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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 induces increased IgE-dependent NF-κB activation but not MAPK, Akt, or Syk. (A) Rcan1+/+ and Rcan1−/− BMMCs were transfected with a pNF-κB-Luc and the control reporter plasmid pRL-TK. After transfection (24 h), cells were sensitized with anti-TNP IgE for 5 h. Cells were then 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 the wild-type group. (B and C) Rcan1+/+ and Rcan1−/− BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA for various times. Total cell lysates were analyzed by Western blotting for various phosphorylated and total proteins. Increased IκB phosphorylation in Rcan1−/− BMMCs was found at 60 min after TNP stimulation when compared with Rcan1+/+ cells. In contrast, the pattern of TNP-induced phosphorylation of p38, JNK, Akt, and Syk is similar between Rcan1−/− and Rcan1+/+ BMMCs.
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fig3: Rcan1 deficiency induces increased IgE-dependent NF-κB activation but not MAPK, Akt, or Syk. (A) Rcan1+/+ and Rcan1−/− BMMCs were transfected with a pNF-κB-Luc and the control reporter plasmid pRL-TK. After transfection (24 h), cells were sensitized with anti-TNP IgE for 5 h. Cells were then 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 the wild-type group. (B and C) Rcan1+/+ and Rcan1−/− BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA for various times. Total cell lysates were analyzed by Western blotting for various phosphorylated and total proteins. Increased IκB phosphorylation in Rcan1−/− BMMCs was found at 60 min after TNP stimulation when compared with Rcan1+/+ cells. In contrast, the pattern of TNP-induced phosphorylation of p38, JNK, Akt, and Syk is similar between Rcan1−/− and Rcan1+/+ BMMCs.

Mentions: In light of a critical role for transcription factor NF-κB in the regulation of gene expression (19), we examined whether Rcan1 is required in FcεRI-mediated NF-κB pathway activation. Rcan1-deficient BMMCs and wild-type cells were transfected with NF-κB luciferase plasmid. After sensitization with anti-TNP IgE, BMMCs were stimulated with TNP-BSA for 5 h. As shown in Fig. 3 A, Rcan1-deficient BMMCs showed significantly enhanced NF-κB activity in response to TNP stimulation, suggesting that Rcan1 is needed for the negative regulation of FcεRI-mediated NF-κB activation. NF-κB activity is regulated by IκB. The effects of Rcan1 on FcεRI-mediated IκB phosphorylation and degradation were examined by Western blotting. Aggregation of FcεRI induced a rapid phosphorylation of IκB (5 min), followed by IκB degradation (5 and 20 min) and subsequent regeneration of this molecule (60, 180, and 360 min; Fig. 3 B). Interestingly, the early events of IκB phosphorylation and degradation at 5 and 20 min were not affected by Rcan1 deficiency. In contrast, phosphorylation at the later time point (60 min) was significantly enhanced in Rcan1-deficient BMMCs (Fig. 3 B). This process was associated with an increased regeneration of IκBα at 60 min after TNP-BSA stimulation (Fig. S5, available at http://www.jem.org/cgi/content/full/jem.20081140/DC1). These results suggest that Rcan1 is likely involved in “turning off” FcεRI-mediated activation signals, a notion consistent with that in NFAT signaling.


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 induces increased IgE-dependent NF-κB activation but not MAPK, Akt, or Syk. (A) Rcan1+/+ and Rcan1−/− BMMCs were transfected with a pNF-κB-Luc and the control reporter plasmid pRL-TK. After transfection (24 h), cells were sensitized with anti-TNP IgE for 5 h. Cells were then 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 the wild-type group. (B and C) Rcan1+/+ and Rcan1−/− BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA for various times. Total cell lysates were analyzed by Western blotting for various phosphorylated and total proteins. Increased IκB phosphorylation in Rcan1−/− BMMCs was found at 60 min after TNP stimulation when compared with Rcan1+/+ cells. In contrast, the pattern of TNP-induced phosphorylation of p38, JNK, Akt, and Syk is similar between Rcan1−/− and Rcan1+/+ BMMCs.
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fig3: Rcan1 deficiency induces increased IgE-dependent NF-κB activation but not MAPK, Akt, or Syk. (A) Rcan1+/+ and Rcan1−/− BMMCs were transfected with a pNF-κB-Luc and the control reporter plasmid pRL-TK. After transfection (24 h), cells were sensitized with anti-TNP IgE for 5 h. Cells were then 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 the wild-type group. (B and C) Rcan1+/+ and Rcan1−/− BMMCs were sensitized with anti-TNP IgE and stimulated with TNP-BSA for various times. Total cell lysates were analyzed by Western blotting for various phosphorylated and total proteins. Increased IκB phosphorylation in Rcan1−/− BMMCs was found at 60 min after TNP stimulation when compared with Rcan1+/+ cells. In contrast, the pattern of TNP-induced phosphorylation of p38, JNK, Akt, and Syk is similar between Rcan1−/− and Rcan1+/+ BMMCs.
Mentions: In light of a critical role for transcription factor NF-κB in the regulation of gene expression (19), we examined whether Rcan1 is required in FcεRI-mediated NF-κB pathway activation. Rcan1-deficient BMMCs and wild-type cells were transfected with NF-κB luciferase plasmid. After sensitization with anti-TNP IgE, BMMCs were stimulated with TNP-BSA for 5 h. As shown in Fig. 3 A, Rcan1-deficient BMMCs showed significantly enhanced NF-κB activity in response to TNP stimulation, suggesting that Rcan1 is needed for the negative regulation of FcεRI-mediated NF-κB activation. NF-κB activity is regulated by IκB. The effects of Rcan1 on FcεRI-mediated IκB phosphorylation and degradation were examined by Western blotting. Aggregation of FcεRI induced a rapid phosphorylation of IκB (5 min), followed by IκB degradation (5 and 20 min) and subsequent regeneration of this molecule (60, 180, and 360 min; Fig. 3 B). Interestingly, the early events of IκB phosphorylation and degradation at 5 and 20 min were not affected by Rcan1 deficiency. In contrast, phosphorylation at the later time point (60 min) was significantly enhanced in Rcan1-deficient BMMCs (Fig. 3 B). This process was associated with an increased regeneration of IκBα at 60 min after TNP-BSA stimulation (Fig. S5, available at http://www.jem.org/cgi/content/full/jem.20081140/DC1). These results suggest that Rcan1 is likely involved in “turning off” FcεRI-mediated activation signals, a notion consistent with that in NFAT signaling.

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