mTORC1通过β-catenin信号通路调控RANKL/OPG和破骨细胞形成
|
摘要
第一部分mTORC1通过β-catenin信号通路调节RANKL/OPG和破骨细胞形成
一、研究背景
骨的代谢是一个动态过程,并随着机械应力、激素及细胞因子等的改变而维持动态的重建,包括骨形成与骨吸收,如此骨组织才具有正常的形态,发挥应有的功能。在骨代谢中骨细胞的主要作用是接收并传递信号给周围的成骨细胞(OB)和破骨细胞(OC),从而启动骨重建。OC主要负责吸收局部骨组织,于此同时OB则形成新骨以替代被吸收的骨组织。OC是由骨髓造血干细胞中的单核/巨噬细胞系分化而成的多核细胞,其活性受多种细胞因子和激素的调节,但具体的机制不清楚。
骨保护素(osteoprotegerin, OPG)、核因子KB受体活化因子(receptor activator of nuclear factor—kappaB, RANK)及核因子KB受体活化因子配体(receptoractivator of nuclear factor—kappa B ligand, RANKL)系统是自1997年以来发现的一组调控破骨细胞分化激活的细胞因子,三者均属肿瘤坏死因子受体超家族(tumomecrosis factor receptor superfamily, TNFRSF)。成骨细胞及骨髓基质细胞表达RANKL,与破骨细胞前体细胞或破骨细胞表面上的RANK结合后,通过激活调节破骨细胞形成的转录因子,从而促进破骨细胞的形成和分化,并抑制破骨细胞的凋亡。由成骨细胞分泌的OPG可以与RANKL结合,竞争性抑制RANKL与RANK之间的结合,从而抑制破骨细胞的增殖和分化,并最终抑制骨吸收。
越来越多的证据证明许多激素、细胞因子直接或间接地调节RANK/RANKL/OPG系统,从而介导破骨细胞的分化和功能,导致多种骨代谢疾病,包括骨质疏松、糖皮质激素导致的骨丢失、多发性骨髓瘤以及风湿性关节炎等。另外,靶向OPG/RANK/RANKL系统的药物比如外源性的OPG、抗RANKL抗体、RANK—Fc等被证实是有效预防和治疗包括骨质疏松、骨肿瘤的骨溶解疾病。
哺乳动物雷帕霉素靶蛋白(mammalian Target Of Rapamycin, mTOR)是一种保守的丝/苏氨酸蛋白质激酶。在细胞多种生理活动的调控中处于核心地位,接受并整合细胞内外的各种刺激如激素、生长因子、营养、能量、氧气、应激等调控着包括基因转录、蛋白质起始翻译、核糖体生物合成、自噬等过程。人类重大疾病如癌症、心血管疾病、移植排斥及自体免疫紊乱、糖尿病、肥胖、神经系统疾病等均涉及mTOR信号的失调,mTOR信号通路成为近年基础与临床研究的热点。
近年来越来越多的文章报导mTOR信号通路参与骨代谢的调节。mTOR/S6K1通过调节il-6和VEGF的合成,进而调节骨形成。也有文章报导,雷帕霉素(rapamycin)增加成骨样细胞的OPN和OCN的mRNA的表达,通过抑制mTOR活性并激活bmp/smad通路促进人胚胎干细胞的成骨分化。另外,BEZ235作为PI3K和mTOR通路的抑制剂能够促进HMSCS向成骨细胞分化。尽管雷帕霉素对成骨细胞分化的影响仍然存在争论,但目前普遍认为抑制mTORC1信号通路可以促进成骨细胞分化。另外,mTOR的激活是破骨细胞前体以及成熟破骨细胞存活所必需的,有文章指出促红细胞生成素(EPO)能激活mTOR的活性,并分别通过上调NFATcl和下调组织蛋白酶K,增加破骨细胞的数量和降低破骨细胞的活性,而Rapa能够抑制EPO的作用。尽管mTOR在骨代谢中的作用被肯定,但是其具体的机制仍然不明了。
