D-type prostanoid receptor enhances the signaling of chemoattractant receptor– homologous molecule expressed on TH2 cells Miriam Sedej, MSc, Ralf Schröder,

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Presentation transcript:

D-type prostanoid receptor enhances the signaling of chemoattractant receptor– homologous molecule expressed on TH2 cells Miriam Sedej, MSc, Ralf Schröder, PhD, Kathrin Bell, MSc, Wolfgang Platzer, BSc, Anela Vukoja, MSc, Evi Kostenis, PhD, Akos Heinemann, MD, Maria Waldhoer, PhD Journal of Allergy and Clinical Immunology Volume 129, Issue 2, Pages 492-500.e9 (February 2012) DOI: 10.1016/j.jaci.2011.08.015 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 The DP receptor modulates CRTH2-induced intracellular Ca2+ release in eosinophils and HEK-CRTH2+DP cells. Intracellular Ca2+ release was measured in eosinophils (A) and HEK-CRTH2+DP, HEK-CRTH2, HEK-DP, and HEK293 cells (B-E) in response to PGD2, DK-PGD2, or BW245c. In Fig 1, E, the Ca2+ ionophore A23187 served as an internal control. Data were normalized to cell numbers (n = 3-5). Veh, Vehicle. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Increasing DP receptor levels enhance the CRTH2-mediated Ca2+ response. Intracellular Ca2+ release was measured in HEK-CRTH2 cells cotransfected with DP (A) or CB2 (B) pcDNA in response to 100 nmol/L DK-PGD2. C and D, Receptor expression levels were determined by means of ELISA against the Flag epitopes of DP and CB2, respectively. Data were normalized to cell numbers (n = 3-5). ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 DP desensitization prevents CRTH2-mediated Ca2+ mobilization in HEK-CRTH2+DP and HEK-DP cells. Intracellular Ca2+ release was measured in HEK-CRTH2+DP (A) and HEK-DP (B) cells. Cells were pretreated with BW245c (10 nmol/L) or vehicle followed by stimulation with 100 nmol/L DK-PGD2 (Fig 3, A) or PGD2 (Fig 3, B; n = 3-5). ∗P < .01 and ∗∗P < .0001. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 The DP antagonist BWA868c prevents agonist-induced intracellular Ca2+ release in HEK-CRTH2+DP and HEK-DP cells. Intracellular Ca2+ release was measured in response to PGD2, DK-PGD2, and BW245c in HEK-CRTH2+DP (A-C) and HEK-DP (D and E) cells pretreated with BWA868c or vehicle. Data were normalized to cell numbers (n = 3-5). DMSO, Dimethyl sulfoxide; Veh, vehicle. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 The Ca2+ signals in HEK-CRTH2+DP cells are mediated by both Gαi and Gαq/11 proteins. Intracellular Ca2+ release was measured in response to PGD2, DK-PGD2, and BW245c in HEK-CRTH2+DP cells treated with 100 ng/mL PTX (A-C) or MH-362-63-8 (D-F). Data were normalized to cell numbers (n = 3-5). Veh, Vehicle. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 CRTH2-mediated Ca2+ responses in eosinophils are affected by inhibition of both CRTH2 and DP receptor pathways. Ca2+ flux in eosinophils stimulated by 3 nmol/L PGD2, DK-PGD2, or BW245c was measured by means of flow cytometry. Cells were pretreated with vehicle or BWA868c or Cay10471 (1 μmol/L each; A), BW245c (10 nmol/L; B), PTX (3 μg/mL; C), or MH-362-63-8 (1 μmol/L; D). Fluorescence units are shown as the percentage of untreated cells (n = 3-5). ∗P < .05 and ∗∗P < .001. Veh, Vehicle. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 CRTH2 and DP receptors form heteromers in HEK-CRTH2+DP cells. A, Myc-CRTH2 and Flag-DP receptors from HEK-CRTH2+DP membranes were coimmunoprecipitated and immunoblotted with anti-Flag or anti-Myc antibodies. B, HEK-CRTH2+DP cells were treated with BWA868c (1 μmol/L, upper panel) before coimmunoprecipitation. Blots of CRTH2 and DP are shown in the middle and lower panels, respectively. Shown are representative blots (n = 3). IB, Immunoblotting; IP, immunoprecipitation. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 Proposed mechanism of CRTH2 and DP signaling. A, In HEK-DP cells activation of the DP receptor with the DP agonist BW245c induces a Gαq/11-mediated Ca2+ response. In contrast, stimulation of HEK-CRTH2 cells with the CRTH2 agonist DK-PGD2 fails to activate intracellular Ca2+ release. B, On CRTH2/DP heteromerization in HEK-CRTH2+DP cells, the BW245c-induced DP response is decreased in the presence of CRTH2. DK-PGD2 induces intracellular Ca2+ release in HEK-CRTH2+DP by transactivating the DP receptor in a Gαi-dependent manner. C and D, Desensitization of the DP receptor by BW245c (Fig 8, C) or the DP antagonist BWA868c (Fig 8, D) abolishes the CRTH2-induced Ca2+ response. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Expression patterns of CRTH2 and DP receptors in HEK293-CRTH2+DP cells and human peripheral blood eosinophils. Myc-CRTH2 and Flag-DP expression in HEK-CRTH2, HEK-DP, and HEK-CRTH2+DP cells was determined by means of fluorescence staining with anti-Myc and anti-Flag antibodies (A; scale bar = 10 μm), flow cytometry with anti-Myc and anti-Flag antibodies (B), and Western blotting with polyclonal rabbit anti-DP and anti-CRTH2 antibodies (D). Plain HEK cells (Con) are shown for comparison. C, DP and CRTH2 expression in eosinophils was shown by using polyclonal rabbit anti-DP and anti-CRTH2 antibodies. Lysate samples immunoblotted for β-actin served as loading controls. Shown is a representative blot (n = 3). Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Effect of the CRTH2 antagonist Cay10471 on agonist-induced intracellular Ca2+ release in HEK-CRTH2+DP and HEK-DP cells. Intracellular Ca2+ release was measured in response to PGD2, DK-PGD2, and BW245c in HEK-CRTH2+DP (A-C) and HEK-DP (D and E) cells pretreated with Cay10471 or vehicle. Data were normalized to cell numbers (n = 3-5). DMSO, Dimethyl sulfoxide; Veh, vehicle. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Effect of the Gαi inhibitor PTX and the Gαq/11 inhibitor MH-362-63-8 on agonist-induced intracellular Ca2+ release in HEK-DP and HEK293 cells. Intracellular Ca2+ release was measured in response to PGD2 or carbachol in HEK-DP (A and B) or HEK293 (C) cells treated with PTX (100 ng/mL), MH-362-63-8 (0.1 nmol/L to 1 μmol/L), or vehicle. Data were normalized to cell numbers (n = 3). Veh, Vehicle. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Ligand-binding properties of receptor monomers and heteromers. Competition-binding experiments were performed with 2 nmol/L tritiated PGD2 on whole HEK-CRTH2 cells (A), HEK-DP cells (B), and HEK-CRTH2+DP cells (C; n = 3-5). Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 CRTH2 but not DP receptors internalize in HEK293 cells and eosinophils. PGD2-, DK-PGD2-, and BW245c-induced CRTH2 and DP internalization was investigated in HEK-CRTH2, HEK-DP, and HEK-CRTH2+DP cells by means of fluorescence staining with anti-Myc and anti-Flag antibodies (A; green, Myc-CRTH2; red, Flag-DP; agonist concentration, 1 μmol/L; scale bar = 10 μm) and (B, C) eosinophils by means of flow cytometry with polyclonal goat anti-DP and rat anti-CRTH2 antibodies (B and C; Fig E5, A, n = 3; Fig E5, B, n = 8-12; and Fig E5, C, n = 3). ∗P < .05 and ∗∗P < .01. Journal of Allergy and Clinical Immunology 2012 129, 492-500.e9DOI: (10.1016/j.jaci.2011.08.015) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions