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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 activation stimulates Elk-1 transactivation in mast  cells. 12 μg LexA OPtk.CAT and 6 μg LexA Elk-1C plasmids were used  per 1 × 107 cells in electroporation at 310 V/960 μF. Cells were recovered as described in Materials and Methods before stimulation for 16 h  with PdBu (50 ng/ml), Ionomycin (500 ng/ml) alone or in combination  (P+I), or IgE priming followed by antigenic cross-linking of the FcεR1  with the indicated dose of KLH-DNP. Control cells (NS) were left unstimulated. (b) Stimulation of LexA OP.tk CAT activity is dependent on  the presence of the LexA Elk-1C fusion protein. 1 × 107 cells per stimulus were electroporated as described with 12 μg LexA OPtk.CAT alone  or in combination with 6 μg LexA Elk-1C. Cells were recovered, and  then stimulated for 16 h as described above. (c) FcεR1 activation of Elk-1  is not PKC-dependent. Cells were transfected with the Elk-1 reporter  system as described, and recovered for 6 h. Preincubation with either 1  μM Ro-318425 or DMSO carrier was 30 min at 37°C before stimulation  with either PdBu (50 ng/ml) or KLH-DNP (250 ng/ml).
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Figure 1: (a) FcεR1 activation stimulates Elk-1 transactivation in mast cells. 12 μg LexA OPtk.CAT and 6 μg LexA Elk-1C plasmids were used per 1 × 107 cells in electroporation at 310 V/960 μF. Cells were recovered as described in Materials and Methods before stimulation for 16 h with PdBu (50 ng/ml), Ionomycin (500 ng/ml) alone or in combination (P+I), or IgE priming followed by antigenic cross-linking of the FcεR1 with the indicated dose of KLH-DNP. Control cells (NS) were left unstimulated. (b) Stimulation of LexA OP.tk CAT activity is dependent on the presence of the LexA Elk-1C fusion protein. 1 × 107 cells per stimulus were electroporated as described with 12 μg LexA OPtk.CAT alone or in combination with 6 μg LexA Elk-1C. Cells were recovered, and then stimulated for 16 h as described above. (c) FcεR1 activation of Elk-1 is not PKC-dependent. Cells were transfected with the Elk-1 reporter system as described, and recovered for 6 h. Preincubation with either 1 μM Ro-318425 or DMSO carrier was 30 min at 37°C before stimulation with either PdBu (50 ng/ml) or KLH-DNP (250 ng/ml).

Mentions: The effects of various stimuli on Elk-1 activity in mast cells were assessed using a CAT reporter gene assay for Elk-1 transactivation. The data in Fig. 1 a shows that antigenic cross-linking of the FcεR1 using KLH-DNP strongly induces Elk-1 transactivation in a dose-dependent manner. In addition, the phorbol ester PdBu that directly activates PKC, induces ninefold induction of Elk-1 transactivation over basal levels. Fig. 1 a also shows that elevation of intracellular calcium levels using the ionophore Ionomycin is not sufficient for Elk-1 activation. Stimulation by FcεR1 or the phorbol ester PdBu does not induce LexA OPtk.CAT activity in the absence of a coexpressed LexA Elk-1C fusion protein (Fig. 1 b).


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 activation stimulates Elk-1 transactivation in mast  cells. 12 μg LexA OPtk.CAT and 6 μg LexA Elk-1C plasmids were used  per 1 × 107 cells in electroporation at 310 V/960 μF. Cells were recovered as described in Materials and Methods before stimulation for 16 h  with PdBu (50 ng/ml), Ionomycin (500 ng/ml) alone or in combination  (P+I), or IgE priming followed by antigenic cross-linking of the FcεR1  with the indicated dose of KLH-DNP. Control cells (NS) were left unstimulated. (b) Stimulation of LexA OP.tk CAT activity is dependent on  the presence of the LexA Elk-1C fusion protein. 1 × 107 cells per stimulus were electroporated as described with 12 μg LexA OPtk.CAT alone  or in combination with 6 μg LexA Elk-1C. Cells were recovered, and  then stimulated for 16 h as described above. (c) FcεR1 activation of Elk-1  is not PKC-dependent. Cells were transfected with the Elk-1 reporter  system as described, and recovered for 6 h. Preincubation with either 1  μM Ro-318425 or DMSO carrier was 30 min at 37°C before stimulation  with either PdBu (50 ng/ml) or KLH-DNP (250 ng/ml).
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Related In: Results  -  Collection

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Figure 1: (a) FcεR1 activation stimulates Elk-1 transactivation in mast cells. 12 μg LexA OPtk.CAT and 6 μg LexA Elk-1C plasmids were used per 1 × 107 cells in electroporation at 310 V/960 μF. Cells were recovered as described in Materials and Methods before stimulation for 16 h with PdBu (50 ng/ml), Ionomycin (500 ng/ml) alone or in combination (P+I), or IgE priming followed by antigenic cross-linking of the FcεR1 with the indicated dose of KLH-DNP. Control cells (NS) were left unstimulated. (b) Stimulation of LexA OP.tk CAT activity is dependent on the presence of the LexA Elk-1C fusion protein. 1 × 107 cells per stimulus were electroporated as described with 12 μg LexA OPtk.CAT alone or in combination with 6 μg LexA Elk-1C. Cells were recovered, and then stimulated for 16 h as described above. (c) FcεR1 activation of Elk-1 is not PKC-dependent. Cells were transfected with the Elk-1 reporter system as described, and recovered for 6 h. Preincubation with either 1 μM Ro-318425 or DMSO carrier was 30 min at 37°C before stimulation with either PdBu (50 ng/ml) or KLH-DNP (250 ng/ml).
Mentions: The effects of various stimuli on Elk-1 activity in mast cells were assessed using a CAT reporter gene assay for Elk-1 transactivation. The data in Fig. 1 a shows that antigenic cross-linking of the FcεR1 using KLH-DNP strongly induces Elk-1 transactivation in a dose-dependent manner. In addition, the phorbol ester PdBu that directly activates PKC, induces ninefold induction of Elk-1 transactivation over basal levels. Fig. 1 a also shows that elevation of intracellular calcium levels using the ionophore Ionomycin is not sufficient for Elk-1 activation. Stimulation by FcεR1 or the phorbol ester PdBu does not induce LexA OPtk.CAT activity in the absence of a coexpressed LexA Elk-1C fusion protein (Fig. 1 b).

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