2型糖尿病大鼠心肌IR、IRS-1的表达与罗格列酮及APP5肽类似物P165的干预效应

目的研究2型糖尿病大鼠心肌胰岛素信号转导通路蛋白胰岛素受体（IR）、胰岛素受体底物-1（IRS-1）的表达与正常SD大鼠的区别,并探讨进行罗格列酮及APP5肽类似物P165干预后对上述蛋白表达的影响。方法60只SD大鼠随机分为正常对照组（C组）、正常对照＋罗格列酮组（C＋RSG组）、2型糖尿病组（T2DM组）、2型糖尿病＋罗格列酮组（T2DM＋RSG组）、糖尿病给予P165小剂量组（T2DM＋P165小剂量组）、糖尿病给予P165大剂量组（T2DM＋P165大剂量组）,其中糖尿病动物采用高脂饮食后给予小剂量STZ腹腔注射的方法造模。后将各组SD大鼠处死,采用免疫组织化学染色和Western blot的方法检测心肌组织IR、IRS-1的表达。结果（1）2型糖尿病组（T2DM组）心肌组织IR、IRS-1的表达水平显著低于对照组（C组）;（2）2型糖尿病＋罗格列酮组（T2DM＋RSG组）心肌组织IR、IRS-1的表达水平显著高于T2DM组;（3）免疫组化染色发现2型糖尿病＋P165小/大剂量组（T2DM＋P165小/大剂量组）心肌组织IR、IRS-1免疫反应阳性颗粒沉着的累积光密度值显著高于T2DM组;Western blot结果显示T2DM＋P165小/大剂量组心肌组织IRS-1的表达水平显著高于T2DM组;而IR的表达水平与T2DM组相比无差别。结论（1）2型糖尿病大鼠心肌存在胰岛素抵抗或信号转导障碍;（2）罗格列酮干预后可以改善2型糖尿病心肌的胰岛素信号转导异常;（3）P165对2型糖尿病大鼠心肌胰岛素信号转导具有调节作用,其作用靶点可能为胰岛素受体底物。     

AdipoRon对2型糖尿病小鼠肾脏损伤的干预作用

目的：研究脂联素受体激动剂（AdipoRon）对2型糖尿病小鼠肾脏损伤的干预作用。方法：将40只SPF级雄性C57/BL6小鼠随机分为正常对照组（n=10）和实验组（n=30）：实验组给予高糖、高脂饲料喂养,联合腹腔注射小剂量链脲佐菌素（STZ）建立2型糖尿病（T2DM）小鼠模型,再随机分为3组（n=10）：模型对照（DM）组、低剂量AdipoRon治疗（DM+L）组及高剂量AdipoRon治疗（DM+H）组。检测血清中葡萄糖含量的变化;采用酶联免疫法检测小鼠血清中胰岛素受体（INSR）、胰岛素受体底物-1（IRS-1）以及肿瘤坏死因子-α（TNF-α）的蛋白质含量;HE染色镜下观察肾组织形态学变化;实时荧光定量PCR法检测肾组织胰岛素促进因子-1（PDX-1）和胰岛素（insulin）mRNA的表达;Western blot检测肾组织内磷酸化胰岛素受体底物-1（p-IRS-1）蛋白质;ELISA试剂盒检测小鼠血胰岛素含量。结果：病理学检查表明,AdipoRon可减轻2型糖尿病所致小鼠肾脏损伤。与DM组小鼠比较,DM+H组和DM+L组小鼠血糖、TNF-α水平均显著降低（P<0.05）,INSR、IRS-1和p-IRS-1表达显著上升,PDX-1和insulin mRNA表达显著上升（P<0.05,P<0.01）。结论：给予AdipoRon治疗的小鼠血糖和血清TNF-α水平显著降低,INSR,IRS-1和p-IRS-1蛋白质含量,PDX-1和insulin mRNA表达均显著上升,表明AdipoRon对2型糖尿病小鼠肾脏损伤有一定的干预作用。     

