α1-adrenoceptors (α1-ARs) and “cannabinoid-like” G Protein Coupled Receptor 55 (GPR55) belong to the G-protein coupled receptor (GPCR) family and play a crucial role in regulating prostate function. Although physi...α1-adrenoceptors (α1-ARs) and “cannabinoid-like” G Protein Coupled Receptor 55 (GPR55) belong to the G-protein coupled receptor (GPCR) family and play a crucial role in regulating prostate function. Although physical and functional interactions between the cannabinoid and adrenergic systems have been reported, analysis of functional interactions between α1-AR and GPR55 in normal and neoplastic prostate has not been reported. Since GPR55 levels are high in rodent adrenal gland, we propose a function link between the adrenergic system and GPR55 receptor. Confocal Laser Scanning Microscopy (CLSM) was employed to examine the endogenous α1-AR and GPR55 expression and their co-localization, expressed as fluorescence, in vitro in human andro-gen-insensitive PC-3 and androgen-sensitive LNCaP prostatic carcinoma cell lines, using the fluo-rescent ligands—Syto 62 (nuclear stain), BODIPY FL-Prazosin (QAPB;fluorescent quinazoline α1-AR ligand) and Tocriflour (T1117;a novel fluorescent diarylpyrazole cannabinoid/GPR55 ligand). Fluorescent ligand binding in untreated PC-3 cells and LNCaP cells and spheroids showed hetero-geneous expression of both α1-ARs and GPR55. A small proportion of cells had both α1-ARs and GPR55 in relatively equal numbers indicating a degree of co-localization. Co-localization of fluo-rescent ligand binding exhibited a stronger correlation in LNCaP (0.87) as compared to PC-3 (0.63) cells. Upregulation of α1-AR was observed in PC-3 cells following chronic doxazosin incubation. Robust T1117 binding, suggestive of GPR55 upregulation, was also observed in these cells. The presence of subtype-rich cells with a degree of co-localization between α1-ARs and GPR55 indicates a possibility for dimerisation or functional interaction and a new paradigm for functional synergism in which interactions may be either between cells or involve converging intracellular signaling processes.展开更多
背景:程序性细胞死亡受体1(programmed death receptor-1,PD-1)在高糖环境下影响骨髓间充质干细胞成骨分化的作用机制尚不清楚。目的:探讨高糖环境中PD-1对大鼠骨髓间充质干细胞成骨分化的影响及其调控机制。方法:将大鼠骨髓间充质干细...背景:程序性细胞死亡受体1(programmed death receptor-1,PD-1)在高糖环境下影响骨髓间充质干细胞成骨分化的作用机制尚不清楚。目的:探讨高糖环境中PD-1对大鼠骨髓间充质干细胞成骨分化的影响及其调控机制。方法:将大鼠骨髓间充质干细胞随机分为正常糖组(5.6 mmol/L)、高糖组(30 mmol/L)、PD-1过表达组、PD-1过表达空载组、PD-1敲低组、PD-1敲低空载组、PI3K/AKT通路抑制剂组(PD-1敲低+5μmol/L LY294002)。通过在高糖培养基中培养大鼠骨髓间充质干细胞来模拟体外糖尿病环境,采用qRT-PCR检测大鼠骨髓间充质干细胞中PD-1及其配体PD-L1和成骨标志物Runx2、OSX的mRNA表达,采用碱性磷酸酶染色和茜素红S染色观察成骨分化能力,采用CCK-8检测细胞增殖情况,采用Western blot检测PD-1、PD-L1、p-PI3K、p-AKT的蛋白表达。结果与结论:①高糖组PD-1及PD-L1表达显著高于正常糖组,高糖组骨髓间充质干细胞的成骨分化能力较正常糖组显著下降;②敲低PD-1表达可以促进骨髓间充质干细胞的成骨分化、增加细胞增殖活性,同时激活PI3K/AKT通路;③加入PI3K/AKT通路抑制剂LY294002后,骨髓间充质干细胞成骨分化能力显著下降。结果表明:PD-1依赖于PI3K/AKT信号通路抑制高糖环境下大鼠骨髓间充质干细胞的成骨分化。展开更多
文摘α1-adrenoceptors (α1-ARs) and “cannabinoid-like” G Protein Coupled Receptor 55 (GPR55) belong to the G-protein coupled receptor (GPCR) family and play a crucial role in regulating prostate function. Although physical and functional interactions between the cannabinoid and adrenergic systems have been reported, analysis of functional interactions between α1-AR and GPR55 in normal and neoplastic prostate has not been reported. Since GPR55 levels are high in rodent adrenal gland, we propose a function link between the adrenergic system and GPR55 receptor. Confocal Laser Scanning Microscopy (CLSM) was employed to examine the endogenous α1-AR and GPR55 expression and their co-localization, expressed as fluorescence, in vitro in human andro-gen-insensitive PC-3 and androgen-sensitive LNCaP prostatic carcinoma cell lines, using the fluo-rescent ligands—Syto 62 (nuclear stain), BODIPY FL-Prazosin (QAPB;fluorescent quinazoline α1-AR ligand) and Tocriflour (T1117;a novel fluorescent diarylpyrazole cannabinoid/GPR55 ligand). Fluorescent ligand binding in untreated PC-3 cells and LNCaP cells and spheroids showed hetero-geneous expression of both α1-ARs and GPR55. A small proportion of cells had both α1-ARs and GPR55 in relatively equal numbers indicating a degree of co-localization. Co-localization of fluo-rescent ligand binding exhibited a stronger correlation in LNCaP (0.87) as compared to PC-3 (0.63) cells. Upregulation of α1-AR was observed in PC-3 cells following chronic doxazosin incubation. Robust T1117 binding, suggestive of GPR55 upregulation, was also observed in these cells. The presence of subtype-rich cells with a degree of co-localization between α1-ARs and GPR55 indicates a possibility for dimerisation or functional interaction and a new paradigm for functional synergism in which interactions may be either between cells or involve converging intracellular signaling processes.