最近发现转录因子CCAAT/enhancer binding protein beta (C/EBPβ)在骨代谢中发挥重要作用。结合转基因小鼠、细胞生物学和药理学方面的研究,发现它不仅可以调控成骨细胞的活性,也可以调节破骨细胞的分化和活性。鉴于RANK/RANKL/OPG系统在骨代谢中的重要作用,特别是对破骨细胞的调控,我们猜想1mTOR是否能通过C/EBPβ调控成骨细胞及其前体释放RANKL/OPG,从而调节破骨细胞的分化和功能。目前,mTOR信号通路对RANKL/OPG的调控仍然存在较大争议,对其中的机制研究尚未见报道。
二、研究结果
1. mTORC1参与去卵巢诱导的破骨细胞形成和骨吸收过程
雌鼠去卵巢可以降低雌激素水平,诱导骨质疏松。首先,将8-12周龄的C57B6小鼠共24只,随机分四组,分别为假手术组(Sham)、去卵巢组(OVX)、假手术+雷帕霉素组、去卵巢+雷帕霉素组。小鼠引颈处死后取材,micro-CT检测分析、骨组织形态计量学分析、骨钙素免疫组化和破骨细胞染色检测表明：在去卵巢组小鼠中,破骨细胞活性增强,骨吸收增加；单纯灌胃雷帕霉素组小鼠破骨细胞活性受抑制,骨吸收减少；此外,雷帕霉素可以逆转去卵巢引起的小鼠骨质疏松。碱性磷酸酶(ALP)和酒石酸酸性磷酸酶(TRAP)分别是成骨细胞分化和破骨细胞分化的特征性外酶,取四组小鼠的血清进行生物化学检测,结果表明抑制mTORC1会增加ALP/TRAP的比例(数据未给出)。
我们进一步检测mTORC1在以上样品中的活性,发现在雷帕霉素组和雷帕霉素+去卵巢组的小鼠标本中,骨吸收的增加都伴随着mTORC1的下游信号分子S6的磷酸化活性降低,说明抑制mTORC1能降低破骨细胞的活性,促进骨吸收过程。
2. mTORCl活性增加下调RANKL的表达
为了进一步验证mTORC1对RANKL表达水平的调节,我们通过体外培养小鼠骨髓基质细胞系OP9、大鼠原代骨髓间充质干细胞BMSC,用mTORC1抑制剂雷帕霉素处理细胞,分别在mRNA水平和蛋白水平检测RANKL的表达,结果表明抑制mTORC1会导致RANKL表达水平的显著下调(P=0.000)。mTORC1的上游抑制基因——结节性硬化症复合物(tuberous sclerosis complex, TSC)由TSC1和TSC2组成,它们二者任一缺失都会导致mTORC1的过度活化,为了进一步验证mTORC1对RANKL的调控作用,我们体外培养TSC2未敲除和TSC2敲除的小鼠胚胎成纤维细胞系MEFTSC2+/+和MEFTSC2-/-,分别在mRNA水平和蛋白水平检测RANKL的表达,发现1mTORC1过度活化可以显著增加RANKL的表达水平(P=0.000)。为了进一步验证以上结果,我们同时用TSC2的siRNA体外敲除OP9细胞系的TSC1(P=0.024)。以上结果均确定,mTORC1对RANKL的调控作用。
3. mTORC1活性增加上调OPG的表达
破骨细胞形成和骨吸收过程受RANKL/OPG系统的调控,除了检测RANKL水平受mTORC1调节的现象,我们同时在体外培养的上述细胞系中分别检测了OPG的mRNA表达水平和蛋白表达水平,结果表明mTORC1活性被雷帕霉素抑制后会导致OPG表达水平的上调(P=0.004,P=0.000),在MEFTSC2+/+和MEFTSC2-/-细胞系和TSC2siRNA干扰实验中均证明OPG的表达受mTORC1负调节(P=0.000,P=0.000)。以上数据均通过统计学检验方法检验,并具有显著性差异。
