染料木黄酮不是通过雌激素受体促进成骨细胞增殖

目的:探讨染料木黄酮对成骨细胞活性的影响及其可能机制,为染料木黄酮防治骨质疏松提供理论依据。方法:实验于2001-05/2003-01在卫生学环境医学研究所完成。无菌条件下用胰蛋白酶和Ⅱ型胶原酶分步消化乳鼠颅盖骨获得成骨细胞,传代后细胞用于实验。染料木黄酮组分别加入10-5～10-7mol/L染料木黄酮,对照组以吐温20为对照。分别用噻唑蓝比色法和3H-胸腺嘧啶掺入法测定成骨细胞增殖和DNA合成,用反转录-聚合酶链式反应和Westernblot测定雌激素受体mRNA和蛋白表达。结果:①成骨细胞培养基中加入(10-5～10-6mol/L)染料木黄酮,培养48h和72h后噻唑蓝的吸光度值与对照组相比均明显升高[培养48h:(0.39±0.03),(0.45±0.03),(0.46±0.02),(0.19±0.05);培养72h:(0.29±0.04),(0.32±0.02),(0.37±0.02),(0.15±0.04)]。②染料木黄酮组3H-胸腺嘧啶掺入量均显著增加[染料木黄酮组为(101.20±10.06),(844.60±366.90),(512.20±197.6);对照组为(68.67±10.39)],未发现成骨细胞雌激素受体基因和蛋白表达。结论:染料木黄酮不是通过促进雌激素受体基因和蛋白的表达来促进成骨细胞增殖。     

染料木黄酮对乳鼠颅盖骨成骨细胞增殖分化和转化生长因子β表达的影响

目的:动物实验、临床研究以及流行病学调查表明染料木黄酮可预防骨质疏松的发生,但其具体机制尚不清楚。观察染料木黄酮对成骨细胞活性的影响及其与转化生长因子β的关系。方法:实验于2001-05/2003-05在卫生学环境医学研究所生化实验室完成。①实验材料:选择出生3d的Wistar大鼠,体质量(10±2)g。②实验方法:无菌条件下分离颅盖骨,剪碎,加入胰蛋白酶和Ⅱ型胶原酶,用含体积分数为0.1胎牛血清的F-12培养基重悬,调整细胞浓度后接种于25mL培养瓶中,Ⅱ代细胞用于实验。实验分为染料木黄酮组、雌激素组和对照组(吐温20),染料木黄酮组浓度分别为0.1,1,10μmol/L,雌激素组的浓度分别为0.1,1nmol/L。③实验评估:培养48,72h应用四甲基偶氮唑盐测定成骨细胞增殖;培养48h3H-胸腺嘧啶掺入实验测定DNA合成;免疫组织化学方法观察转化生长因子β的表达。结果:①染料木黄酮对成骨胞增殖的影响:与对照组相比,培养48h后0.1,1,10μmol/L染料木黄酮四甲基偶氮唑盐的吸光度值分别增加1.43,1.36,1.05倍;0.1,1nmol/L雌激素组分别增加1.00倍和0.84倍;培养72h,染料木黄酮3个浓度组分别增加1.46,1.13倍和0.93倍,雌激素组分别增加2.26倍和2.30倍;各组与对照组相比差异均有显著性(P<0.05)。②3H-胸腺嘧啶掺入实验:0.1,1,10μmol/L染料木黄酮3H-胸腺嘧啶掺入量分别比对照组增加6.45,11.29,0.47倍;0.1,1nmol/L雌激素组增加16.5倍和15.4倍,各组与对照组相比差异均有显著性(P<0.05)。③成骨细胞转化生长因子β的表达:各组成骨细胞周围均有较强的呈棕色的阳性颗粒,但染料木黄酮组与对照组相比差异无显著性(P>0.05)。结论:不同浓度染料木黄酮和雌激素一样可以促进成骨细胞增殖和DNA合成,但染料木黄酮不是通过影响转化生长因子β的表达来促进成骨细胞增殖与分化,详细机制还需进一步研究。     

