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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: We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells.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 stimulation of IL-4 NFAT is potentiated by active  V12Rac, and is inhibited by the presence of dominant negative N17Rac.  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  V12Rac (broken line, filled diamonds), 15 μg dominant inhibitory N17Rac  (solid line, open circles), or 15 μg active V14Rho (broken line, open triangles).  Cells were recovered for 6 h before IgE priming and stimulation with the  indicated concentrations of KLH-DNP. (b) FcεR1 stimulation of Elk-1  activity, a Raf-1/MEK-dependent process, is insensitive to N17Rac expression. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone (control), or in combination with 15 μg of pEFN17Rac.  Cells were recovered and left unstimulated (NS), or exposed to IgE/ KLH-DNP as described. (c) Stimulation of NFAT by the FcεR1 alone or  in concert with V12Rac is sensitive to CsA. 1 × 107 cells per point were  transfected with 15 μg IL-4 NFAT CAT reporter alone (solid line, open  squares), or in combination with 15 μg active V12Rac (broken line, filled diamonds). Cells were recovered, primed with IgE anti-DNP, and preincubated for 30 min with either vehicle (control) or the indicated dose of CsA  before FcεR1 stimulation as described.
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Figure 5: (a) FcεR1 stimulation of IL-4 NFAT is potentiated by active V12Rac, and is inhibited by the presence of dominant negative N17Rac. 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 V12Rac (broken line, filled diamonds), 15 μg dominant inhibitory N17Rac (solid line, open circles), or 15 μg active V14Rho (broken line, open triangles). Cells were recovered for 6 h before IgE priming and stimulation with the indicated concentrations of KLH-DNP. (b) FcεR1 stimulation of Elk-1 activity, a Raf-1/MEK-dependent process, is insensitive to N17Rac expression. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone (control), or in combination with 15 μg of pEFN17Rac. Cells were recovered and left unstimulated (NS), or exposed to IgE/ KLH-DNP as described. (c) Stimulation of NFAT by the FcεR1 alone or in concert with V12Rac is sensitive to CsA. 1 × 107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter alone (solid line, open squares), or in combination with 15 μg active V12Rac (broken line, filled diamonds). Cells were recovered, primed with IgE anti-DNP, and preincubated for 30 min with either vehicle (control) or the indicated dose of CsA before FcεR1 stimulation as described.

Mentions: The Rho family GTPase Rac-1 has been shown to play a role in Ras regulation of fibroblast transformation (19) and in T cell antigen receptor regulation of NFAT (34). We therefore investigated the effects of Rho family GTPases in FcεR1 signaling to NFAT. Active mutants of Rac-1 and Rho (V12Rac and V14Rho, respectively) were used to assay whether these GTPases can regulate IL-4 NFAT activation in the mast cell. Fig. 5 a shows that expression of the active V12Rac mutant induced an increase in the basal activity of the NFAT reporter gene, and highly potentiated the FcεR1 activation of IL-4 NFAT. To demonstrate the specificity of this effect, we showed that an activated V14Rho had no discernable potentiating effect on IL-4 NFAT induction (Fig. 5 a).


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 stimulation of IL-4 NFAT is potentiated by active  V12Rac, and is inhibited by the presence of dominant negative N17Rac.  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  V12Rac (broken line, filled diamonds), 15 μg dominant inhibitory N17Rac  (solid line, open circles), or 15 μg active V14Rho (broken line, open triangles).  Cells were recovered for 6 h before IgE priming and stimulation with the  indicated concentrations of KLH-DNP. (b) FcεR1 stimulation of Elk-1  activity, a Raf-1/MEK-dependent process, is insensitive to N17Rac expression. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone (control), or in combination with 15 μg of pEFN17Rac.  Cells were recovered and left unstimulated (NS), or exposed to IgE/ KLH-DNP as described. (c) Stimulation of NFAT by the FcεR1 alone or  in concert with V12Rac is sensitive to CsA. 1 × 107 cells per point were  transfected with 15 μg IL-4 NFAT CAT reporter alone (solid line, open  squares), or in combination with 15 μg active V12Rac (broken line, filled diamonds). Cells were recovered, primed with IgE anti-DNP, and preincubated for 30 min with either vehicle (control) or the indicated dose of CsA  before FcεR1 stimulation as described.
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Figure 5: (a) FcεR1 stimulation of IL-4 NFAT is potentiated by active V12Rac, and is inhibited by the presence of dominant negative N17Rac. 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 V12Rac (broken line, filled diamonds), 15 μg dominant inhibitory N17Rac (solid line, open circles), or 15 μg active V14Rho (broken line, open triangles). Cells were recovered for 6 h before IgE priming and stimulation with the indicated concentrations of KLH-DNP. (b) FcεR1 stimulation of Elk-1 activity, a Raf-1/MEK-dependent process, is insensitive to N17Rac expression. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone (control), or in combination with 15 μg of pEFN17Rac. Cells were recovered and left unstimulated (NS), or exposed to IgE/ KLH-DNP as described. (c) Stimulation of NFAT by the FcεR1 alone or in concert with V12Rac is sensitive to CsA. 1 × 107 cells per point were transfected with 15 μg IL-4 NFAT CAT reporter alone (solid line, open squares), or in combination with 15 μg active V12Rac (broken line, filled diamonds). Cells were recovered, primed with IgE anti-DNP, and preincubated for 30 min with either vehicle (control) or the indicated dose of CsA before FcεR1 stimulation as described.
Mentions: The Rho family GTPase Rac-1 has been shown to play a role in Ras regulation of fibroblast transformation (19) and in T cell antigen receptor regulation of NFAT (34). We therefore investigated the effects of Rho family GTPases in FcεR1 signaling to NFAT. Active mutants of Rac-1 and Rho (V12Rac and V14Rho, respectively) were used to assay whether these GTPases can regulate IL-4 NFAT activation in the mast cell. Fig. 5 a shows that expression of the active V12Rac mutant induced an increase in the basal activity of the NFAT reporter gene, and highly potentiated the FcεR1 activation of IL-4 NFAT. To demonstrate the specificity of this effect, we showed that an activated V14Rho had no discernable potentiating effect on IL-4 NFAT induction (Fig. 5 a).

Bottom Line: We observe that Elk-1 and NFAT are targeted by distinct Ras effector pathways in mast cells.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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