4. mTORC1调节RANKL/OPG的转录活性
由于mTORC1活性调节了RANKL/OPG的表达水平,我们通过荧光素酶报告系统来进一步验证mTORC1是否调节了RANKL/OPG的转录活性。我们通过体外转染含有荧光素酶报告基因的质粒给OP9细胞,并用mTORC1抑制剂雷帕霉素处理,通过荧光素酶报告基因检测仪检测和分析证实：抑制mTORC1活性会显著抑制RANKL的转录活性(P=0.000),和增强OPG的转录活性(P=0.011)。以上数据均通过统计学检验方法检验,并具有显著性差异。
5. mTORCl通过抑制Akt降低β-catenin的mRNA的稳定性
在上述结果中,我们证实TSC缺失导致的mTORC1过度活化可以引起RANKL/OPG的比例降低,从而抑制骨吸收。
在前期实验中,我们用雷帕霉素体外处理OP9细胞系,通过mRNA芯片筛选出其中表达差异显著的转录因子之一：C/EBPp (p-catenin),介于目前已报导的β-catenin对RANKL/OPG和骨代谢的调节作用,我们推测mTORC1通过对mTORC1/2负反馈途径的下游分子Akt磷酸化的调节来调控β-catenin,进而调节RANKL/OPG的比例。
我们通过体外培养大鼠骨髓间充质干细胞BMSC、小鼠骨髓基质细胞系OP9,用雷帕霉素处理细胞和敲除细胞的TSC基因,得到mRNA水平和蛋白水平检测结果符合我们的预期：抑制mTORC1活性,会引起β-catenin的表达上调(P=0.000),同时伴随1mTORC2下游信号分子Akt的Ser473位点磷酸化的活性增强。为了进一步验证,我们通过用显著性抑制性Akt的腺病毒(Dominant negative Akt Ad virus)感染骨髓间充质干细胞,并用雷帕霉素处理细胞,结果证实：当Akt被抑制时,能完全抵抗mTORC1活性受阻所引起的β-catenin的表达。这说明,1mTORC1很可能是通过Akt来维持β-catenin的表达稳定性的。以上数据均通过统计学检验方法检验,并具有显著性差异。
6. mTORC1通过β-catenin信号通路调节RANKL/OPG
根据1mTORC1对RANKL/OPG的调节作用和β-catenin对骨代谢的调控作用,我们通过体外实验对以上分子机制进行探讨,当抑制β-catenin信号时,mTORC1对RANKL/OPG的影响,从而验证mTORC1能通过β-catenin来调节RANKL/OPG.我们对体外培养的细胞用同种属的β-catenin的siRNA干扰实验来敲除β-catenin的表达,同时用雷帕霉素处理细胞,所得结果证明：当β-catenin被抑制时,mTORC1失去了对RANKL/OPG的调控作用。
三、结论
综上所述,我们的研究揭示了一条新的涉及mTORC1、p-catenin、Akt与RANKL/OPG系统对骨代谢调节的信号转导通路。通过这条通路,mTORC1被抑制,进而通过mTORC1/2负反馈抑制途径激活Akt (Ser473)的磷酸化,激活的磷酸化Akt通过减少β-catenin的降解来改变RANKL/OPG的比例,进而调控破骨细胞的发生和骨吸收过程。mTORC1-Akt-β-catenin-RANKL/OPG途径对骨代谢的调控作用为雷帕霉素治疗骨质疏松病人的分子机制增添了新的内容。目前,我们通过可诱导的全身性敲除TSC1/Raptor和成骨细胞特异性敲除TSC1/Raptor的转基因小鼠,对于mTOR成为治疗骨质疏松的靶点及其治疗效果进行进一步的研究。