染料木黄酮对乳鼠颅盖骨成骨细胞增殖分化的影响及其与白细胞介素6的关系

目的:观察染料木黄酮对成骨细胞活性的影响及其相关机制。方法:实验于2001-05/2003-05在卫生学环境医学研究所实验室完成。①成骨细胞培养:无菌条件下分离出生3d的乳鼠颅盖骨,剪碎,加入胰蛋白酶和Ⅱ型胶原酶,用含体积分数为0.1的胎牛血清的F-12培养基重悬,调整细胞浓度后接种于25mL细胞培养瓶,Ⅱ代细胞用于实验。②实验方法:实验分为染料木黄酮组、雌激素组和对照组(吐温20),染料木黄酮剂量分别为10-5,10-6、10-7mol/L,雌激素剂量分别为10-9,10-10mol/L,培养48,72h后,应用MTT和3H-TdR掺入实验观察不同剂量的染料木黄酮和雌激素对成骨细胞活性影响,免疫组化的方法测定白细胞介素6表达。结果:①A570nm值:培养48h,染料木黄酮10-5,10-6,10-7mol/L组分别比对照组增加105%,136%和143%;10-9,10-10mol/L雌激素组分别比对照组增加84%和100%;培养72h,染料木黄酮3个剂量组分别比对照组增加93%,113%和146%,各剂量组与对照组相比均有显著性差异(P<0.05)。②3H-TdR掺入量:10-5,10-6,10-7mol/L染料木黄酮组分别比对照组增加0.47,11.29,6.45倍;10-9和10-10mol/L雌激素组增加15.4倍和16.5倍(P<0.05)。③白细胞介素6表达:各组成骨细胞周围均有较强的呈棕色的阳性颗粒,但染料木黄酮组组与对照组相比无明显差别。结论:①10-5～10-7mol/L染料木黄酮和雌激素一样可促进成骨细胞增殖和DNA合成。②染料木黄酮不是通过促进白细胞介素6表达来促进成骨细胞增殖与分化。     

植物雌激素染料木素对狗体外冠状血管的舒张作用及其机制

目的：以血管张力变化为指标，对染料木素和17β-雌二醇的心血管保护作用及相关机制进行探讨和比较。方法：①实验于2005—04／10在湖南学院机能实验室完成。选用雄性杂交家狗心脏，把狗心放在冰冷的K—H液中从屠宰场取回进行实验。染料木索（存在于大豆的植物雌激素），17β-雌二醇，吲哚美辛，Nω-L-硝基精氨酸。放线菌酮，前列腺索F2α和缓激肽，正矾酸钠，均购自Sigma公司；甲烯蓝由上海润捷化学试剂有限公司提供。用蒸馏水溶解，它莫西酚、染料术紊、17β-雌二醇用二甲亚砜溶解配制，吲哚美辛用体积分数0.2乙醇溶解。②制备狗冠状动脉血管环，固定于恒温肌槽内，观察染料木紊与17β-雌二醇对KCl预收缩离体冠状血管环的舒张作用：血管环稳定后，向浴槽中加入50mmol／LKCl。待血管环收缩张力达坪值后。用K-H液冲洗。然后，20min换液1次，平衡30min后重复上述实验，待血管环张力达高峰并稳定后，分别加入染料木索与17β-雌二醇，累积浓度为1，4，8，40和80μmol／L，观察血管环张力变化。观察阻断剂及内皮细胞对染料木紊与17β-雌二醇舒血管作用的影响：重复前述实验内容后，用K-H液冲洗，每20min换液1次，温育1,5h，血管环稳定后，向浴槽中分别加入Nω-L-硝基精氨酸1&;#215;10^-4mol／L，它奠西酚10^-5mol／L，吲哚美辛1&;#215;10^-5mol／L，正矾酸钠1&;#215;10^-5mol／L或放线菌酮1&;#215;10^-5mol／L，甲烯蓝1&;#215;10^-5mol／L，温育15min或用棉签擦除内皮细胞后，重复前述实验内容。对比染料木紊与17β-雌二醇对KCl50mmol／L预收缩血管平滑肌的舒张作用的变化，并随时记录张力的变化值。③用线性回归法计算染料木素与17β-雌二醇的使激动剂效应降低50％时所采用的拮抗剂的浓度。两组间计量资料差异比较采用t检验，3组或3组以上资料差异比较采用单因素方差分析。结果：①染料木素与17β-雌二醇作用相似，均可使50mmol／L KCl预收缩冠状动脉血管环产生剂量依赖性舒张作用（r=0．96，P〈0．01），使激动剂效应降低50％时所采用的拮抗剂的浓度分别为（16．37&;#177;5．19）μmol／L和（22．64&;#177;8．26）μmol／L。②除内皮细胞或加入一氧化氮合酶抑制剂Nω-L-硝基精氨酸1&;#215;10^-4mol/L，甲烯蓝1&;#215;10^-4mol／L或正矾酸钠1&;#215;10^-5mol／L温育，染料木素对50mmol／LKCl预收缩动脉血管环产生的量效舒张作用无明显改变（P〉0．05）。但17β-雌二醇对50mmol／LKCl预收缩动脉血管环产生的量效舒张作用有明显改变（P〈0．01），1&;#215;10^-5mol／L吲哚美辛，它莫西酚与放线菌酮对两者量效舒张血管的作用均无明显影响（P〉0．05）。结论：①染料木索和17β-雌二醇对冠状动脉毗管的舒张作用与前列腺素类物质的合成和血管壁传统雌激素受体无关，与雌激素经典核内作用途径无关。②17β-雌二醇的作用部分与内皮细胞释放的一氧化氮有关。③酪氨酸蛋白激酶系统可能参与了冠状动脉血管张力的调节。     

