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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) PD098059 inhibits MEK activation of Erk in mast cells. 1 ×  106 RBL2H3 per point were incubated for 30 min at 37°C with DMSO  (control) or 25 μM PD098059. Stimulations were for 10 min with either  50 ng/ml PdBu or, after priming with IgE anti DNP, 10 min 500 ng/ml  KLH-DNP. Western blotting for Erk was carried out as described in Materials and Methods. (b) Induction of Elk-1 via activated Ras and Raf-1 is  sensitive to inhibition of MEK. 1 × 107 cells per point were transfected  with either the Elk-1 reporter system alone, or in combination with 10 μg  pEF V12Ras or 10 μg pEF Raf-CAAX. Control cells were left unstimulated (NS) or exposed to IgE/KLH-DNP. Cotransfected cells were left  unstimulated. Preincubation with 25 μM PD098059 or DMSO was for  30 min before stimulation. (c) Induction of Elk-1 activity by FcεR1 or  PdBu is sensitive to inhibition of MEK. 1 × 107 cells per point were  transfected with the Elk-1 reporter system 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 PdBu as described.
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Figure 3: (a) PD098059 inhibits MEK activation of Erk in mast cells. 1 × 106 RBL2H3 per point were incubated for 30 min at 37°C with DMSO (control) or 25 μM PD098059. Stimulations were for 10 min with either 50 ng/ml PdBu or, after priming with IgE anti DNP, 10 min 500 ng/ml KLH-DNP. Western blotting for Erk was carried out as described in Materials and Methods. (b) Induction of Elk-1 via activated Ras and Raf-1 is sensitive to inhibition of MEK. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone, or in combination with 10 μg pEF V12Ras or 10 μg pEF Raf-CAAX. Control cells were left unstimulated (NS) or exposed to IgE/KLH-DNP. Cotransfected cells were left unstimulated. Preincubation with 25 μM PD098059 or DMSO was for 30 min before stimulation. (c) Induction of Elk-1 activity by FcεR1 or PdBu is sensitive to inhibition of MEK. 1 × 107 cells per point were transfected with the Elk-1 reporter system 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 PdBu as described.

Mentions: The specificity of PD098059 as a MEK inhibitor has been previously described (33). Nevertheless, in initial experiments we verified that PD098059 was an effective MEK inhibitor in RBL2H3 cells. The phosphorylation of Erk by MEK results in decreased electrophoretic mobility of Erk in SDS-PAGE. Fig. 3 a shows an Erk mobility shift assay visualized by pan-Erk Western blot. In control cells (left), PdBu or FcεR1 stimulation cause the appearance of an hyperphosphorylated Erk population. The application of 25 μM PD098059 (right) efficiently inhibits the activation of Erk by MEK in mast cells. Accordingly, we examined the effect of PD098059 on induction of Elk-1 transactivation in mast cells. As shown in Fig. 3 b, the activation of Elk-1 induced by cotransfection of Raf-CAAX and V12Ras constructs is inhibited by PD098059. Hence, these mutants are indeed reflecting the use of a Raf-1/Erk pathway to target Elk-1. Fig. 3 c shows that the induction of Elk-1 transactivation by both PdBu and FcεR1 cross-linking is inhibited by PD098059 in a dose-dependent manner. These data confirm that the Raf-1/MEK cascade is the critical Ras effector pathway for FcεR1 activation of Elk-1.


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) PD098059 inhibits MEK activation of Erk in mast cells. 1 ×  106 RBL2H3 per point were incubated for 30 min at 37°C with DMSO  (control) or 25 μM PD098059. Stimulations were for 10 min with either  50 ng/ml PdBu or, after priming with IgE anti DNP, 10 min 500 ng/ml  KLH-DNP. Western blotting for Erk was carried out as described in Materials and Methods. (b) Induction of Elk-1 via activated Ras and Raf-1 is  sensitive to inhibition of MEK. 1 × 107 cells per point were transfected  with either the Elk-1 reporter system alone, or in combination with 10 μg  pEF V12Ras or 10 μg pEF Raf-CAAX. Control cells were left unstimulated (NS) or exposed to IgE/KLH-DNP. Cotransfected cells were left  unstimulated. Preincubation with 25 μM PD098059 or DMSO was for  30 min before stimulation. (c) Induction of Elk-1 activity by FcεR1 or  PdBu is sensitive to inhibition of MEK. 1 × 107 cells per point were  transfected with the Elk-1 reporter system 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 PdBu as described.
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Figure 3: (a) PD098059 inhibits MEK activation of Erk in mast cells. 1 × 106 RBL2H3 per point were incubated for 30 min at 37°C with DMSO (control) or 25 μM PD098059. Stimulations were for 10 min with either 50 ng/ml PdBu or, after priming with IgE anti DNP, 10 min 500 ng/ml KLH-DNP. Western blotting for Erk was carried out as described in Materials and Methods. (b) Induction of Elk-1 via activated Ras and Raf-1 is sensitive to inhibition of MEK. 1 × 107 cells per point were transfected with either the Elk-1 reporter system alone, or in combination with 10 μg pEF V12Ras or 10 μg pEF Raf-CAAX. Control cells were left unstimulated (NS) or exposed to IgE/KLH-DNP. Cotransfected cells were left unstimulated. Preincubation with 25 μM PD098059 or DMSO was for 30 min before stimulation. (c) Induction of Elk-1 activity by FcεR1 or PdBu is sensitive to inhibition of MEK. 1 × 107 cells per point were transfected with the Elk-1 reporter system 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 PdBu as described.
Mentions: The specificity of PD098059 as a MEK inhibitor has been previously described (33). Nevertheless, in initial experiments we verified that PD098059 was an effective MEK inhibitor in RBL2H3 cells. The phosphorylation of Erk by MEK results in decreased electrophoretic mobility of Erk in SDS-PAGE. Fig. 3 a shows an Erk mobility shift assay visualized by pan-Erk Western blot. In control cells (left), PdBu or FcεR1 stimulation cause the appearance of an hyperphosphorylated Erk population. The application of 25 μM PD098059 (right) efficiently inhibits the activation of Erk by MEK in mast cells. Accordingly, we examined the effect of PD098059 on induction of Elk-1 transactivation in mast cells. As shown in Fig. 3 b, the activation of Elk-1 induced by cotransfection of Raf-CAAX and V12Ras constructs is inhibited by PD098059. Hence, these mutants are indeed reflecting the use of a Raf-1/Erk pathway to target Elk-1. Fig. 3 c shows that the induction of Elk-1 transactivation by both PdBu and FcεR1 cross-linking is inhibited by PD098059 in a dose-dependent manner. These data confirm that the Raf-1/MEK cascade is the critical Ras effector pathway for FcεR1 activation of Elk-1.

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.

Show MeSH