that two GTPases, Ras and Rac-1, are important regulators of NFAT, and therefore of cytokine expression in mast cells.

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(a) FcεR1 cross-linking induces IL-4 NFAT CAT activity in  a dose-dependent manner. 1 × 107 cells per point were transfected with  15 μg IL-4 NFAT CAT reporter. Cells were recovered for 6 h before  IgE priming and stimulation with the indicated concentrations of KLHDNP. (b) FcεR1 stimulation of IL-4 NFAT is potentiated by active  V12Ras, but not Raf-CAAX, and is inhibited by the presence of dominant negative N17Ras. 1 × 107 cells per point were transfected with 15 μg  IL-4 NFAT CAT reporter alone (solid line, filled squares), or in combination with 15 μg active V12Ras (solid line, open circles), 15 μg dominant inhibitory N17Ras (broken line, filled diamonds), or 15 μg Raf-CAAX (broken  line, open triangles). Cells were recovered for 6 h before IgE priming, and then  stimulation with the indicated concentrations of KLH-DNP. (c) FcεR1  stimulation of IL-4 NFAT CAT activity is insensitive to PD098059. 1 ×  107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter  alone, and recovered for 6 h. Cells were preincubated for 30 min with either DMSO or the indicated concentrations of PD098059. Cells were left  unstimulated (NS), exposed to IgE/KLH-DNP, or treated with 50 ng/ml  PdBu and 500 ng/ml ionomycin.
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Figure 4: (a) FcεR1 cross-linking induces IL-4 NFAT CAT activity in a dose-dependent manner. 1 × 107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter. Cells were recovered for 6 h before IgE priming and stimulation with the indicated concentrations of KLHDNP. (b) FcεR1 stimulation of IL-4 NFAT is potentiated by active V12Ras, but not Raf-CAAX, and is inhibited by the presence of dominant negative N17Ras. 1 × 107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter alone (solid line, filled squares), or in combination with 15 μg active V12Ras (solid line, open circles), 15 μg dominant inhibitory N17Ras (broken line, filled diamonds), or 15 μg Raf-CAAX (broken line, open triangles). Cells were recovered for 6 h before IgE priming, and then stimulation with the indicated concentrations of KLH-DNP. (c) FcεR1 stimulation of IL-4 NFAT CAT activity is insensitive to PD098059. 1 × 107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter alone, and recovered for 6 h. Cells were preincubated for 30 min with either DMSO or the indicated concentrations of PD098059. Cells were left unstimulated (NS), exposed to IgE/KLH-DNP, or treated with 50 ng/ml PdBu and 500 ng/ml ionomycin.

Mentions: Fig. 4 a shows that FcεR1 cross-linking induces IL-4 NFAT activity in a manner dose dependent on antigen. The PKC inhibitor Ro-318425 did not affect the FcεR1 activation of NFAT (data not shown). To address whether there is a role for Ras in the FcεR1 regulation of NFAT, we cotransfected active and dominant inhibitory Ras mutants with the IL-4 NFAT-CAT reporter gene. Expression of active V12Ras induced a weak increase in the basal activity of the NFAT reporter gene, and robustly potentiated FcεR1 induction of NFAT. Conversely, expression of N17Ras inhibited the NFAT response to FcεR1 (Fig. 4 b). Hence, cotransfected activated Ras (V12Ras) causes a potentiation of FcεR1 activation of IL-4 NFAT that potently increases the sensitivity of NFAT responses to antigen. The presence of N17Ras consistently inhibits the antigen dose response for IL-4 NFAT CAT induction to half-maximal levels, and supresses the antigen sensitivity of the mast cells for NFAT activation.


Distinct Ras effector pathways are involved in Fc epsilon R1 regulation of the transcriptional activity of Elk-1 and NFAT in mast cells.

Turner H, Cantrell DA - J. Exp. Med. (1997)