雌激素、促性腺激素与大鼠成骨细胞雌激素受体β的表达

目的：观察不同剂量雌激素，卵泡雌激素及黄体生成素对大鼠成骨细胞雌激素受体β表达的影响。方法：实验于2004-02／08在哈尔滨医科大学第二临床医学院科研中心完成。①取三四个月龄雌性Wistar大鼠28只用于大鼠成骨细胞体外培养。采用二次酶消化、反复贴壁法分离纯化培养大鼠成骨细胞。观察其形态、改良钙钴法碱性磷酸酶染色，培养上清碱性磷酸酶、骨钙素测定来鉴定成骨细胞。②取2代培养的成骨细胞用无血清培养基培养24h，分为10组：对照组，1&;#215;10^10，1&;#215;10^-8，1&;#215;10^-6mol/L 17β-雌二醇组，10，100，1000U／L黄体生成素组，1&;#215;10^-7,1&;#215;10^-6，1&;#215;10^-5g／L卵泡刺激素组。每组3个孔。对照组加入基础培养基，其他各组分别加入相应终浓度药物，培养24-48h。大鼠成骨细胞雌激素受体表达阳性细胞数检测应用免疫组织化学法测定，以细胞浆有棕黄色颗粒沉积为阳性。成骨细胞雌激素受体β表达检测应用半定量免疫印渍酶促底物染色法以及化学发光法，以A值越高，说明雌激素受体β表达越多。结果：①成骨细胞形态及特征性鉴定：成骨细胞随培养时间的延长，细胞呈指数增长，分泌碱性磷酸酶和骨钙素，碱性磷酸酶染色阳性率为90％，表现出旺盛的分泌功能。②成骨细胞雌激素受体表达阳性细胞数检测结果：不同剂量卵泡刺激素组和黄体生成素组对雌激素受体表达无明显影响；雌激素1&;#215;10^-8mol／L组和1&;#215;10^-6mol/L组显著高于对照组[(90．95&;#177;5．89)，(95．12&;#177;6．17)，(81．44&;#177;5.37)个数／100个细胞，τ=2．95，P&;lt;0．05；τ=3．50,P&;lt;0．01]。③成骨细胞雌激素受体β表达结果：不同剂量卵泡刺激素及黄体生成素对雌激素受体β的表达无明显影响；随雌激素浓度的增加成骨细胞雌激素受体β表达呈上升趋势。结论：雌激素能促进成骨细胞雌激素受体β的表达，且表现出剂量依赖性。而黄体生成素、卵泡刺激素对雌激素受体β表达无直接影响。     

