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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 enhanced IgE-dependent production of IL-6, IL-13, and TNF. After sensitization with anti-TNP IgE for 24 h, Rcan1+/+ and Rcan1−/− BMMCs were either not treated (NT) or stimulated with TNP-BSA for various times. Cell-free supernatants were collected for the detection of IL-6 (A), IL-13 (D), and TNF (G) by ELISA. Cell pellets were used for RNA isolation and analysis for cytokine mRNA expression. Real-time quantitative PCR was performed to determine IL-6 (B), IL-13 (E), and TNF (H) expression. IL-6, IL-13, and TNF expression was normalized to endogenous control GAPDH. The PCR products for IL-6 (C), IL-13 (F), and TNF (I) were also separated by agarose gel and stained with ethidium bromide. Untreated BMMCs (NT) showed little cytokine expression, whereas TNP induced enhanced cytokine production in Rcan1−/− BMMCs. Error bars represent SEs from six independent experiments (A, B, D, E, G, and H). Results presented in C, F, and I are representative of three to six separate experiments. *, P < 0.05 compared with the wild-type group.
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fig4: Rcan1 deficiency leads to enhanced IgE-dependent production of IL-6, IL-13, and TNF. After sensitization with anti-TNP IgE for 24 h, Rcan1+/+ and Rcan1−/− BMMCs were either not treated (NT) or stimulated with TNP-BSA for various times. Cell-free supernatants were collected for the detection of IL-6 (A), IL-13 (D), and TNF (G) by ELISA. Cell pellets were used for RNA isolation and analysis for cytokine mRNA expression. Real-time quantitative PCR was performed to determine IL-6 (B), IL-13 (E), and TNF (H) expression. IL-6, IL-13, and TNF expression was normalized to endogenous control GAPDH. The PCR products for IL-6 (C), IL-13 (F), and TNF (I) were also separated by agarose gel and stained with ethidium bromide. Untreated BMMCs (NT) showed little cytokine expression, whereas TNP induced enhanced cytokine production in Rcan1−/− BMMCs. Error bars represent SEs from six independent experiments (A, B, D, E, G, and H). Results presented in C, F, and I are representative of three to six separate experiments. *, P < 0.05 compared with the wild-type group.

Mentions: Several cytokines such as IL-6, IL-13, and TNF play important roles in allergy and are regulated by the transcription factors NFAT and NF-κB. To determine whether Rcan1 is involved in the regulation of FcεRI-mediated cytokine production, Rcan1-deficient and wild-type BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA for various times. IL-6, IL-13, and TNF production in cell-free supernatants was determined by ELISA. Expression of these cytokine mRNAs was analyzed by real-time quantitative PCR using total RNA isolated from cell pellets. Production of IL-6 was significantly enhanced by 87, 170, and 115% at the time points of 3, 6, and 18 h, respectively, in Rcan1-deficient BMMCs compared with wild-type BMMCs (Fig. 4 A). Importantly, real-time PCR analysis revealed that TNP-BSA–induced IL-6 mRNA expression was prolonged in Rcan1-deficient BMMCs (Fig. 4 B). PCR-amplified products separated by agarose gel electropheresis also revealed a prolonged TNP-BSA–induced IL-6 expression in Rcan1-deficient BMMCs (Fig. 4 C). Similarly, TNP-BSA–induced IL-13 and TNF production was enhanced at the protein level, whereas the mRNA expression of these cytokines was prolonged in Rcan1-deficient cells (Fig. 4, D–I). Enhanced production of IL-6, IL-13, and TNF at the protein level is likely caused by the prolonged mRNA expression of these cytokines. The prolonged mRNA expression of these cytokines supports the notion that Rcan1 functions as a negative regulator by turning off FcεRI-activated signals. This is consistent with the negative role of Rcan1 in the regulation of FcεRI-induced NFAT and IκB–NF-κB 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 enhanced IgE-dependent production of IL-6, IL-13, and TNF. After sensitization with anti-TNP IgE for 24 h, Rcan1+/+ and Rcan1−/− BMMCs were either not treated (NT) or stimulated with TNP-BSA for various times. Cell-free supernatants were collected for the detection of IL-6 (A), IL-13 (D), and TNF (G) by ELISA. Cell pellets were used for RNA isolation and analysis for cytokine mRNA expression. Real-time quantitative PCR was performed to determine IL-6 (B), IL-13 (E), and TNF (H) expression. IL-6, IL-13, and TNF expression was normalized to endogenous control GAPDH. The PCR products for IL-6 (C), IL-13 (F), and TNF (I) were also separated by agarose gel and stained with ethidium bromide. Untreated BMMCs (NT) showed little cytokine expression, whereas TNP induced enhanced cytokine production in Rcan1−/− BMMCs. Error bars represent SEs from six independent experiments (A, B, D, E, G, and H). Results presented in C, F, and I are representative of three to six separate experiments. *, P < 0.05 compared with the wild-type group.
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fig4: Rcan1 deficiency leads to enhanced IgE-dependent production of IL-6, IL-13, and TNF. After sensitization with anti-TNP IgE for 24 h, Rcan1+/+ and Rcan1−/− BMMCs were either not treated (NT) or stimulated with TNP-BSA for various times. Cell-free supernatants were collected for the detection of IL-6 (A), IL-13 (D), and TNF (G) by ELISA. Cell pellets were used for RNA isolation and analysis for cytokine mRNA expression. Real-time quantitative PCR was performed to determine IL-6 (B), IL-13 (E), and TNF (H) expression. IL-6, IL-13, and TNF expression was normalized to endogenous control GAPDH. The PCR products for IL-6 (C), IL-13 (F), and TNF (I) were also separated by agarose gel and stained with ethidium bromide. Untreated BMMCs (NT) showed little cytokine expression, whereas TNP induced enhanced cytokine production in Rcan1−/− BMMCs. Error bars represent SEs from six independent experiments (A, B, D, E, G, and H). Results presented in C, F, and I are representative of three to six separate experiments. *, P < 0.05 compared with the wild-type group.
Mentions: Several cytokines such as IL-6, IL-13, and TNF play important roles in allergy and are regulated by the transcription factors NFAT and NF-κB. To determine whether Rcan1 is involved in the regulation of FcεRI-mediated cytokine production, Rcan1-deficient and wild-type BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA for various times. IL-6, IL-13, and TNF production in cell-free supernatants was determined by ELISA. Expression of these cytokine mRNAs was analyzed by real-time quantitative PCR using total RNA isolated from cell pellets. Production of IL-6 was significantly enhanced by 87, 170, and 115% at the time points of 3, 6, and 18 h, respectively, in Rcan1-deficient BMMCs compared with wild-type BMMCs (Fig. 4 A). Importantly, real-time PCR analysis revealed that TNP-BSA–induced IL-6 mRNA expression was prolonged in Rcan1-deficient BMMCs (Fig. 4 B). PCR-amplified products separated by agarose gel electropheresis also revealed a prolonged TNP-BSA–induced IL-6 expression in Rcan1-deficient BMMCs (Fig. 4 C). Similarly, TNP-BSA–induced IL-13 and TNF production was enhanced at the protein level, whereas the mRNA expression of these cytokines was prolonged in Rcan1-deficient cells (Fig. 4, D–I). Enhanced production of IL-6, IL-13, and TNF at the protein level is likely caused by the prolonged mRNA expression of these cytokines. The prolonged mRNA expression of these cytokines supports the notion that Rcan1 functions as a negative regulator by turning off FcεRI-activated signals. This is consistent with the negative role of Rcan1 in the regulation of FcεRI-induced NFAT and IκB–NF-κB 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