AdipoRon对2型糖尿病小鼠的治疗及其可能的肝脏机制

目的：观察口服脂联素受体激动剂（AdipoRon）对2型糖尿病小鼠的治疗效果及对肝脏的影响。方法：40只SPF级雄性C57/BL6小鼠随机分为正常对照组和实验组,实验组给予高糖高脂饲养联合腹腔注射小剂量链脲佐菌素建立二型糖尿病（T2DM）小鼠模型,随机分为模型对照（DM）组,低剂量AdipoRon治疗（DM+L）组,高剂量AdipoRon治疗（DM+H）组（n=10）。检测血清中丙氨酸转氨酶（ALT）、天门冬氨酸转氨酶（AST）、碱性磷酸酶（ALP）的变化;HE染色镜下观察肝细胞形态学变化;实时定量荧光PCR法检测肝脏中肝糖类相关基因（PEPCK）的表达。结果：与DM组小鼠比较,DM+H组和DM+L组小鼠ALT、AST、ALP、甘油三酯（TG）、葡萄糖（GLU）水平均降低（P<0.05）;与DM组小鼠比较,DM+H组小鼠和DM+L组小鼠血清游离脂肪酸（FFA）浓度显著下降（P<0.05）,而肝组织葡萄糖-6-磷酸酶（G-6-P）活性DM+L组小鼠显著下降,DM+H组小鼠无显著差异;与DM组小鼠比较,DM+H组小鼠肝组织磷酸烯醇式丙酮酸羧激酶（PEPCK） mRNA表达显著降低（P<0.05）,而DM+L组小鼠无显著差异。结论：给予AdipoRon治疗的小鼠血糖降低,ALT、AST、ALP的水平及G-6-P和PEPCK的表达下降,表明AdipoRon对2型糖尿病具有显著的治疗效果,对糖尿病小鼠肝脏有一定的保护作用。     

口服活性AdipoRon对2型糖尿病小鼠肝脏氧化应激的干预作用

目的：研究口服活性AdipoRon对2型糖尿病小鼠肝脏氧化应激是否有干预作用,为临床应用提供基础资料。方法：将健康雄性C57BL/6小鼠分为正常组（n=8）,糖尿病组（n=8）,AdipoRon高剂量治疗组（n=8）,Adi-poRon低剂量治疗组（n=8）,以高脂饲料喂养6周后腹腔注射40 mg/kg链脲佐菌素（STZ）诱导2型糖尿病模型,用高、低剂量的口服活性AdipoRon分别对治疗组灌胃治疗10 d后,检测相关生化指标,Western-blot法检测肝脏组织中IRS-1蛋白的表达;实时荧光定量PCR检测胰腺组织中PDX-1 mRNA的表达。结果：DM组小鼠血糖值明显高于NC组（P < 0.05）,DM+L组和DM+H组小鼠血糖值显著低于DM组。DM组小鼠肝脏组织中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性显著低于NC组（P < 0.05）,丙二醛（MDA）及一氧化氮合酶（NOS）活性显著高于NC组（P <0.05）;DM+L组和DM+H组SOD、CAT活性明显高于DM组（P < 0.05）,MDA及NOS活性显著低于DM组（P <0.05）。肝脏组织中IRS-1的蛋白表达及胰腺PDX-1 mRNA表达显著升高,存在统计学意义（P < 0.05）。结论：口服活性AdipoRon对糖尿病小鼠肝脏组织氧化应激有一定的干预作用,能降低小鼠的血糖水平。     

花生油对2型糖尿病大鼠海马CA1区神经生长因子及胆碱乙酰转移酶表达的影响

目的通过观察2型糖尿病大鼠海马CA1区神经生长因子（NGF）和胆碱乙酰转移酶（ChAT）表达的改变,研究花生油对2型糖尿病大鼠海马神经元NGF及ChAT表达的影响,探讨花生油在防治糖尿病脑病中的作用。方法 60只健康雄性SD大鼠随机分为4组：正常对照组（C组）、2型糖尿病组（T2DM组）、2型糖尿病给予2 mL花生油组（T2DM＋2 mL组）及2型糖尿病给予5 mL花生油组（T2DM＋5 mL组）。其中C组给予正常饮食,糖尿病组大鼠给予高脂饮食喂养,2个月后,按25 mg/kg体质量腹腔注射链脲佐菌素（STZ）制成2型糖尿病模型,T2DM组、T2DM＋2 mL组及T2DM＋5 mL组大鼠继续给予高脂饮食。糖尿病造模1个月后处死全部大鼠,行脑冰冻切片,用免疫组织化学方法检测各组大鼠海马CA1区NGF和ChAT的表达。结果 （1）T2DM组大鼠海马CA1区NGF表达比C组明显降低（P〈0.05）,T2DM＋2 mL组及T2DM＋5 mL组大鼠海马CA1区NGF表达均明显高于未给予花生油的T2DM组（P〈0.05）。（2）T2DM组大鼠海马CA1区ChAT表达显著低于C组（P〈0.05）,T2DM＋2 mL组和T2DM＋5 mL组大鼠海马CA1区ChAT表达均明显高于未给予花生油的T2DM组（P〈0.05）。结论 2型糖尿病大鼠海马CA1区神经生长因子表达降低,胆碱能神经元数量减少,这可能是2型糖尿病脑病发生的原因之一。花生油能增加2型糖尿病大鼠海马区内神经生长因子表达,促进胆碱能神经元存活,表明花生油具有一定的保护大鼠糖尿病脑病的作用。     

