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Rcan1 negatively regulates Fc epsilonRI-mediated signaling and mast cell function.

Yang YJ, Chen W, Edgar A, Li B, Molkentin JD, Berman JN, Lin TJ - J. Exp. Med. (2009)

Bottom Line: Forced expression of Rcan1 in wild-type or Rcan1-deficient mast cells reduced Fc epsilonRI-mediated cytokine production.Analysis of the Rcan1 promoter identified a functional Egr1 binding site.Our results identified Rcan1 as a novel inhibitory signal in Fc epsilonRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in Fc epsilonRI signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia B3K 6R8, Canada.

ABSTRACT
Aggregation of the high affinity IgE receptor (Fc epsilonRI) activates a cascade of signaling events leading to mast cell activation. Subsequently, inhibitory signals are engaged for turning off activating signals. We identified that regulator of calcineurin (Rcan) 1 serves as a negative regulator for turning off Fc epsilonRI-mediated mast cell activation. Fc epsilonRI-induced Rcan1 expression was identified by suppression subtractive hybridization and verified by real-time quantitative polymerase chain reaction and Western blotting. Deficiency of Rcan1 led to increased calcineurin activity, increased nuclear factor of activated T cells and nuclear factor kappaB activation, increased cytokine production, and enhanced immunoglobulin E-mediated late-phase cutaneous reactions. Forced expression of Rcan1 in wild-type or Rcan1-deficient mast cells reduced Fc epsilonRI-mediated cytokine production. Rcan1 deficiency also led to increased Fc epsilonRI-mediated mast cell degranulation and enhanced passive cutaneous anaphylaxis. Analysis of the Rcan1 promoter identified a functional Egr1 binding site. Biochemical and genetic evidence suggested that Egr1 controls Rcan1 expression. Our results identified Rcan1 as a novel inhibitory signal in Fc epsilonRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in Fc epsilonRI signaling.

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Rcan1 deficiency leads to increased IgE-dependent NFAT activation. (A) Rcan1+/+ and Rcan1−/− BMMCs were transfected with a pNFAT-Luc and the control reporter plasmid pRL-TK. After transfection (24 h), cells were sensitized with anti-TNP IgE for 18 h. Then cells were either left untreated (NT) or treated with 10 ng/ml TNP-BSA for 18 h (TNP). Firefly and Renilla activities were sequentially quantified using a dual-luciferase reporter assay system. Data are means ± SEM (n = 3 independent experiments). *, P < 0.05 compared with wild-type group. (B) NFAT binding concensus sequence (N) on mouse IL-13 promoter 5′-AAGGTGTTTCCCCAAGCCTTTCCC-3′ was labeled with 32P for EMSA. After sensitization with anti-TNP IgE, Rcan1+/+ and Rcan1−/− BMMCs were either not treated (NT) or stimulated with 10 ng/ml TNP-BSA for 5, 20, 60, 180, and 360 min. Nuclear proteins were isolated and subjected to EMSA. Shown is a representative from six independent experiments. (C) Densitometry analysis of NFAT activation by EMSA was performed based on six experiments. *, P < 0.05 compared with the same time point of the wild-type group (180 or 360 min). (D) Nuclear extracts from Rcan1+/+ and Rcan1−/− BMMCs were used for competition assays. 50× concentrated unlabeled NFAT probe (N) was used to compete with the 32P-labeled NFAT probe, whereas 50× concentrated unlabeled mutant NFAT probe (Nm; 5′-AAGGTGTCCATCCAAGCCTCCTAC-3′) was used as a control. 1 μl of nonradiolabeled wild-type NFAT probe (N) or mutant probe Nm were added and incubated for 15 min before the addition of the radiolabeled probe. (E) Antibody blockade of the DNA–protein complex formation (supershift assay). Nuclear proteins from BMMCs treated for 1 h with 10 ng/ml TNP-BSA were incubated with or without specific antibodies to NFATc1 or NFATc2 for 30 min on ice before EMSA experiments using the 32P-labeled NFAT probe (N). Shown is a representative from three (D) or two (E) independent experiments. (F) Rcan1+/+ and Rcan1−/− BMMCs were treated with 10 ng/ml TNP-BSA for 6 h or left untreated. Cells were lysed, and calcineurin activity was analyzed by using a calcineurin assay kit according to the manufacturer's instructions. Error bars represent SE (n = 4). *, P < 0.05 compared with the TNP-treated Rcan1+/+ group.
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fig2: Rcan1 deficiency leads to increased IgE-dependent NFAT activation. (A) Rcan1+/+ and Rcan1−/− BMMCs were transfected with a pNFAT-Luc and the control reporter plasmid pRL-TK. After transfection (24 h), cells were sensitized with anti-TNP IgE for 18 h. Then cells were either left untreated (NT) or treated with 10 ng/ml TNP-BSA for 18 h (TNP). Firefly and Renilla activities were sequentially quantified using a dual-luciferase reporter assay system. Data are means ± SEM (n = 3 independent experiments). *, P < 0.05 compared with wild-type group. (B) NFAT binding concensus sequence (N) on mouse IL-13 promoter 5′-AAGGTGTTTCCCCAAGCCTTTCCC-3′ was labeled with 32P for EMSA. After sensitization with anti-TNP IgE, Rcan1+/+ and Rcan1−/− BMMCs were either not treated (NT) or stimulated with 10 ng/ml TNP-BSA for 5, 20, 60, 180, and 360 min. Nuclear proteins were isolated and subjected to EMSA. Shown is a representative from six independent experiments. (C) Densitometry analysis of NFAT activation by EMSA was performed based on six experiments. *, P < 0.05 compared with the same time point of the wild-type group (180 or 360 min). (D) Nuclear extracts from Rcan1+/+ and Rcan1−/− BMMCs were used for competition assays. 50× concentrated unlabeled NFAT probe (N) was used to compete with the 32P-labeled NFAT probe, whereas 50× concentrated unlabeled mutant NFAT probe (Nm; 5′-AAGGTGTCCATCCAAGCCTCCTAC-3′) was used as a control. 1 μl of nonradiolabeled wild-type NFAT probe (N) or mutant probe Nm were added and incubated for 15 min before the addition of the radiolabeled probe. (E) Antibody blockade of the DNA–protein complex formation (supershift assay). Nuclear proteins from BMMCs treated for 1 h with 10 ng/ml TNP-BSA were incubated with or without specific antibodies to NFATc1 or NFATc2 for 30 min on ice before EMSA experiments using the 32P-labeled NFAT probe (N). Shown is a representative from three (D) or two (E) independent experiments. (F) Rcan1+/+ and Rcan1−/− BMMCs were treated with 10 ng/ml TNP-BSA for 6 h or left untreated. Cells were lysed, and calcineurin activity was analyzed by using a calcineurin assay kit according to the manufacturer's instructions. Error bars represent SE (n = 4). *, P < 0.05 compared with the TNP-treated Rcan1+/+ group.

Mentions: To determine the specific signaling pathways regulated by Rcan1 in FcεRI-mediated mast cell activation, we examined activation of NFAT, NF-κB, and MAPK pathways in Rcan1-deficient BMMCs. For the study of NFAT pathway activation, an NFAT luciferase assay, EMSA, and an immunofluorescence assay were used. Rcan1-deficient and wild-type BMMCs were transfected with NFAT luciferase plasmid. Cells were then sensitized with anti-TNP IgE and activated with TNP-BSA for 18 h. A significant increase in NFAT luciferase activity was seen in Rcan1-deficient BMMCs after TNP-BSA stimulation compared with that in wild-type BMMCs (Fig. 2 A). These data suggests that Rcan1 is a negative regulator of IgE-dependent NFAT activation.


Rcan1 negatively regulates Fc epsilonRI-mediated signaling and mast cell function.

