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 binds to and transactivates the Rcan1 promoter. (A) RNA from TNP-BSA–treated BMMCs was analyzed by real-time quantitative PCR for Egr1 and Rcan1. Egr1 and Rcan1 expression was normalized to endogenous control GAPDH. The data are expressed as relative mRNA levels compared with the mean expression level in BMMCs treated with TNP-BSA for 15 min (=1; Egr1) or for 60 min (=1; Rcan1), because at this time point Egr1 or Rcan1 showed the highest expression level, respectively (n = 3 experiments). (B) The location and sequence of the Egr1 binding site on the Rcan1 promoter. (C) BMMCs were transfected with various plasmids generated from pGL4 containing different lengths of the Egr1 binding sequence and the control reporter plasmid pRL-TK. All constructs start at −67 bp relative to the ATG codon. The 5′ end of each construct is shown at the left. Firefly and Renilla activities were sequentially quantified using a dual-luciferase reporter assay system. Results are means ± SEM (n = 3). (D) Nuclear proteins from untreated (NT) or TNP-BSA–treated BMMCs (TNP-BSA 1 h) were subjected to EMSA. An Egr1 DNA probe was synthesized based on the Egr1 binding sequences on the Rcan1 promoter (E). A mutant Egr1 DNA probe with two nucleotide mutations was used for a competition assay (Em). Strong TNP-BSA–induced Egr1 binding was seen and was competed by the specific cold probe (E) but not by the mutant probe (Em). Results are representative of three independent experiments. (E) ChIP assay for the association of Egr1 with Rcan1 promoter in vivo. BMMCs were stimulated with TNP-BSA for 60 min or left unstimulated. Protein–DNA complexes were extracted and precipitated with anti-Egr1 antibody (Egr1) or control IgG (IgG). DNA from samples before immunoprecipitation (IP) was used as an input control (Input). PCR was performed using primers based on the Rcan1 promoter sequence. Amplified DNA was resolved in agarose gel. Specific Egr1 binding to the Rcan1 promoter was seen in TNP-stimulated cells (lane 8) but not in unstimulated cells (lane 7). Results are representative of three independent experiments. M, molecular marker.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Egr1 binds to and transactivates the Rcan1 promoter. (A) RNA from TNP-BSA–treated BMMCs was analyzed by real-time quantitative PCR for Egr1 and Rcan1. Egr1 and Rcan1 expression was normalized to endogenous control GAPDH. The data are expressed as relative mRNA levels compared with the mean expression level in BMMCs treated with TNP-BSA for 15 min (=1; Egr1) or for 60 min (=1; Rcan1), because at this time point Egr1 or Rcan1 showed the highest expression level, respectively (n = 3 experiments). (B) The location and sequence of the Egr1 binding site on the Rcan1 promoter. (C) BMMCs were transfected with various plasmids generated from pGL4 containing different lengths of the Egr1 binding sequence and the control reporter plasmid pRL-TK. All constructs start at −67 bp relative to the ATG codon. The 5′ end of each construct is shown at the left. Firefly and Renilla activities were sequentially quantified using a dual-luciferase reporter assay system. Results are means ± SEM (n = 3). (D) Nuclear proteins from untreated (NT) or TNP-BSA–treated BMMCs (TNP-BSA 1 h) were subjected to EMSA. An Egr1 DNA probe was synthesized based on the Egr1 binding sequences on the Rcan1 promoter (E). A mutant Egr1 DNA probe with two nucleotide mutations was used for a competition assay (Em). Strong TNP-BSA–induced Egr1 binding was seen and was competed by the specific cold probe (E) but not by the mutant probe (Em). Results are representative of three independent experiments. (E) ChIP assay for the association of Egr1 with Rcan1 promoter in vivo. BMMCs were stimulated with TNP-BSA for 60 min or left unstimulated. Protein–DNA complexes were extracted and precipitated with anti-Egr1 antibody (Egr1) or control IgG (IgG). DNA from samples before immunoprecipitation (IP) was used as an input control (Input). PCR was performed using primers based on the Rcan1 promoter sequence. Amplified DNA was resolved in agarose gel. Specific Egr1 binding to the Rcan1 promoter was seen in TNP-stimulated cells (lane 8) but not in unstimulated cells (lane 7). Results are representative of three independent experiments. M, molecular marker.

Mentions: Next, we examined how the negative Rcan1 signal is activated during FcεRI activation. Our SSH assay using RNAs from TNP-BSA–stimulated BMMCs identified one clone (2A10) that matched the Egr1 gene (Table S1). Real-time quantitative PCR analysis showed that Egr1 gene expression peaked at 15 min after TNP-BSA stimulation. In contrast, Rcan1 expression began to increase at 15 min and peaked at 60 min (Fig. 7 A). These data suggested a sequential gene expression relationship between Egr1 and Rcan1 in mast cells and prompted us to analyze the promoter sequence of Rcan1, where we identified a putative Egr1 binding domain (Fig. 7 B).


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 binds to and transactivates the Rcan1 promoter. (A) RNA from TNP-BSA–treated BMMCs was analyzed by real-time quantitative PCR for Egr1 and Rcan1. Egr1 and Rcan1 expression was normalized to endogenous control GAPDH. The data are expressed as relative mRNA levels compared with the mean expression level in BMMCs treated with TNP-BSA for 15 min (=1; Egr1) or for 60 min (=1; Rcan1), because at this time point Egr1 or Rcan1 showed the highest expression level, respectively (n = 3 experiments). (B) The location and sequence of the Egr1 binding site on the Rcan1 promoter. (C) BMMCs were transfected with various plasmids generated from pGL4 containing different lengths of the Egr1 binding sequence and the control reporter plasmid pRL-TK. All constructs start at −67 bp relative to the ATG codon. The 5′ end of each construct is shown at the left. Firefly and Renilla activities were sequentially quantified using a dual-luciferase reporter assay system. Results are means ± SEM (n = 3). (D) Nuclear proteins from untreated (NT) or TNP-BSA–treated BMMCs (TNP-BSA 1 h) were subjected to EMSA. An Egr1 DNA probe was synthesized based on the Egr1 binding sequences on the Rcan1 promoter (E). A mutant Egr1 DNA probe with two nucleotide mutations was used for a competition assay (Em). Strong TNP-BSA–induced Egr1 binding was seen and was competed by the specific cold probe (E) but not by the mutant probe (Em). Results are representative of three independent experiments. (E) ChIP assay for the association of Egr1 with Rcan1 promoter in vivo. BMMCs were stimulated with TNP-BSA for 60 min or left unstimulated. Protein–DNA complexes were extracted and precipitated with anti-Egr1 antibody (Egr1) or control IgG (IgG). DNA from samples before immunoprecipitation (IP) was used as an input control (Input). PCR was performed using primers based on the Rcan1 promoter sequence. Amplified DNA was resolved in agarose gel. Specific Egr1 binding to the Rcan1 promoter was seen in TNP-stimulated cells (lane 8) but not in unstimulated cells (lane 7). Results are representative of three independent experiments. M, molecular marker.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Egr1 binds to and transactivates the Rcan1 promoter. (A) RNA from TNP-BSA–treated BMMCs was analyzed by real-time quantitative PCR for Egr1 and Rcan1. Egr1 and Rcan1 expression was normalized to endogenous control GAPDH. The data are expressed as relative mRNA levels compared with the mean expression level in BMMCs treated with TNP-BSA for 15 min (=1; Egr1) or for 60 min (=1; Rcan1), because at this time point Egr1 or Rcan1 showed the highest expression level, respectively (n = 3 experiments). (B) The location and sequence of the Egr1 binding site on the Rcan1 promoter. (C) BMMCs were transfected with various plasmids generated from pGL4 containing different lengths of the Egr1 binding sequence and the control reporter plasmid pRL-TK. All constructs start at −67 bp relative to the ATG codon. The 5′ end of each construct is shown at the left. Firefly and Renilla activities were sequentially quantified using a dual-luciferase reporter assay system. Results are means ± SEM (n = 3). (D) Nuclear proteins from untreated (NT) or TNP-BSA–treated BMMCs (TNP-BSA 1 h) were subjected to EMSA. An Egr1 DNA probe was synthesized based on the Egr1 binding sequences on the Rcan1 promoter (E). A mutant Egr1 DNA probe with two nucleotide mutations was used for a competition assay (Em). Strong TNP-BSA–induced Egr1 binding was seen and was competed by the specific cold probe (E) but not by the mutant probe (Em). Results are representative of three independent experiments. (E) ChIP assay for the association of Egr1 with Rcan1 promoter in vivo. BMMCs were stimulated with TNP-BSA for 60 min or left unstimulated. Protein–DNA complexes were extracted and precipitated with anti-Egr1 antibody (Egr1) or control IgG (IgG). DNA from samples before immunoprecipitation (IP) was used as an input control (Input). PCR was performed using primers based on the Rcan1 promoter sequence. Amplified DNA was resolved in agarose gel. Specific Egr1 binding to the Rcan1 promoter was seen in TNP-stimulated cells (lane 8) but not in unstimulated cells (lane 7). Results are representative of three independent experiments. M, molecular marker.
Mentions: Next, we examined how the negative Rcan1 signal is activated during FcεRI activation. Our SSH assay using RNAs from TNP-BSA–stimulated BMMCs identified one clone (2A10) that matched the Egr1 gene (Table S1). Real-time quantitative PCR analysis showed that Egr1 gene expression peaked at 15 min after TNP-BSA stimulation. In contrast, Rcan1 expression began to increase at 15 min and peaked at 60 min (Fig. 7 A). These data suggested a sequential gene expression relationship between Egr1 and Rcan1 in mast cells and prompted us to analyze the promoter sequence of Rcan1, where we identified a putative Egr1 binding domain (Fig. 7 B).

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