槟榔碱对2型糖尿病大鼠肝脏胰岛素抵抗的作用

目的：探讨槟榔碱对2型糖尿病大鼠肝脏胰岛素抵抗的影响及其机制。方法：采用高果糖饲料饲养Wistar大鼠12周制备2型糖尿病大鼠模型,实验动物随机分为5组（n=8）：对照组、模型组、模型＋不同浓度的槟榔碱（0,0．5,1,5mg／kg）组。4周后通过检测血糖、血脂、胰岛素、RT-PCR检测肝脏组成型雄甾烷受体（CAR）、孕甾烷x受体（PXR）、糖代谢相关基因：葡萄糖-6-磷酸酶（G6Pase）、磷酸烯醇式丙酮酸羧激酶（PEPCK）和炎症相关因子：白细胞介素-6（IL-6）、肿瘤坏死因子α（TNF-α）mRNA表达,Western blot检测大鼠肝内p-AKT和葡萄糖转运体4（GLUT4）蛋白表达。结果：1,5mg／kg槟榔碱显著降低糖尿病大鼠体重、空腹血糖、空腹胰岛素、血脂和糖代谢相关基因及炎症相关因子mRNA水平,提高CAR、PXR mRNA水平及p-AKT、GLUT4蛋白水平。结论：槟榔碱可能通过提高CAR和PXR的表达,导致肝脏糖代谢关键酶PEPCK、G6Pase基因表达或者炎性因子肿瘤坏死因子-α（TNF-α）、白介素-6（n-6）表达降低,改善2型糖尿病大鼠肝脏胰岛素抵抗。     

二氢杨梅素对2型糖尿病小鼠认知功能障碍的影响

目的：观察二氢杨梅素（DHM）对2型糖尿病（T2DM）小鼠认知功能障碍及海马中BDNF蛋白表达的影响。方法：将40只C57BL/6J小鼠首先随机分为两组：正常对照组（n=8）：普通饲料喂养;2型糖尿病模型组（n=32）：高糖高脂联合100 mg/kg的STZ处理（造模过程中死亡5只,不成功3只）。24只建模成功的小鼠随机分成3组：T2DM组、T2DM+L-DHM组和T2DM+H-DHM组,3组小鼠高糖高脂喂养,同时分别用等体积生理盐水、125 mg/（kg·d）的DHM和250 mg/（kg·d）的DHM （1次/天,灌胃）处理16周。正常对照小鼠继续普通饲料喂养,同时用等体积生理盐水（1次/天,灌胃）处理16周。16周后测定小鼠体重、空腹血糖、进行腹腔注射葡萄糖耐量实验和相关行为学实验。最后,Western blot检测各组小鼠海马中BDNF蛋白的表达。结果：高糖高脂联合100 mg/kg的STZ成功建立2型糖尿病小鼠模型。16周后,与正常对照组相比,T2DM组小鼠体重明显下降,空腹血糖显著升高,糖耐量显著异常;而T2DM+DHM组相比T2DM组小鼠体重却显著增加、空腹血糖降低,且H-DHM可显著改善T2DM小鼠糖耐量异常。行为学实验结果显示：与正常对照组相比,T2DM组小鼠学习记忆能力明显下降;与T2DM组相比,T2DM+DHM组小鼠学习记忆能力得到改善,且H-DHM组更为明显。Western blot结果显示：与对照组相比,T2DM组小鼠海马中BDNF蛋白表达显著下降,而DHM组相比T2DM组小鼠其BDNF蛋白的表达明显增加。结论：二氢杨梅素可改善2型糖尿病小鼠认知功能障碍,其机制可能通过降血糖作用,并激活海马中BDNF蛋白表达。     

西红花水提物对实验性糖尿病小鼠血糖水平的影响

目的：观察西红花水提物对链脲佐菌素（STZ）诱导的糖尿病小鼠血糖、血脂及胰腺组织的影响。方法：采用STZ （60 mg/kg）连续2 d腹腔注射建立糖尿病小鼠模型。将造模成功后的小鼠随机分为3组（n=10）：糖尿病模型（DM）组、西红花水提物（SE）组、阳性对照二甲双胍（MH）组。另取10只正常小鼠设为正常对照（NC）组。给药组每天灌胃1次,连续6周,模型组和正常对照组灌胃生理盐水。给药期间每周测定小鼠进食量、饮水量及体重,给药6周后测定空腹血糖（FBG）、口服糖耐量（OGTT）、糖化血清蛋白（GSP）、血清胰岛素（INS）和血脂等指标的变化情况;HE染色观察胰腺组织病理变化。结果：与NC组相比,DM组进食量、饮水量、线下曲线面积、FBG、GSP以及血脂中的总胆固醇（TC）均显著升高,空腹体重、血清胰岛素（INS）及高密度脂蛋白胆固醇（HDL-c）均显著降低;与DM组相比,SE组小鼠饮水量、FBG、线下曲线面积、TC显著降低,HDL-c以及INS显著升高。病理学显示DM组胰岛结构破坏、胰岛细胞数量明显减少、胰岛血管增生、形态不规则等变化,SE能明显修复受损胰腺组织。结论：SE对链脲佐菌素诱导的糖尿病小鼠有一定降血糖、降血脂作用,可以有效改善胰腺病变的情况,提示西红花可能用于糖尿病的防治。     

桑叶黄酮调控糖尿病模型小鼠心肌线粒体功能和纤维化进展的机制

摘要 目的：研究桑叶黄酮对糖尿病小鼠心肌线粒体功能和心肌纤维化的影响,并探讨其作用的具体分子机制。方法：45只ICR小鼠,随机分为3组,即正常组、模型组和桑叶黄酮组。模型组和桑叶黄酮组通过腹腔注射四氧嘧啶生理盐水溶液建立糖尿病模型,正常组小鼠腹腔注射生理盐水。桑叶黄酮组糖尿病小鼠在模型成功建立后灌胃给予桑叶黄酮（1.0 g/kg/天）治疗,正常组和模型组给予等量生理盐水治疗。治疗6周后,测定并比较三组小鼠血糖、血胰岛素、肝糖原、肝己糖激酶（HK）和丙酮酸激酶（PK）含量,心肌线粒体谷胱甘肽（GSH）、超氧化物歧化酶（SOD）、总钙和ATP含量,以及心脏CD31、α-SMA和Collagen I mRNA表达。结果：研究期间,模型组和桑叶黄酮组小鼠分别死亡3只和1只。经桑叶黄酮治疗6周后,糖尿病小鼠血糖显著降低,血胰岛素水平、肝糖原、肝HK和肝PK含量,心肌线粒体GSH、SOD、总钙和ATP含量均显著增高（P<0.05）。心肌纤维化指标：糖尿病小鼠心肌CD31 mRNA表达水平经桑叶黄酮治疗后显著增高,而α-SMA和Collagen I mRNA表达水平却显著降低（P<0.05）。结论：桑叶黄酮可显著降低糖尿病小鼠血糖水平,改善其糖代谢和心肌线粒体损伤,延缓心肌纤维化进展。     