第二部分去甲二氢化愈创木酸(NDGA)以mTORC1为靶点抑制乳腺癌的生长
一、研究背景
去甲二氢化愈创木酸Nordihydroguaiaretic acid (NDGA)是一种来源于石炭酸灌木Larrea divaricatta的天然的酚类化合物,目前文献报道它在体外和体内均具有抗肿瘤活性。据许多临床研究报道得知,NDGA(去甲二氢化愈创木酸)的许多化学同型物正被开发为难治性实体瘤的临床药物。虽然NDGA(去甲二氢化愈创木酸)具有很好的临床药用价值,但其发挥抗肿瘤作用的分子机制目前仍然没有得到清晰的解释,这对其能得到广泛应用是一个极大的限制。
哺乳动物雷帕霉素靶蛋白(mammalian Target Of Rapamycin, mTOR)是一种保守的丝／苏氨酸蛋白质激酶。在细胞多种生理活动的调控中处于核心地位,接受并整合细胞内外的各种刺激如激素、生长因子、营养、能量、氧气、应激等调控着包括基因转录、蛋白质起始翻译、核糖体生物合成、自噬等过程。人类重大疾病如癌症、心血管疾病、移植排斥及自体免疫紊乱、糖尿病、肥胖、神经系统疾病等均涉及mTOR信号的失调,mTOR信号通路成为近年基础与临床研究的热点。在本实验室前期的研究成果中发现,不饱和脂肪酸花生四烯酸(Arachidonic acid, AA)及其代谢产物能以mTORC1为靶点促进乳腺癌的发生发展,而花生四烯酸的代谢途径之一脂氧合酶途径在其中发挥了重要作用,NDGA(去甲二氢化愈创木酸)做为一种强抗氧化剂是抑制脂氧合酶途径的重要物质。我们推测mTORC1能作为NDGA(去甲二氢化愈创木酸)具有抗肿瘤和抑制乳腺癌生长作用的分子机制的潜在靶点。
在我们的研究中,我们发现并证明了哺乳动物雷帕霉素靶标复合物1(mTORC1)是NDGA(去甲二氢化愈创木酸)的一个靶点,这个结论在体外培养的乳腺癌细胞和体内移植瘤小鼠身上都得到了验证。
二、研究结果
1. NDGA(去甲二氢化愈创木酸)在乳腺癌细胞中抑制mTORC1而不是mTORC2
我们体外培养三种乳腺癌细胞系,在不同时间点(时间梯度)给细胞加入同一浓度的NDGA(去甲二氢化愈创木酸)和在同一时间点给细胞加入不同浓度(浓度梯度)的NDGA(去甲二氢化愈创木酸),药物处理完成后,我们通过用免疫印迹(western blot)检测mTORC1和mTORC2下游途径信号分子的表达水平和活化水平来验证NDGA(去甲二氢化愈创木酸)的作用。
我们发现NDGA(去甲二氢化愈创木酸)能抑制mTORC1的活性而不是mTORC2的活性,并具有一定的时间梯度和浓度梯度。
说明NDGA(去甲二氢化愈创木酸)能特异性地以mTORC1而不是mTORC2为靶点而发挥作用。
2. NDGA(去甲二氢化愈创木酸)抑制mTORCl下游信号分子和抑制乳腺癌细胞的增殖
细胞周期蛋白D1(cyclinD1),缺氧诱导因子(HIF-α),血管内皮生长因子(VEGF)都是mTORC1下游重要的信号分子,它们的表达水平直接受到mTORCl活性的调节并与由于mTORC1过度活化导致的肿瘤发生发展具有重要关系。我们在体外培养的乳腺癌细胞中证明,NDGA(去甲二氢化愈创木酸)能抑制以上信号分子的表达水平,并在体外培养的条件下,抑制不同的乳腺癌细胞系的增殖水平(P=0.000)。
以上数据同样提示了mTORC1作为NDGA(去甲二氢化愈创木酸)的靶标,对于乳腺癌的治疗具有重要意义。
3. NDGA(去甲二氢化愈创木酸)抑制mTORC1通过部分活化AMPK/TSC2