(a) FcεR1 cross-linking induces IL-4 NFAT CAT activity in  a dose-dependent manner. 1 × 107 cells per point were transfected with  15 μg IL-4 NFAT CAT reporter. Cells were recovered for 6 h before  IgE priming and stimulation with the indicated concentrations of KLHDNP. (b) FcεR1 stimulation of IL-4 NFAT is potentiated by active  V12Ras, but not Raf-CAAX, and is inhibited by the presence of dominant negative N17Ras. 1 × 107 cells per point were transfected with 15 μg  IL-4 NFAT CAT reporter alone (solid line, filled squares), or in combination with 15 μg active V12Ras (solid line, open circles), 15 μg dominant inhibitory N17Ras (broken line, filled diamonds), or 15 μg Raf-CAAX (broken  line, open triangles). Cells were recovered for 6 h before IgE priming, and then  stimulation with the indicated concentrations of KLH-DNP. (c) FcεR1  stimulation of IL-4 NFAT CAT activity is insensitive to PD098059. 1 ×  107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter  alone, and recovered for 6 h. Cells were preincubated for 30 min with either DMSO or the indicated concentrations of PD098059. Cells were left  unstimulated (NS), exposed to IgE/KLH-DNP, or treated with 50 ng/ml  PdBu and 500 ng/ml ionomycin.
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Figure 4: (a) FcεR1 cross-linking induces IL-4 NFAT CAT activity in a dose-dependent manner. 1 × 107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter. Cells were recovered for 6 h before IgE priming and stimulation with the indicated concentrations of KLHDNP. (b) FcεR1 stimulation of IL-4 NFAT is potentiated by active V12Ras, but not Raf-CAAX, and is inhibited by the presence of dominant negative N17Ras. 1 × 107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter alone (solid line, filled squares), or in combination with 15 μg active V12Ras (solid line, open circles), 15 μg dominant inhibitory N17Ras (broken line, filled diamonds), or 15 μg Raf-CAAX (broken line, open triangles). Cells were recovered for 6 h before IgE priming, and then stimulation with the indicated concentrations of KLH-DNP. (c) FcεR1 stimulation of IL-4 NFAT CAT activity is insensitive to PD098059. 1 × 107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter alone, and recovered for 6 h. Cells were preincubated for 30 min with either DMSO or the indicated concentrations of PD098059. Cells were left unstimulated (NS), exposed to IgE/KLH-DNP, or treated with 50 ng/ml PdBu and 500 ng/ml ionomycin.
Mentions: Fig. 4 a shows that FcεR1 cross-linking induces IL-4 NFAT activity in a manner dose dependent on antigen. The PKC inhibitor Ro-318425 did not affect the FcεR1 activation of NFAT (data not shown). To address whether there is a role for Ras in the FcεR1 regulation of NFAT, we cotransfected active and dominant inhibitory Ras mutants with the IL-4 NFAT-CAT reporter gene. Expression of active V12Ras induced a weak increase in the basal activity of the NFAT reporter gene, and robustly potentiated FcεR1 induction of NFAT. Conversely, expression of N17Ras inhibited the NFAT response to FcεR1 (Fig. 4 b). Hence, cotransfected activated Ras (V12Ras) causes a potentiation of FcεR1 activation of IL-4 NFAT that potently increases the sensitivity of NFAT responses to antigen. The presence of N17Ras consistently inhibits the antigen dose response for IL-4 NFAT CAT induction to half-maximal levels, and supresses the antigen sensitivity of the mast cells for NFAT activation.

Bottom Line: Ras mediates diverse effects via its diverse effector pathways, which may include other members of the Ras GTPase family such as RhoA and Rac-1.The effector pathway for Ras activation of NFAT is not Raf-1/MEK.We identify that the Rac-1 GTPase is critical in Fc epsilon R1 regulation of NFAT, acting either in parallel with or as an effector of Ras.

View Article: PubMed Central - PubMed

Affiliation: Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London, United Kingdom.

ABSTRACT
Activation of Ras GTPases is a conserved feature of antigen receptor signaling, including Fc epsilon R1 activation of mast cells. Antigenic cross-linking of the Fc epsilon R1 on mast cells results in secretion of allergic mediators and induction of immediate early and cytokine genes. Here we examine the role of Ras in coupling the Fc epsilon R1 to transcriptional regulation. The transcription factors Elk-1, an immediate early gene regulator and the nuclear factor of activated T cells (NFAT), in the context of the IL-4 gene, are identified as Ras targets in mast cells. Ras mediates diverse effects via its diverse effector pathways, which may include other members of the Ras GTPase family such as RhoA and Rac-1. We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells. Activation of the "classical" Ras/Raf-1/MEK/ ERK cascade is necessary and sufficient for Fc epsilon R1 induction of Elk-1. Ras function is required, but not sufficient for Fc epsilon R1 induction of NFAT. However, activation or inhibition of Ras markedly shifts the antigen dose-response for Fc epsilon R1 induction of NFAT. The effector pathway for Ras activation of NFAT is not Raf-1/MEK. We identify that the Rac-1 GTPase is critical in Fc epsilon R1 regulation of NFAT, acting either in parallel with or as an effector of Ras. These data place Ras in a crucial position in mast cells, regulating disparate nuclear targets. Moreover, we identify that two GTPases, Ras and Rac-1, are important regulators of NFAT, and therefore of cytokine expression in mast cells.

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