口服AdipoRon对2型糖尿病小鼠脾脏和胰腺功能的影响

目的:观察口服AdipoRon对2型糖尿病小鼠脾脏和胰腺功能的影响,为AdipoRon的临床应用提供基础资料。方法:将40只C57/BL6雄性小鼠随机分为正常对照组(NC,n=10)和造模组(n=30),并分别给予普通饲料和高脂高糖饲料喂养。4周后,造模组小鼠腹腔注射链脲佐菌素(STZ,40 mg/kg)以诱导建立2型糖尿病模型。造模成功后将糖尿病模型小鼠随机分为糖尿病模型组(DM)、高剂量AdipoRon(50 mg/kg)(DM+H)组、低剂量AdipoRon(20 mg/kg)(DM+L)组,每组10只。DM+L组和DM+H组灌胃相应浓度的AdipoRon(使用去离子水溶解AdipoRon),NC组和DM组灌胃等体积去离子水,每日灌胃1次,灌胃10 d。末次干预后禁食12 h,处死小鼠取血液、胰腺和脾脏。HE染色光镜下观察胰腺的病理改变; ELISA法检测小鼠胰腺和脾脏组织中胰岛素受体(INSR)、胰岛素受体底物1(IRS-1)和肿瘤坏死因子-α(TNF-α)蛋白质含量;小鼠脾脏系数; Western blot法检测胰腺组织中pIRS-1蛋白质水平;实时荧光定量PCR检测胰腺组织中insulin mRNA表达。结果:光镜下可见正常组小鼠胰腺组织排列紧密、饱满、胰岛体积大,DM组小鼠胰腺组织排列较为疏散、胰岛体积较小,口服AdipoRon组小鼠胰腺组织基本紧密、饱满、胰岛体积略小。与NC比较,DM组小鼠胰腺和脾脏TNF-α水平明显升高,INSR、IRS-1水平均降低,脾脏系数、胰腺p-IRS-1蛋白质水平和insulin mRNA表达均降低,均具有统计学意义(P<0.05);与DM组比较,口服AdipoRon组小鼠胰腺和脾脏TNF-α水平明显下降,INSR和IRS-1水平均升高,脾脏系数升高,DM+H组胰腺p-IRS-1蛋白质水平和insulin mRNA表达均升高(P<0.05);与DM+L组比较,DM+H组小鼠TNF-α水平明显下降,INSR和IRS-1水平均升高(P<0.05)。结论:口服AdipoRon可通过减弱糖尿病小鼠炎症反应,上调INSR表达、提高p-IRS-1水平,从而对糖尿病小鼠脾脏和胰腺组织有一定的保护作用。     

KIT receptor activation by autocrine and paracrine stem cell factor stimulates growth of merkel cell carcinoma in vitro

The co‐expression of KIT receptor and its ligand stem cell factor (SCF) has been reported in biopsy specimens of Merkel cell carcinoma (MCC). However, the functional role of SCF/KIT in the pathogenesis of this aggressive tumor has not been elucidated. The present study reports expression and effects of SCF and KIT in the Merkel cell carcinoma cell line MCC‐1 in vitro. SCF and KIT were endogenously co‐expressed in MCC‐1 cells. Exogenous soluble SCF modulated KIT receptor mRNA and protein expression, stimulated growth of MCC‐1 cells, upregulated endogenous activation of KIT, AKT, and of extracellular signal‐regulated kinase (ERK) 1/2 signaling pathway. On the contrary, an inhibitory antibody that neutralized the KIT ligand binding site, reduced growth of MCC‐1 cells, as did high doses of the KIT kinase inhibitors imatinib and nilotinib. Also, inhibitors of KIT downstream effectors, U0126 that blocks MEK1/2 as well as wortmannin and LY294002 that inhibit phosphatidylinositol 3‐kinase‐dependent AKT phosphorylation, inhibited the proliferation of MCC‐1 cells. These data support the hypothesis that KIT is activatable by paracrine or autocrine tumor cell‐derived SCF and stimulates growth of Merkel cell carcinoma in vitro. Blockade of KIT and the downstream signaling cascade at various levels results in inhibition of Merkel cell carcinoma growth in vitro, suggesting targets for therapy of this cancer. J. Cell. Physiol. 226: 1099–1109, 2011. © 2010 Wiley‐Liss, Inc.     