AMP激活蛋白激酶(AMPK)和结节性硬化症复合物2(TSC2)都是mTORC1上游的抑制基因。能量缺乏和其它多种信号刺激或药物都能引起AMPK的磷酸化从而抑制mTORC1活性；活化的AMPK能直接磷酸化TSC2并抑制mTORC1活性。为了探讨NDGA(去甲二氢化愈创木酸)作用mTORC1的分子机制,我们在体外敲除乳腺癌细胞的AMPK或TSC2发现,NDGA(去甲二氢化愈创木酸)能部分通过AMPK或TSC2途径来抑制：mTORC1的活性。
4. NDGA(去甲二氢化愈创木酸)抑制氨基酸和胰岛素引起的mTORC1活化,并破坏mTOR-Raptor相互作用
胰岛素对mTORC1信号通路的激活是依赖于TSC途径的,而氨基酸对mTORC1信号通路的激活是不依赖于TSC2的。有趣的是,NDGA(去甲二氢化愈创木酸)不但能抑制氨基酸引起的mTORC1活化,它同样能抑制胰岛素引起的mTORC1活性升高。因此,我们对NDGA(去甲二氢化愈创木酸)是否能直接作用mTORC1复合物本身进行验证。
(Rapamycin)一样干扰mTOR-Raptor直接的相互作用并能抑制mTORC1的体外激酶活性。这个结果让我们明确,NDGA(去甲二氢化愈创木酸)抗肿瘤的分子机制,可能是直接以mTORC1为靶标来发挥作用的。
5. NDGA(去甲二氢化愈创木酸)以mTORCl为靶点抑制乳腺癌荷瘤小鼠肿瘤的生长
体外水平的实验结果给了我们很好的提示,因此我们建立了乳腺癌体外移植瘤动物模型来验证NDGA能否以mTORC1为靶点,并抑制乳腺癌的生长。
我们用NDGA(去甲二氢化愈创木酸)腹腔注射体外移植乳腺癌细胞的裸鼠,给药期间测量肿瘤大小,给药一个月后取材,测量,称重,鉴定。结果证明,NDGA(去甲二氢化愈创木酸)在体内水平能有效抑制乳腺癌的生长,并显著下调mTORC1信号通路的下游信号分子的活性。以上数据均通过统计学检验方法检验,并具有显著性差异,P=0.000。
三、结论
综上所述,清楚NDGA(去甲二氢化愈创木酸)抑制肿瘤发生发展过程的分子机制对临床应用具有重要的意义。我们的研究揭示了一条新的NDGA(去甲二氢化愈创木酸)以(?)mTORC1而不是mTORC2为靶标的治疗乳腺癌发展的信号转导通路。通过这条通路,NDGA(去甲二氢化愈创木酸)能抑制mTORC1活性和乳腺癌细胞的增殖；能部分通过AMPK/TSC2发挥抑制作用；NDGA(去甲二氢化愈创木酸)能打破mTOR和Raptor的相互作用并抑制(?)mTORC1的体外激酶反应；NDGA(去甲二氢化愈创木酸)能以mTORC1为靶点并显著抑制乳腺癌体外移植瘤小鼠的乳腺癌的发展。这为NDGA(去甲二氢化愈创木酸)治疗乳腺癌,发挥抗肿瘤作用的分子机制增添了新的内容。
PART ONE
mTORCl regulates RANKL/OPG and osteoclastogenesis through β-catenin signaling pathway
Background
Bone remodeling is in a dynamic process where bone resorption and bone formation take responsibilities. Under internal or external stimuli such as mechanical strength, hormones, cytokines, the variational activities of osteoblasts and osteoclasts would maintain coordinated balance in healthy adults.