雌激素受体基因ERβ与小鼠成骨细胞的增殖和分化

背景:雌激素受体ERs表达于所有关系到骨形成和骨吸收的细胞成分中。目的:观察雌激素受体ERβ对成骨细胞增殖、分化能力的调控作用。方法:以小鼠成骨细胞株MC3T3-E1为对象,设立3组:实验组转染雌激素受体ERβ RNAi载体、阴性对照组转染ERL RNAi载体、空白对照组不进行转染,3组在相同条件下培养。采用MTT法绘制各组细胞的生长曲线;流式细胞仪分析各组细胞的细胞周期。结果与结论:实验组成骨细胞的增殖能力明显高于空白对照组和阴性对照组,G1期细胞百分率明显少于空白对照组和阴性对照组,而S期和G2期细胞百分率明显高于空白对照组和阴性对照组(P均<0.05)。根据雌激素受体ERβ沉默后检测的结果可以反向推断,ERβ的表达对成骨细胞的增殖、分化具有抑制作用。     

雌激素受体基因ERβ与小鼠成骨细胞的增殖和分化

背景：雌激素受体ERs表达于所有关系到骨形成和骨吸收的细胞成分中。目的：观察雌激素受体ERβ对成骨细胞增殖、分化能力的调控作用。方法：以小鼠成骨细胞株MC3T3-E1为对象,设立3组：实验组转染雌激素受体ERβ RNAi载体、阴性对照组转染ERL RNAi载体、空白对照组不进行转染,3组在相同条件下培养。采用MTT法绘制各组细胞的生长曲线;流式细胞仪分析各组细胞的细胞周期。结果与结论：实验组成骨细胞的增殖能力明显高于空白对照组和阴性对照组,G1期细胞百分率明显少于空白对照组和阴性对照组,而S期和G2期细胞百分率明显高于空白对照组和阴性对照组（P均〈0.05）。根据雌激素受体ERβ沉默后检测的结果可以反向推断,ERβ的表达对成骨细胞的增殖、分化具有抑制作用。     

雌激素、促性腺激素与大鼠成骨细胞雌激素受体β的表达

目的:观察不同剂量雌激素,卵泡雌激素及黄体生成素对大鼠成骨细胞雌激素受体β表达的影响。方法:实验于2004-02/08在哈尔滨医科大学第二临床医学院科研中心完成。①取三四个月龄雌性Wistar大鼠28只用于大鼠成骨细胞体外培养。采用二次酶消化、反复贴壁法分离纯化培养大鼠成骨细胞。观察其形态、改良钙钴法碱性磷酸酶染色,培养上清碱性磷酸酶、骨钙素测定来鉴定成骨细胞。②取2代培养的成骨细胞用无血清培养基培养24h,分为10组:对照组,1×10-10,1×10-8,1×10-6mol/L17β-雌二醇组,10,100,1000U/L黄体生成素组,1×10-7,1×10-6,1×10-5g/L卵泡刺激素组。每组3个孔。对照组加入基础培养基,其他各组分别加入相应终浓度药物,培养24～48h。大鼠成骨细胞雌激素受体表达阳性细胞数检测应用免疫组织化学法测定,以细胞浆有棕黄色颗粒沉积为阳性。成骨细胞雌激素受体β表达检测应用半定量免疫印渍酶促底物染色法以及化学发光法,以A值越高,说明雌激素受体β表达越多。结果:①成骨细胞形态及特征性鉴定:成骨细胞随培养时间的延长,细胞呈指数增长,分泌碱性磷酸酶和骨钙素,碱性磷酸酶染色阳性率为90%,表现出旺盛的分泌功能。②成骨细胞雌激素受体表达阳性细胞数检测结果:不同剂量卵泡刺激素组和黄体生成素组对雌激素受体表达无明显影响;雌激素1×10-8mol/L组和1×10-6mol/L组显著高于对照组[(90.95±5.89),(95.12±6.17),(81.44±5.37)个数/100个细胞,t=2.95,P<0.05;t=3.50,P<0.01]。③成骨细胞雌激素受体β表达结果:不同剂量卵泡刺激素及黄体生成素对雌激素受体β的表达无明显影响;随雌激素浓度的增加成骨细胞雌激素受体β表达呈上升趋势。结论:雌激素能促进成骨细胞雌激素受体β的表达,且表现出剂量依赖性。而黄体生成素、卵泡刺激素对雌激素受体β表达无直接影响。     