RIN3 Is a Negative Regulator of Mast Cell Responses to SCF

Stimulation of the receptor tyrosine kinase KIT by Stem Cell Factor (SCF) triggers activation of RAS and its downstream effectors. Proper KIT activation is essential for the maturation, survival and proliferation of mast cells. In addition, SCF activation of KIT is critical for recruiting mast cells to sites of infection or injury, where they release a mix of pro-inflammatory substances. RIN3, a RAS effector and RAB5-directed guanine nucleotide exchange factor (GEF), is highly expressed and enriched in human mast cells. SCF treatment of mast cells increased the amount of GTP-bound RAB5, and the degree of RAB5 activation correlated with the expression level of RIN3. At the same time, SCF caused the dissociation of a pre-formed complex of RIN3 with BIN2, a membrane bending protein implicated in endocytosis. Silencing of RIN3 increased the rate of SCF-induced KIT internalization, while persistent RIN3 over-expression led to KIT down regulation. These observations strongly support a role for RIN3 in coordinating the early steps of KIT endocytosis. Importantly, RIN3 also functioned as an inhibitor of mast cell migration toward SCF. Finally, we demonstrate that elevated RIN3 levels sensitize mastocytosis cells to treatment with a KIT tyrosine kinase inhibitor, suggesting the value of a two-pronged inhibitor approach for this difficult to treat malignancy. These findings directly connect KIT activation with a mast cell-specific RAS effector that regulates the cellular response to SCF and provide new insight for the development of more effective mastocytosis treatments.     

Similar developmental patterns in immunolocalisation of stem cell factor and KIT in bovine meso- and metanephros

The mesonephros is often regarded as a simplified version of the terminal renal organ, the metanephros. Both renal organs result from an epithelio-mesenchymal interaction between the Wolffian duct and the nephrogenic ridge. It appears that the epithelio-mesenchymal interaction makes use of similar signal cascades for both renal organs and that key events required for the development of the metanephros occur at earlier stages. In murine metanephroi, the stem cell factor (SCF)/-KIT-signal transduction pathway has recently been shown to regulate ureteric bud branching and epithelial cell differentiation. We immunohistochemically defined the time-sequence of KIT and SCF presence in both renal organs using bovine embryos/foetuses with crown rump length (CRL) of 1.7–24 cm. In the mesonephroi, epithelial cells with strong KIT staining were scattered in distal tubules, and SCF was expressed in the epithelial wall of corpuscles and proximal tubules. KIT positivity occurred in the metanephroi of embryos prior to SCF; KIT was predominantly localised at the ureteric bud tips in the nephrogenic zone. In foetuses of 13 cm and more CRL, the SCF/KIT profile of developmentally advanced nephrons mirrored the situation in the mesonephros. Epithelial cells with strong KIT staining were scattered in the cortical areas of distal tubules, while SCF was expressed in the epithelial wall of corpuscles and proximal tubules. Our morphological findings agree with a potential role of KIT at the ureteric bud tips and demonstrate a similar expression of KIT and SCF along the areas of developmentally advanced mesonephric and metanephric nephrons.     

Fibronectin Combined with Stem Cell Factor Plays an Important Role in Melanocyte Proliferation,Differentiation and Migration in Cultured Mouse Neural Crest Cells

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild‐type mice, we previously demonstrated that KIT‐positive and/or L ‐3, 4‐dihydroxyphenylalanine (DOPA)‐positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT‐positive cells and DOPA‐positive cells were detected in the presence of SCF on ECM‐coated wells than on non‐coated wells. A statistically significant increase in DOPA‐positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA‐positive cells and KIT‐positive cells were detected on either the ECM‐coated or non‐coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA‐positive cells decreased with RGDS concentration in a dose‐dependent fashion. Immunohistochemical staining revealed the presence of integrin a5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.     

Structural basis for stem cell factor-KIT signaling and activation of class III receptor tyrosine kinases

Stem cell factor (SCF) binds to and activates the KIT receptor, a class III receptor tyrosine kinase (RTK), to stimulate diverse processes including melanogenesis, gametogenesis and hematopoeisis. Dysregulation of KIT activation is associated with many cancers. We report a 2.5 A crystal structure of the functional core of SCF bound to the extracellular ligand-binding domains of KIT. The structure reveals a 'wrapping' SCF-recognition mode by KIT, in which KIT adopts a bent conformation to facilitate each of its first three immunoglobulin (Ig)-like domains to interact with SCF. Three surface epitopes on SCF, an extended loop, the B and C helices, and the N-terminal segment, contact distinct KIT domains, with two of the epitopes undergoing large conformational changes upon receptor binding. The SCF/KIT complex reveals a unique RTK dimerization assembly, and a novel recognition mode between four-helix bundle cytokines and Ig-family receptors. It serves as a framework for understanding the activation mechanisms of class III RTKs.     

