- Email: [email protected]
Antigen presentation action of human bronchial epithelial cells mediated by bombesin receptor subtype 3 activation
Abstracts
(HBECs), which indicated that CTNNAL1 might be involved in the proliferation and adhesion of HBECs. Aims: To explore the effect of CTNNAL1 on proliferation and adhesion of HBECs. Methods: Overexpression plasmid (pcDNA3.1/CTNNAL1) was constructed and transfected into HBECs. The efficiency of recombinant plasmid pcDNA3.1/CTNNAL1 was identified by Real-time PCR and Western-blot. Cell cycle of HBECs was detected by flow cytometry (FCM). The adhesion rate of HBECs on rat-tail collagen was detected by MTT. The percentage of inflammatory cell adhesion with HBECs was detected by FCM, and adhesion molecule (E-cadherin, ICAM-1 and ITGβ4) expressions were observed by RT-PCR. Results: The expression of CTNNAL1 was significantly increased (189.7%) in pcDNA3.1/CTNNAL1 transfected HBECs. FCM results demonstrated that overexpression of CTNNAL1 increased G2 phase and G2/G1 of cell cycle by 20.75% and 98.5% respectively, while decreased S phase by 180.9%. MTT results showed that the CTNNAL1 can promote the rate of the matrix adhesion with HBECs. The percentage of inflammatory cell adhesion with HBECs was increased which was detected by FCM. The expression of E-cadherin, ICAM-1 and ITGβ4 was significantly decreased by CTNNAL1. Discussion: CTNNAL1 could promote the proliferation of HBECs, increase matrix adhesion and inflammatory cell adhesion to HBECs, and decrease the expressions of adhesion molecules.
doi:10.1016/j.regpep.2012.05.054
Antigen presentation action of human bronchial epithelial cells mediated by bombesin receptor subtype 3 activation H.J. Liu, C. Liu ,.Y. Xiang, X.P. Qu, X.Q. Qin Physiology Department, Xiangya Medical School, Central South University, Changsha, Hunan 410078, People's Republic of China Introduction: Bombesin receptor subtype 3 (BRS-3) is one member of bombesin‐like peptide receptor family, its native ligand has currently not yet been identified. Our studies have showed that BRS-3 was involved in regulating B7 constimulatory ligand expression on human airway epithelial cells (HBECs). Aims: To confirm the antigen presenting ability of HBECs and determine the role of BRS-3 activation in the process. Methods: The activation of antigen presentation process on HBECs was realized by using P3513 (a specific agonist of BRS-3). The expressions of B7 costimulatory molecules CD80, CD86,B7H1 and B7DC on HBECs were examined by flow cytometry. Uptake of FITCtagged OVA by HBECs was analyzed with confocal microscopy and flow cytometry respectively. The proliferation of T lymphocyte cell after antigen presentation by HBECs was measured by MTT. The levels of IFN-γ and IL-4 in supernate were examined by ELISA. Results: HBECs express CD80, B7H1 and B7DC, but not CD86; After pretreatment with P3513, significantly increased, the expressions of CD80 and B7H1, while decreased B7DC. The fluorescent intensity of FITC-tagged OVA in activated HBECs by P3513 and the proliferation of lymphocyte were enhanced after P3513 pretreatment. Besides, P3513 increased the level of IFN-γ, but with no effect on IL-4 in coculture of HBECs and T lymphocyte cell. Discussion: Our data demonstrated that HBECs can act as effective antigen presenting cells. BRS-3 activation might influence both the antigen-presenting function of HBECs and the derivation of T lymphocyte cells through increasing expression of CD80, B7H1 and decreased expression of B7DC.
doi:10.1016/j.regpep.2012.05.055
S27
Interaction between bombesin receptor activated protein and bombesin receptor subtype 3 Y. Liu, H.J. Liu, M.L. Li, C.X. Liu, X.P. Qu, Y. Xiang, C. Liu, M. Guo, X.Q. Qin Xiangya Medical School, Central South University, Changsha 410078, China Introduction: Bombesin receptor subtype 3 (BRS-3), the orphan bombesin receptor belongs to the GPCR (G protein coupled receptor) family, and could play a role in the regulation of stress responses in airway epithelium. By bacterial two-hybird method, we found a novel protein name Bombesin receptor activated protein (BRAP) which may interact with BRS-3. In addition, expression of BRAP was up-regulated especially in bronchial epithelium in the ozone-stressed airway hyperresponsiveness animal model. Aims: This study was designed to confirm that BRAP could interact with BRS-3 under physiological conditions. Methods: We chose a unique peptide in BRAP to product a polyantibody from rabbit since BRAP is a novel protein and has no commercial antibody. Using this antibody as primary antibody, we captured complex of BRAP and its interacted protein which comes from human bronchial epithelial cells (HBECs) under mild lysis conditions in Co-IP experiment. Then BRS-3 antibody was used as primary antibody to stain the captured complex by western blot. Results: Western Blot results demonstrated that BRS-3 exists in the captured complex in Co-IP experiment. Discussion: BRAP could interact with BRS-3 under physiological conditions. We predicted structure of BRAP, and found that there is a jmjC-domain (fold into eight β-sheets, forming an enzymatically active pocket that coordinates Fe(II) and α-ketoglutarate) in Cterminal. According to the Subcellular localization of BRAP, it may play a role in Krebs cycle.
doi:10.1016/j.regpep.2012.05.056
Distinct conformational states of the CCK2R are associated with β-arrestin-2 recruitment or phospholipase-C activation Rémi Magnana, Chantal Escrieuta, Véronique Gigouxa, Kavita Dea, Pascal Clerca, Fan Niua, Joelle Azemab,d, Bernard Masric,d, Arnau Cordomia, Michel Baltasb,d, Irina G. Tikhonova⁎e, Daniel Fourmy⁎a a Université de Toulouse 3, EA 4552, Toulouse, France b CNRS, LSPCMIB, UMR-5068, Toulouse, France c INSERM U1037, Cancer Research Center of Toulouse, Toulouse, France d Université de Toulouse 3, Toulouse, France e Queen's University of Belfast, School of Pharmacy, Belfast, United Kingdom Aim: Seven-transmembrane receptors (7TMRs) recruit β-arrestins to trigger intracellular signals independent from G protein coupling in addition to undergoing desensitization. 7TMRs adopt multiple conformations upon agonist binding, and biased ligands are believed to stabilize conformations preferentially activating either G‐ protein- or β-arrestin-dependent signaling pathways. We examined whether conformational states responsible for the Gαq proteindependent signal or recruiting β-arrestins are structurally distinguishable in the CCK2R. Methods: States of the CCK2R activating Gq-protein-dependent pathway or recruiting β-arrestin2 were monitored using inositol phosphate production assay or confocal microscopy and Bioluminescence resonance Energy Transfer, respectively. Structure of CCK2R and docking of pharmacological compounds were investigated using molecular modelling/site directed mutagenesis. Results: We discovered a non-peptide ligand (GV150,013X), acting as a competitive antagonist on CCK2R inducing phospholipase-C activation but inefficient at inhibiting CCK2R inducing β-arrestin-2
(HBECs), which indicated that CTNNAL1 might be involved in the proliferation and adhesion of HBECs. Aims: To explore the effect of CTNNAL1 on proliferation and adhesion of HBECs. Methods: Overexpression plasmid (pcDNA3.1/CTNNAL1) was constructed and transfected into HBECs. The efficiency of recombinant plasmid pcDNA3.1/CTNNAL1 was identified by Real-time PCR and Western-blot. Cell cycle of HBECs was detected by flow cytometry (FCM). The adhesion rate of HBECs on rat-tail collagen was detected by MTT. The percentage of inflammatory cell adhesion with HBECs was detected by FCM, and adhesion molecule (E-cadherin, ICAM-1 and ITGβ4) expressions were observed by RT-PCR. Results: The expression of CTNNAL1 was significantly increased (189.7%) in pcDNA3.1/CTNNAL1 transfected HBECs. FCM results demonstrated that overexpression of CTNNAL1 increased G2 phase and G2/G1 of cell cycle by 20.75% and 98.5% respectively, while decreased S phase by 180.9%. MTT results showed that the CTNNAL1 can promote the rate of the matrix adhesion with HBECs. The percentage of inflammatory cell adhesion with HBECs was increased which was detected by FCM. The expression of E-cadherin, ICAM-1 and ITGβ4 was significantly decreased by CTNNAL1. Discussion: CTNNAL1 could promote the proliferation of HBECs, increase matrix adhesion and inflammatory cell adhesion to HBECs, and decrease the expressions of adhesion molecules.
doi:10.1016/j.regpep.2012.05.054
Antigen presentation action of human bronchial epithelial cells mediated by bombesin receptor subtype 3 activation H.J. Liu, C. Liu ,.Y. Xiang, X.P. Qu, X.Q. Qin Physiology Department, Xiangya Medical School, Central South University, Changsha, Hunan 410078, People's Republic of China Introduction: Bombesin receptor subtype 3 (BRS-3) is one member of bombesin‐like peptide receptor family, its native ligand has currently not yet been identified. Our studies have showed that BRS-3 was involved in regulating B7 constimulatory ligand expression on human airway epithelial cells (HBECs). Aims: To confirm the antigen presenting ability of HBECs and determine the role of BRS-3 activation in the process. Methods: The activation of antigen presentation process on HBECs was realized by using P3513 (a specific agonist of BRS-3). The expressions of B7 costimulatory molecules CD80, CD86,B7H1 and B7DC on HBECs were examined by flow cytometry. Uptake of FITCtagged OVA by HBECs was analyzed with confocal microscopy and flow cytometry respectively. The proliferation of T lymphocyte cell after antigen presentation by HBECs was measured by MTT. The levels of IFN-γ and IL-4 in supernate were examined by ELISA. Results: HBECs express CD80, B7H1 and B7DC, but not CD86; After pretreatment with P3513, significantly increased, the expressions of CD80 and B7H1, while decreased B7DC. The fluorescent intensity of FITC-tagged OVA in activated HBECs by P3513 and the proliferation of lymphocyte were enhanced after P3513 pretreatment. Besides, P3513 increased the level of IFN-γ, but with no effect on IL-4 in coculture of HBECs and T lymphocyte cell. Discussion: Our data demonstrated that HBECs can act as effective antigen presenting cells. BRS-3 activation might influence both the antigen-presenting function of HBECs and the derivation of T lymphocyte cells through increasing expression of CD80, B7H1 and decreased expression of B7DC.
doi:10.1016/j.regpep.2012.05.055
S27
Interaction between bombesin receptor activated protein and bombesin receptor subtype 3 Y. Liu, H.J. Liu, M.L. Li, C.X. Liu, X.P. Qu, Y. Xiang, C. Liu, M. Guo, X.Q. Qin Xiangya Medical School, Central South University, Changsha 410078, China Introduction: Bombesin receptor subtype 3 (BRS-3), the orphan bombesin receptor belongs to the GPCR (G protein coupled receptor) family, and could play a role in the regulation of stress responses in airway epithelium. By bacterial two-hybird method, we found a novel protein name Bombesin receptor activated protein (BRAP) which may interact with BRS-3. In addition, expression of BRAP was up-regulated especially in bronchial epithelium in the ozone-stressed airway hyperresponsiveness animal model. Aims: This study was designed to confirm that BRAP could interact with BRS-3 under physiological conditions. Methods: We chose a unique peptide in BRAP to product a polyantibody from rabbit since BRAP is a novel protein and has no commercial antibody. Using this antibody as primary antibody, we captured complex of BRAP and its interacted protein which comes from human bronchial epithelial cells (HBECs) under mild lysis conditions in Co-IP experiment. Then BRS-3 antibody was used as primary antibody to stain the captured complex by western blot. Results: Western Blot results demonstrated that BRS-3 exists in the captured complex in Co-IP experiment. Discussion: BRAP could interact with BRS-3 under physiological conditions. We predicted structure of BRAP, and found that there is a jmjC-domain (fold into eight β-sheets, forming an enzymatically active pocket that coordinates Fe(II) and α-ketoglutarate) in Cterminal. According to the Subcellular localization of BRAP, it may play a role in Krebs cycle.
doi:10.1016/j.regpep.2012.05.056
Distinct conformational states of the CCK2R are associated with β-arrestin-2 recruitment or phospholipase-C activation Rémi Magnana, Chantal Escrieuta, Véronique Gigouxa, Kavita Dea, Pascal Clerca, Fan Niua, Joelle Azemab,d, Bernard Masric,d, Arnau Cordomia, Michel Baltasb,d, Irina G. Tikhonova⁎e, Daniel Fourmy⁎a a Université de Toulouse 3, EA 4552, Toulouse, France b CNRS, LSPCMIB, UMR-5068, Toulouse, France c INSERM U1037, Cancer Research Center of Toulouse, Toulouse, France d Université de Toulouse 3, Toulouse, France e Queen's University of Belfast, School of Pharmacy, Belfast, United Kingdom Aim: Seven-transmembrane receptors (7TMRs) recruit β-arrestins to trigger intracellular signals independent from G protein coupling in addition to undergoing desensitization. 7TMRs adopt multiple conformations upon agonist binding, and biased ligands are believed to stabilize conformations preferentially activating either G‐ protein- or β-arrestin-dependent signaling pathways. We examined whether conformational states responsible for the Gαq proteindependent signal or recruiting β-arrestins are structurally distinguishable in the CCK2R. Methods: States of the CCK2R activating Gq-protein-dependent pathway or recruiting β-arrestin2 were monitored using inositol phosphate production assay or confocal microscopy and Bioluminescence resonance Energy Transfer, respectively. Structure of CCK2R and docking of pharmacological compounds were investigated using molecular modelling/site directed mutagenesis. Results: We discovered a non-peptide ligand (GV150,013X), acting as a competitive antagonist on CCK2R inducing phospholipase-C activation but inefficient at inhibiting CCK2R inducing β-arrestin-2