染料木黄酮诱导人结肠癌SW480细胞凋亡及机制

目的 探讨染料木黄酮（GEN）诱导人结肠癌SW480细胞凋亡的生物学效应及抗肿瘤机制。方法 通过MTT法检测GEN对该细胞系生长的影响；应用PI染色流式细胞术（FCM）分析GEN处理前后的细胞周期分布的变化；SP免疫组化法检测细胞内bcl-2、Bax蛋白的表达。结果 GEN能抑制SW480细胞增殖，呈浓度依赖性。流式细胞仪示不同浓度GEN作用于SW480细胞，出现G2／M阻滞；SP免疫组化结果表明GEN处理细胞72h后，Bax蛋白表达升高，bcl-2蛋白表达下降。结论 GEN有诱导人结肠癌SW480细胞凋亡的作用，其机制与bcl-2／Bax比值下降有关。     

染料木素对人子宫内膜癌细胞系HEC-1B体内外增殖抑制作用的研究

目的研究染料木素(genistein,GEN)对人子宫内膜癌细胞系HEC-1B细胞体内外增殖的抑制作用,探讨其抗癌作用的机制。方法采用四甲基偶氮唑蓝比色分析(MTT)、病理组织切片HE染色等方法,分别检测体内外GEN对人子宫内膜癌细胞系HEC-1B增殖的抑制效果。结果体外实验:GEN浓度为0~25μmol/L时,对HEC-1B细胞有明显促进增殖作用(P<0.05),随着GEN浓度升高至50μmol/L时,对HEC-1B细胞则呈现抑制作用,当GEN浓度为100μmol/L时,则显示了明显的生长抑制作用(P<0.05),其抑制率达到55%。体内实验:人子宫内膜癌裸鼠模型经100μmol/L染料木素处理后体内平均瘤质量明显减小(P<0.05),抑瘤率可达39%,且处理后肿瘤组织微血管数量减少,部分区域坏死明显。结论染料木素能够明显抑制人子宫内膜癌细胞及其移植瘤的生长,其机理可能为通过和雌激素竞争结合雌激素受体,减轻雌激素促细胞增殖作用和抑制肿瘤血管生成导致肿瘤细胞坏死,从而发挥抗肿瘤作用。     

Immunoassay for estrogen receptor does not detect inactivated receptor

Accurate quantification of estrogen receptor (ER) is essential for optimal clinical characterization of individual cases of breast cancer. If breast tumors are mishandled, the relatively labile ER protein may lose its steroid-binding capacity (become inactivated) and not be measurable by the routine steroid-binding assay. We tested whether the commercial enzyme immunoassay of Abbott Laboratories could quantify inactivated ER. Samples of powdered breast tumors from humans were exposed to various temperature and homogenization conditions known to inactivate ER, and any remaining ER was quantified by both the immunoassay and the steroid-binding assay. For all inactivation conditions tested, the two assay methods detected the same proportions of remaining ER. We conclude that the inactivation reaction for ER also alters one or both of the antigenic site(s) necessary for the immunoassay. Hence, for breast tumors mishandled to the extent of inactivating ER, the immunoassay offers no advantage over the more conventional steroid-binding assay for quantifying any remaining ER.     