The osteoclast, which takes responsibilities for local bone resorption is derived from hematopoietic stem cells through myeloid progenitor cells to mono/macrophage and into osteoclast lineage. The molecular mechanism of the activation of osteoclast activity is not clear. Osteoclast differentiation and activation are considered to be under the regulation of RANK/RANKL/OPG system since1997. Receptor activator of nuclear factor-kappaB (RANK), receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG) is belongs to tumornecrosis factor receptor super-family (TNFRSF). Osteoblast and bone marrow stromal cells both express RANKL, while they bind to RANK which express on the surface of osteoclast progenitors and mature osteoclast, they will promote the formation and differiation of osteoclast and inhibit the apoptosis through activating the transcriptional factor. OPG is secreted by osteoblast and able to bind to RANKL, which competitively inhibit the combining of RANKL and RANK, as a result of the suppression of osteoclast proliferation, differentiation and bone resorption.
At present, several evidences demonstrate that multiple hormone, cytokines can regulate RANK/RANKL/OPG system directly or indirectly, lead to many bone metabolism diseases, including osteoporosis, bone loss induced by glucocorticoids, multiple myeloma, rheumatoid arthritis and so on. Beside these, which target OPG/RANK/RANKL system such as exogenetic OPG, anti-RANKL antibody and RANK-Fc have been confirmed as potent and effectual medicines to prevent and treat osteolytic diseases.
Mammalian target of rapamycin (mTOR), is a protein Ser-Thr kinase that functions as a central element in a signaling pathway involved in the control of cell growth and proliferation. Disregulation of mTOR signaling associated to human major diseases, for instance, cancer, cardiovascular diseases, graft rejection and autoimmune diseases, diabetes, adiposity, nerve system diseases. mTOR signaling pathway have been become to a certain target in recent years.
mTOR signaling pathway take part in the regulation of bone metaboslism is definitely confirmed:mTOR/S6K1regulates il-6and VEGF to affect bone formation; rapamycin promote mRNA expression of OPN and OCN in osteoblast-like cells, and promote osteogenic differentiation of human embryonic stem cell through inhibit mTOR activity and activate bmp/smad pathways. What's more, BEZ235as an inhibitor of PI3K/mTOR pathway, is able to stimulate human bone marrow mesenchymal stem cells (hMSC) differentiate to osteoblast. Rapamycin promote osteoblast differentiation is still in argument, but most consideration about mTORC1signaling pathway inhibition can promote osteoblast formation were supported. On the other hand, mTOR activation is necessary for the survival of osteoclast progenitor cells and mature osteoclast. Someone pointed out erythropoletin hematopoietin (EPO) can activate mTOR and up-or down-regulate NFATcl or cathepsin k to enhance osteoclast formation and decrease osteoclast activity, however rapamycin can reverse these. Despite mTOR has been considered as a regulator in bone metabolism, the concrete mechanism is still unclear.
Recently published articles have found that the transcriptional factor CCAAT/enhancer binding protein beta (C/EBPβ) plays a key role in bone metabolism. C/EBPβ not only regulates osteoblast activity, but also regulates osteoclast differentiation and activity under the investigation on transgenic mice, cell biology and pharmacology. In view of the importance of RANK/RANKL/OPG system, especially the regulation of osteoclast, we considered that wether mTOR can control osteoblast and its progenitors to secrete RANKL/OPG via C/EBPβ, and modulate osteoclast differentiation and function.
Results
1mTORCl is involved in OVX-induced osteoclastogenesis and bone resorption
Ovariectomized (OVX) female mice will keep low estrogen level and lead to osteoporosis. Firstly, we randomly grouped24C57B6mice into four, respectively, including Sham, OVX, Sham+rapamycin, OVX+rapamycin. Mice were executed then detected and analysed bone samples for bone mineral density (BMD), micro-CT, bone histomorphometry, osteocalcin immunohistochemistry and osteoclast tartaric acid and acid phosphatase (TRAP) staining. These data demonstrated that, osteoclast activity and bore resorption enhanced in OVX mice, and osteoclast activity and bone resorption decreased in rapamycin-treated mice, besides this, rapamycin can dominantly rescure osteoporosis in OVX mice.
Alkaline phosphatase (ALP) and tartaric acid and acid phosphatase (TRAP) are respectively the characterized exoenzymes of osteoblast and osteoclast differentiation. We collected serum of four groups mice for biochemical detection and found that mTORC1inhibition will increase ALP/TRAP (data not shown). We further assay mTORC1activity in above samples, as a result of activated mTORC1lead to activated bone resorption progression.
