Limits...
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.

Show MeSH

Related in: MedlinePlus

Egr1 is required for Rcan1 expression. (A and B) After sensitization with anti-TNP IgE for 24 h, Egr1+/+ and Egr1−/− BMMCs were either not treated (NT) or stimulated with TNP-BSA for 1 h. RNA was isolated and analyzed by real-time quantitative PCR for Rcan1. Rcan1 expression was normalized to endogenous control GAPDH. The data are expressed as relative mRNA levels compared with the mean expression level in Egr1+/+ BMMCs treated with TNP-BSA for 60 min (=1), because in this group Rcan1 showed the highest expression level (A). Error bars represent SEs from three independent experiments. *, P < 0.05 compared with the wild-type group. The PCR products were also separated by agarose gel and stained with ethidium bromide (B). A representative gel from three independent experiments is shown. M, molecular marker.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2626669&req=5

fig8: Egr1 is required for Rcan1 expression. (A and B) After sensitization with anti-TNP IgE for 24 h, Egr1+/+ and Egr1−/− BMMCs were either not treated (NT) or stimulated with TNP-BSA for 1 h. RNA was isolated and analyzed by real-time quantitative PCR for Rcan1. Rcan1 expression was normalized to endogenous control GAPDH. The data are expressed as relative mRNA levels compared with the mean expression level in Egr1+/+ BMMCs treated with TNP-BSA for 60 min (=1), because in this group Rcan1 showed the highest expression level (A). Error bars represent SEs from three independent experiments. *, P < 0.05 compared with the wild-type group. The PCR products were also separated by agarose gel and stained with ethidium bromide (B). A representative gel from three independent experiments is shown. M, molecular marker.

Mentions: To evaluate whether Egr1 is required for FcεRI-induced Rcan1 expression, we generated Egr1-deficient and wild-type BMMCs. Egr1-deficient BMMCs mature normally and express similar levels of FcεRI compared with wild-type BMMCs. BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA. Total RNA was isolated and analyzed by real-time quantitative PCR for Rcan1 (isoform Rcan1-4) expression. TNP-BSA–induced Rcan1 expression was significantly reduced in Egr1-deficient BMMCs (Fig. 8 A). A PCR-amplified Rcan1 product (Rcan1-4) was also separated on an agarose gel. A representative gel is presented in Fig. 8 B. Reduced Rcan1 expression in Egr1-deficient BMMCs can be seen after TNP-BSA stimulation. Thus, Egr1 is required for FcεRI-induced Rcan1 expression.


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)

Egr1 is required for Rcan1 expression. (A and B) After sensitization with anti-TNP IgE for 24 h, Egr1+/+ and Egr1−/− BMMCs were either not treated (NT) or stimulated with TNP-BSA for 1 h. RNA was isolated and analyzed by real-time quantitative PCR for Rcan1. Rcan1 expression was normalized to endogenous control GAPDH. The data are expressed as relative mRNA levels compared with the mean expression level in Egr1+/+ BMMCs treated with TNP-BSA for 60 min (=1), because in this group Rcan1 showed the highest expression level (A). Error bars represent SEs from three independent experiments. *, P < 0.05 compared with the wild-type group. The PCR products were also separated by agarose gel and stained with ethidium bromide (B). A representative gel from three independent experiments is shown. M, molecular marker.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2626669&req=5

fig8: Egr1 is required for Rcan1 expression. (A and B) After sensitization with anti-TNP IgE for 24 h, Egr1+/+ and Egr1−/− BMMCs were either not treated (NT) or stimulated with TNP-BSA for 1 h. RNA was isolated and analyzed by real-time quantitative PCR for Rcan1. Rcan1 expression was normalized to endogenous control GAPDH. The data are expressed as relative mRNA levels compared with the mean expression level in Egr1+/+ BMMCs treated with TNP-BSA for 60 min (=1), because in this group Rcan1 showed the highest expression level (A). Error bars represent SEs from three independent experiments. *, P < 0.05 compared with the wild-type group. The PCR products were also separated by agarose gel and stained with ethidium bromide (B). A representative gel from three independent experiments is shown. M, molecular marker.
Mentions: To evaluate whether Egr1 is required for FcεRI-induced Rcan1 expression, we generated Egr1-deficient and wild-type BMMCs. Egr1-deficient BMMCs mature normally and express similar levels of FcεRI compared with wild-type BMMCs. BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA. Total RNA was isolated and analyzed by real-time quantitative PCR for Rcan1 (isoform Rcan1-4) expression. TNP-BSA–induced Rcan1 expression was significantly reduced in Egr1-deficient BMMCs (Fig. 8 A). A PCR-amplified Rcan1 product (Rcan1-4) was also separated on an agarose gel. A representative gel is presented in Fig. 8 B. Reduced Rcan1 expression in Egr1-deficient BMMCs can be seen after TNP-BSA stimulation. Thus, Egr1 is required for FcεRI-induced Rcan1 expression.

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