Insulin receptor substrate 2 plays a crucial role in beta cells and the hypothalamus

We previously demonstrated that insulin receptor substrate 2 (Irs2) KO mice develop diabetes associated with hepatic insulin resistance, lack of compensatory beta cell hyperplasia, and leptin resistance. To more precisely determine the roles of Irs2 in beta cells and the hypothalamus, we generated beta cell-specific Irs2 KO and hypothalamus-specific Irs2 knockdown (betaHT-IRS2) mice. Expression of Irs2 mRNA was reduced by approximately 90% in pancreatic islets and was markedly reduced in the arcuate nucleus of the hypothalamus. By contrast, Irs2 expression in liver, muscle, and adipose tissue of betaHT-IRS2 mice was indistinguishable from that of control mice. The betaHT-IRS2 mice displayed obesity and leptin resistance. At 4 weeks of age, the betaHT-IRS2 mice showed normal insulin sensitivity, but at 8 and 12 weeks, they were insulin resistant with progressive obesity. Despite their normal insulin sensitivity at 8 weeks with caloric restriction, the betaHT-IRS2 mice exhibited glucose intolerance and impaired glucose-induced insulin secretion. beta Cell mass and beta cell proliferation in the betaHT-IRS2 mice were reduced significantly at 8 and 12 weeks but not at 10 days. Insulin secretion, normalized by cell number per islet, was significantly increased at high glucose concentrations in the betaHT-IRS2 mice. We conclude that, in beta cells and the hypothalamus, Irs2 is crucially involved in the regulation of beta cell mass and leptin sensitivity.     

The Eph receptor A4 plays a role in demyelination and depression-related behavior

Proper myelination of axons is crucial for normal sensory, motor, and cognitive function. Abnormal myelination is seen in brain disorders such as major depressive disorder (MDD), but the molecular mechanisms connecting demyelination with the pathobiology remain largely unknown. We observed demyelination and synaptic deficits in mice exposed to either chronic, unpredictable mild stress (CUMS) or LPS, 2 paradigms for inducing depression-like states. Pharmacological restoration of myelination normalized both synaptic deficits and depression-related behaviors. Furthermore, we found increased ephrin A4 receptor (EphA4) expression in the excitatory neurons of mice subjected to CUMS, and shRNA knockdown of EphA4 prevented demyelination and depression-like behaviors. These animal data are consistent with the decrease in myelin basic protein and the increase in EphA4 levels we observed in postmortem brain samples from patients with MDD. Our results provide insights into the etiology of depressive symptoms in some patients and suggest that inhibition of EphA4 or the promotion of myelination could be a promising strategy for treating depression.     

渐进延长后适应通过MAPK途径减轻再灌注损伤

目的 探讨后适应不同模式对大鼠急性心肌缺血-再灌注损伤的影响,并进一步研究丝裂原活化蛋白激酶途径(MAPK)在其中的作用.方法 60只SD大鼠随机(随机数字法)分为假手术组(Sham)、缺血-再灌注组(I/R)、渐进缩短后适应组(GDR,后适应处理方案短暂再灌注/缺血时间为30/10-25/15-15/25-10/30s)、标准后适应组(ER,后适应处理方案为20/20s×4)和渐进延长后适应组(GIR,后适应处理方案短暂再灌注/缺血时间为10/30-15/25-25/15-30/10s)5组,建立急性心肌梗死和缺血后适应模型.再灌注6h后每组取3只处死,取心肌组织用Western Blot方法测定磷酸化细胞外信号调节蛋白激酶(P-ERK)、磷酸化应激活化蛋白激酶(stress activated protein kinase,P-JNK)、磷酸化p38 MAPK (P-p38)、肿瘤坏死因子-α(TNF-α)、半胱氨酸天冬氨酸蛋白酶-8 (Caspase-8)在心肌组织中的表达及细胞色素c在胞浆中的表达;各组其余大鼠再灌注24 h后测定血流动力学,抽血测心肌酶,取心脏进行TUNEL凋亡检测.统计学多组间比较应用单因素方差分析,组间差异两两比较应用q检验.结果 三种后适应处理组均有显著的心肌保护作用(P＜0.05),其中GIR组最为明显,其次是ER组,GDR组最不明显.GIR组同ER组相比,细胞凋亡指数和血清标志物水平更低;P-ERK表达水平高于ER组[(1.82±0.22)vs.(1.54±0.32),P＜0.05],同时P-p38[(0.82±0.26) vs.(1.63±0.24)],P-JNK[(0.76±0.28) vs.(1.33±0.21),TNF-α[(0.62±0.20)vs.(1.00±0.12)],Caspase-8[(0.61±0.21)vs.(1.00±0.30)],Cyt-c[(0.66±0.16) vs.(1.68±0.22)]表达水平低(以上P＜0.05).在以上指标中渐进延长后适应组均显著优于渐进缩短后适应组.结论 渐进延长后适应较标准后适应能显著减轻心肌再灌注损伤,MAPK途径在其中发挥了重要作用.     