2RANKL expression is positively regulated by mTORCl activity
To confirm mTORC1regulate RANKL expression level, we in vitro culture mouse bone marrow stromal cell line OP9, rat primary bone marrow stem cell BMSC and treated cells with mTORCl inhibitor rapamycin, then detected mRNA and protein expression levels of RANKL, the data showed us mTORC1inhibition dominantly down-regulate RANKL expression level(P=0.000).
mTORCl upstream suppressor------Tuberous Sclerosis Complex (TSC) is organized with TSC1and TSC2, either of them lost will over-activate mTORCl. In orter to confirm mTORC1take part in RANKL regulation, we cultured mouse embryo fibroblast cell lines MEFTSC2+/+and MEFTSC2-/-, and use TSC2siRNA to knockdown TSC1in OP9cell line, then detected RANKL mRNA and protein expression levels(P=0.000, P=0.024). All data were detected and analysed by statistical method, and with significant differences.
3OPG expression is negatively regulated by mTORCl activity
Osteoclastogenesis and bone resorption is regulated by RANKL/OPG system.
Except for measuring mTORCl regulates RANKL, we have also detected OPG expression on mRNA and protenin level in above cell lines. Data showed us mTORC1activity is suppressed by rapamycin while up-regulating OPG expression(P=0.004, P=0.000), and in embryo fibroblast cell lines or TSC2knockdown OP9cell line, OPG expression is denifitely negatively regulated by mTORC1(P=0.000, P=0.000). All data were detected and analysed by statistical method, and with significant differences.
4mTORCl regulates transcriptional activity of RANKL/OPG
We next investigated how mTORC1regulated the expression of RANKL/OPG. Luciferase reporter gene system is a useful system for detecting transcriptional factor activity. To determine the role of mTORC1activity in RANKL/OPG regulation, we use luciferase reporter gene plasmids which have been constructed with RANKL or OPG genes, and companied by rapamycin treatment in OP9cell line. These results suggest that mTORC1activity is required for RANKL and OPG transcriptional activity. All data were detected and analysed by statistical method, and with significant differences (P=0.000, P=0.011).
5mTORC1decreases mRNA stability of beta-catenin via inhibition of Akt
In previous experiments, a screening test via mRNA chip to find a signifinantly variable transcriptional factor:C/EBPβ (β-catenin), in rapamycin-treated OP9cell line. β-catenin regulated RANKL/OPG has been shown recently, we supposed mTORC1is able to regulate RANKL/OPG through β-catenin signaling pathway while affacting the phosphorylation level of Akt (Ser473), which is a downstream signal of mTORC2.
Indeed, we found that mTORC1activity inhibition raised an induction of β-catenin expression, meanwhile, mTORC2downstream signal Akt (Ser473) was phosphorylated enhanced in rapamycin-treated or TSC knockdowning cell lines.
To further investigate mTORC1/Akt/β-catenin signaling pathway, we use dominant negtice Akt adenovirus to knockdown Akt activity, then treat cells with rapamycin. The data show us when Akt was totally blocked, β-catenin expression is not affected by mTORCl activity. We proposed that mTORC1may decreases mRNA stability of β-catenin via Akt inhibition. All data were detected and analysed by statistical method, and with significant differences.(P=0.000)
6mTORCl regulates RANKL/OPG through beta-catenin signaling
As we known, mTORC1and β-catenin mainly in the regulation of RANKL/OPG system, we performed in vitro measuring the molecular mechanism. Interestingly, β-catenin siRNA knockdown interrupted the regulation of mTORC1to RANKL/OPG expression level.