Dose-dependent and time-limited proliferation of cultured murine interstitial cells of Cajal in response to stem cell factor

Interstitial cells of Cajal (ICCs) play a role as pacemakers for gastrointestinal movement. Although some in vivo experiments showed that the c-kit receptor tyrosine kinase (KIT) and its ligand, stem cell factor (SCF), might be required for the development of murine ICCs near birth, in vitro experiments would be useful to clarify the role of SCF-KIT system for the development of ICCs. We attempted to establish a culture system in order to investigate the proliferation of ICCs. Murine gastrointestinal cells from embryos or neonates were cultured with SCF and stained with anti-KIT antibody and/or alcian-blue. The numbers of KIT+ cells a n d alcian-blue+ cells we re counted, and the number of KIT+.alcian-blue- cells, which represent ICCs was calculated. Clusters containing KIT+ cells were formed in culture. The number of KIT+.alcian-blue- cells from day-18 post coitum embryos increased in response to SCF up to a concentration of 50 ng/ml or for 8 days. The number of cells from day-2 post-partum neonates increased for 4 days, and then remained constant in the presence of SCF. In contrast, the number of cells from day-6 post-partum neonates did not increase and remained constant, even in the presence of SCF. ICCs showed a dose-dependent and time-limited proliferation in response to SCF in the in vitro culture system used here in.     

Role of c-KIT expression level and phosphatidylinositol 3-kinase activation in survival and proliferative responses of early myeloid cells

The receptor tyrosine kinase c-KIT and its ligand Stem Cell Factor (SCF) are critical in haemopoiesis but pathways linking receptor activation to specific responses in progenitor cells are still unclear. We have investigated the role of c-KIT expression level and the phosphatidylinositol 3-kinase (PI3-K) pathway in survival and cell division of early myeloid cells in response to SCF. Two factor-dependent murine early myeloid cell lines, FDC-P1 and Myb-immortalised haemopoietic cells (MIHC), were transduced to express wild-type c-KIT or a mutant form of the receptor (Y721F) that lacks the major recruitment site for the p85 regulatory subunit of PI3-K. Several clones expressing different receptor levels were analysed in each case. Growth of cells expressing either the wild-type or Y721F mutant KIT was strongly dependent on receptor level within the physiological range. Using an assay that allows quantitative measurement of the contributions of cell survival and cell division, diminished cell growth in response to SCF under limiting conditions of receptor copy number or PI3-K recruitment was shown to be almost entirely due to decreased cell survival. Further studies with the PI3-K inhibitor LY294002 indicated that PI3-K activation was also required for cell division. Alternate binding and/or indirect activation of PI3-K could support cell division mediated by Y721F mutant KIT, but was insufficient for the survival response.     

Alterations of the interstitial cells of Cajal and the microstructure of the gastrointestinal tract in KIT distal kinase mutant mice

The development and maintenance of interstitial cells of Cajal (ICC) are closely associated with SCF/KIT signal activity. In this study, we evaluate the distribution of ICC in KIT distal kinase domain mutant mice (Wads) and determine whether the loss-of-function mutations in KIT easily lead to gastrointestinal (GI) disorders. ICC were examined by anti-KIT immunohistochemistry and western blotting. The GI microstructure of wild-type (WT) and Wads mice in normal intestines and incomplete intestinal obstruction was evaluated by hematoxylin and eosin staining. The results in Wads^m/m mice were as follows. Myenteric ICC were obviously decreased in the stomach and colon and were totally absent in the small intestine. Intramuscular ICC were nearly absent in the stomach and irregularly distributed in the colon. Moreover, the smooth muscle thickness of the small intestine was increased 1.3-fold in Wads^m/m, compared to WT and Wads^m/+ mice and the diameter of the intestinal lumen was also enlarged in Wads^m/m mice. When constructing an incomplete intestinal obstruction model, the extent of distention involved was greater in Wads mice (1.6-fold in Wads^m/+ mice and 1.8-fold in Wads^m/m mice vs. WT mice). Meanwhile, the intestinal lumen expansion and decrease in ICC were more pronounced in Wads mice than in WT mice. Our results suggest that the KIT distal kinase domain mutation leads to an ICC loss in a subtype and location-specific pattern in Wads^m/m mice. The injury of the KIT signaling in mutant mice results in more serious pathological manifestations after being exposed to pathogenic factors.     

Structural basis for activation of the receptor tyrosine kinase KIT by stem cell factor

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4 interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.