Yang YJ, Chen W, Edgar A, Li B, Molkentin JD, Berman JN, Lin TJ - J. Exp. Med. (2009)

Rcan1 deficiency leads to increased IgE-dependent NFAT activation. (A) Rcan1+/+ and Rcan1−/− BMMCs were transfected with a pNFAT-Luc and the control reporter plasmid pRL-TK. After transfection (24 h), cells were sensitized with anti-TNP IgE for 18 h. Then cells were either left untreated (NT) or treated with 10 ng/ml TNP-BSA for 18 h (TNP). Firefly and Renilla activities were sequentially quantified using a dual-luciferase reporter assay system. Data are means ± SEM (n = 3 independent experiments). *, P < 0.05 compared with wild-type group. (B) NFAT binding concensus sequence (N) on mouse IL-13 promoter 5′-AAGGTGTTTCCCCAAGCCTTTCCC-3′ was labeled with 32P for EMSA. After sensitization with anti-TNP IgE, Rcan1+/+ and Rcan1−/− BMMCs were either not treated (NT) or stimulated with 10 ng/ml TNP-BSA for 5, 20, 60, 180, and 360 min. Nuclear proteins were isolated and subjected to EMSA. Shown is a representative from six independent experiments. (C) Densitometry analysis of NFAT activation by EMSA was performed based on six experiments. *, P < 0.05 compared with the same time point of the wild-type group (180 or 360 min). (D) Nuclear extracts from Rcan1+/+ and Rcan1−/− BMMCs were used for competition assays. 50× concentrated unlabeled NFAT probe (N) was used to compete with the 32P-labeled NFAT probe, whereas 50× concentrated unlabeled mutant NFAT probe (Nm; 5′-AAGGTGTCCATCCAAGCCTCCTAC-3′) was used as a control. 1 μl of nonradiolabeled wild-type NFAT probe (N) or mutant probe Nm were added and incubated for 15 min before the addition of the radiolabeled probe. (E) Antibody blockade of the DNA–protein complex formation (supershift assay). Nuclear proteins from BMMCs treated for 1 h with 10 ng/ml TNP-BSA were incubated with or without specific antibodies to NFATc1 or NFATc2 for 30 min on ice before EMSA experiments using the 32P-labeled NFAT probe (N). Shown is a representative from three (D) or two (E) independent experiments. (F) Rcan1+/+ and Rcan1−/− BMMCs were treated with 10 ng/ml TNP-BSA for 6 h or left untreated. Cells were lysed, and calcineurin activity was analyzed by using a calcineurin assay kit according to the manufacturer's instructions. Error bars represent SE (n = 4). *, P < 0.05 compared with the TNP-treated Rcan1+/+ group.
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fig2: Rcan1 deficiency leads to increased IgE-dependent NFAT activation. (A) Rcan1+/+ and Rcan1−/− BMMCs were transfected with a pNFAT-Luc and the control reporter plasmid pRL-TK. After transfection (24 h), cells were sensitized with anti-TNP IgE for 18 h. Then cells were either left untreated (NT) or treated with 10 ng/ml TNP-BSA for 18 h (TNP). Firefly and Renilla activities were sequentially quantified using a dual-luciferase reporter assay system. Data are means ± SEM (n = 3 independent experiments). *, P < 0.05 compared with wild-type group. (B) NFAT binding concensus sequence (N) on mouse IL-13 promoter 5′-AAGGTGTTTCCCCAAGCCTTTCCC-3′ was labeled with 32P for EMSA. After sensitization with anti-TNP IgE, Rcan1+/+ and Rcan1−/− BMMCs were either not treated (NT) or stimulated with 10 ng/ml TNP-BSA for 5, 20, 60, 180, and 360 min. Nuclear proteins were isolated and subjected to EMSA. Shown is a representative from six independent experiments. (C) Densitometry analysis of NFAT activation by EMSA was performed based on six experiments. *, P < 0.05 compared with the same time point of the wild-type group (180 or 360 min). (D) Nuclear extracts from Rcan1+/+ and Rcan1−/− BMMCs were used for competition assays. 50× concentrated unlabeled NFAT probe (N) was used to compete with the 32P-labeled NFAT probe, whereas 50× concentrated unlabeled mutant NFAT probe (Nm; 5′-AAGGTGTCCATCCAAGCCTCCTAC-3′) was used as a control. 1 μl of nonradiolabeled wild-type NFAT probe (N) or mutant probe Nm were added and incubated for 15 min before the addition of the radiolabeled probe. (E) Antibody blockade of the DNA–protein complex formation (supershift assay). Nuclear proteins from BMMCs treated for 1 h with 10 ng/ml TNP-BSA were incubated with or without specific antibodies to NFATc1 or NFATc2 for 30 min on ice before EMSA experiments using the 32P-labeled NFAT probe (N). Shown is a representative from three (D) or two (E) independent experiments. (F) Rcan1+/+ and Rcan1−/− BMMCs were treated with 10 ng/ml TNP-BSA for 6 h or left untreated. Cells were lysed, and calcineurin activity was analyzed by using a calcineurin assay kit according to the manufacturer's instructions. Error bars represent SE (n = 4). *, P < 0.05 compared with the TNP-treated Rcan1+/+ group.
Mentions: To determine the specific signaling pathways regulated by Rcan1 in FcεRI-mediated mast cell activation, we examined activation of NFAT, NF-κB, and MAPK pathways in Rcan1-deficient BMMCs. For the study of NFAT pathway activation, an NFAT luciferase assay, EMSA, and an immunofluorescence assay were used. Rcan1-deficient and wild-type BMMCs were transfected with NFAT luciferase plasmid. Cells were then sensitized with anti-TNP IgE and activated with TNP-BSA for 18 h. A significant increase in NFAT luciferase activity was seen in Rcan1-deficient BMMCs after TNP-BSA stimulation compared with that in wild-type BMMCs (Fig. 2 A). These data suggests that Rcan1 is a negative regulator of IgE-dependent NFAT activation.