The third transmembrane helix of the cannabinoid receptor plays a role in the selectivity of aminoalkylindoles for CB2, peripheral cannabinoid receptor.

Two subtypes of the human cannabinoid receptor have been identified. The CB1 receptor is primarily distributed in the central nervous system, whereas the CB2 receptor is associated with peripheral tissue, including the spleen. These two subtypes are also distinguished by their ligand-binding profiles. The goal of this study was to identify critical residues in transmembrane region III (TM3) of the receptors that contribute to subtype specificity in ligand binding. For this purpose, a chimeric cannabinoid receptor [CB1/2(TM3)] was generated in which the TM3 of CB1 was replaced with the corresponding region of CB2. These receptors were stably expressed in Chinese hamster ovary cells for evaluation. The binding affinities of CB1/2(TM3) and the wild-type CB1 receptor to several prototype ligands were similar with one notable exception: the chimeric receptor exhibited a 4-fold enhancement in binding affinity to WIN 55,212-2 (K(d) = 4.8 nM) relative to that observed with CB1 (K(d) = 21.7 nM). Two additional aminoalkylindoles, JWH 015 and JWH 018, also bound the chimeric receptor (K(i) = 1.0 microM and 1.4 nM, respectively) with higher affinity compared with the wild-type CB1 (K(i) = 5.2 microM and 9.8 nM, respectively). Furthermore, the increase in binding affinities of the aminoalkylindoles were reflected in the EC(50) values for the ligand-induced inhibition of intracellular cAMP levels mediated by the chimeric receptor. This pattern mirrors the selectivity of WIN 55,212-2 binding to CB2 compared with CB1. Site-specific mutagenesis of the most notable amino acid changes in the chimeric receptor, Gly195 to Ser and Ala198 to Met, revealed that the enhancement in WIN 55,212-2 binding is contributed to by the Ser but not by the Met residue. The data indicate that the amino acid differences in TM3 between CB1 and CB2 play a critical role in subtype selectivity for this class of compounds.     

Orphan G protein-coupled receptor GPR56 plays a role in cell transformation and tumorigenesis involving the cell adhesion pathway

GPR56 is an orphan G protein - coupled receptor, mutations of which have recently been associated with bilateral frontoparietal polymicrogyria, a rare neurologic disease that has implications in brain development. However, no phenotype beyond central nervous system has yet been described for the GPR56-null mutations despite abundant GPR56 expression in many non - central nervous system adult tissues. In the present study, we show that higher GPR56 expression is correlated with the cellular transformation phenotypes of several cancer tissues compared with their normal counterparts, implying a potential oncogenic function. RNA interference-mediated GPR56 silencing results in apoptosis induction and reduced anchorage-independent growth of cancer cells via increased anoikis, whereas cDNA overexpression resulted in increased foci formation in mouse fibroblast NIH3T3 cell line. When GPR56 silencing was induced in vivo in several xenograft tumor models, significant tumor responses (including regression) were observed, suggesting the potential of targeting GPR56 in the development of tumor therapies. The expression profiling of GPR56-silenced A2058 melanoma cell line revealed several genes whose expression was affected by GPR56 silencing, particularly those in the integrin-mediated signaling and cell adhesion pathways. The potential role of GPR56 in cancer cell adhesion was further confirmed by the observation that GPR56 silencing also reduced cell adhesion to the extracellular matrix, which is consistent with the observed increase in anoikis and reduction in anchorage-independent growth phenotypes. The oncogenic potential and apparent absence of physiologic defects in adult human tissues lacking GPR56, as well as the targetable nature of G protein - coupled receptor by small molecule or antibody, make GPR56 an attractive drug target for the development of cancer therapies.