Bottom Line: Forced expression of Rcan1 in wild-type or Rcan1-deficient mast cells reduced Fc epsilonRI-mediated cytokine production.Analysis of the Rcan1 promoter identified a functional Egr1 binding site.Our results identified Rcan1 as a novel inhibitory signal in Fc epsilonRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in Fc epsilonRI signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia B3K 6R8, Canada.

ABSTRACT
Aggregation of the high affinity IgE receptor (Fc epsilonRI) activates a cascade of signaling events leading to mast cell activation. Subsequently, inhibitory signals are engaged for turning off activating signals. We identified that regulator of calcineurin (Rcan) 1 serves as a negative regulator for turning off Fc epsilonRI-mediated mast cell activation. Fc epsilonRI-induced Rcan1 expression was identified by suppression subtractive hybridization and verified by real-time quantitative polymerase chain reaction and Western blotting. Deficiency of Rcan1 led to increased calcineurin activity, increased nuclear factor of activated T cells and nuclear factor kappaB activation, increased cytokine production, and enhanced immunoglobulin E-mediated late-phase cutaneous reactions. Forced expression of Rcan1 in wild-type or Rcan1-deficient mast cells reduced Fc epsilonRI-mediated cytokine production. Rcan1 deficiency also led to increased Fc epsilonRI-mediated mast cell degranulation and enhanced passive cutaneous anaphylaxis. Analysis of the Rcan1 promoter identified a functional Egr1 binding site. Biochemical and genetic evidence suggested that Egr1 controls Rcan1 expression. Our results identified Rcan1 as a novel inhibitory signal in Fc epsilonRI-induced mast cell activation and established a new link of Egr1 and Rcan1 in Fc epsilonRI signaling.

Show MeSH
Related in: MedlinePlus