Conclusion
In summary, our study reveals a novel regulatory pathway that involves RANKL/OPG system and bone resorption progress. Through this novel pathway, the key regulator Akt drives β-catenin to regulate RANKL/OPG balance dependent of mTORC1. The activated Akt in turn increases mRNA stability of β-catenin through a mechanism involved mTORC1activation to reduce RANKL secretion and induce OPG expression, finally regulate osteoclastogenesis and bone resorption.
mTORCl-Akt-catenin-RANKL/OPG pathway is meaningful to provide a new molecular mechanism to treat osteoporosis patient with rapamycin. At present, we determine the further investigation using inducible systemic-specific knockout TSC1/Raptor transgenic mice and osteoblast-specific knockout TSC1/Raptor mice, to confirm mTORC1as a key regulator in osteoclastogenesis and bone metabolism.
PART TWO
mTORCl is a target of nordihydroguaiaretic acid to prevent breast tumor growth in vitro and in vivo
Background
Nordihydroguaiaretic acid (NDGA) is a natural phenolic compound isolated from the creosote bush Larrea divaricatta that has anti-tumor activities both in vitro and in vivo. Its analogues are in clinical development for use in refractory solid tumors. But the mechanisms underlying the anti-cancer effect of NDGA are not fully understood. In this study, we identified mammalian target of rapamycin complex1(mTORCl) as a target of NDGA both in cultured breast cancer cells and in xenograft models.
Mammalian target of rapamycin (mTOR), is a protein Ser-Thr kinase that functions as a central element in a signaling pathway involved in the control of cell growth and proliferation. Disregulation of mTOR signaling associated to human major diseases, for instance, cancer, cardiovascular diseases, graft rejection and autoimmune diseases, diabetes, adiposity, nerve system diseases. mTOR signaling pathway have been become to a certain target in recent years. In our previous research data we have found that unsaturated fatty acid arachidonic acid (AA) and its metabolites can target mTORC1and promote breast cancers. Lipoxygenase pathway is a critical way in arachidodic acid metabolism, NDGA as a strong antioxidant against to lipoxygenase and dominantly inhibits arachidonic acid production. We suppose that mTORC1can be a potent target of NDGA in prevention of breast cancer growth and carcinogenesis.
Together our data provide a novel mechanism for NDGA activity which could help explain its anti-cancer activity. NDGA repressed breast tumor growth and targeted mTORC1and its downstream signaling in vitro and in vivo.
Results
1NDGA inhibits mTORCl in breast cance cells
To investigate the potential effect of NDGA on mTOR signaling in breast cancer cells, we cultured several breast cancer cell lines in vitro and treated them with NDGA, we found that NDGA effectively suppressed basal levels of phosphorylation of S6(S235/236) and4E-BP1(downstream target of S6K1and mTORC1) in a dose-and time-dependent manner in all tested cells. NDGA can particularly target mTORC1but not mTORC2.
2NDGA inhibits mTORCl downstream signaling and suppresses proliferation in breast cancer cells
Cyclin D1, HIF-α and VEGF are three key mTORC1downstream signaling molecules whose expression are positively regulated by mTORC1and responsible for mTORCl overactivation-related carcinogenesis and progression of cancer. we next examine the effect of NDGA on Cyclin D1, HIF-α and VEGF protein levels in MCF-7cells.
Taken together, these results further confirmed mTORC1signaling is a target of NDGA and is involved in prevention of breast cancer proliferation. All data were detected and analysed by statistical method, and with significant differences.(P=0.000)
3NDGA inhibits mTORCl in part through activation of AMPK/TSC2
AMP-activated protein kinase (AMPK) and tuberous sclerosis complex1/2(TSC1/2) are upstream signals that negatively regulate mTORCl. Energy starvation and various stimuli/drugs have been shown to induce phosphorylation and activation of AMPK, activated AMPK phosphorylates TSC directly and inhibit mTORC1. To understand the mechanism through which NDGA inhibits mTORCl, the roles of AMPK/TSC in this process were examined. Interestingly, NDGA increased phosphorylation of AMPK, and knockdown of AMPK or TSC2by siRNA, partially repressed NDGA-induced mTORC1activity decreased.
4NDGA suppresses amino acid-and insulin-stimulated mTORCl activity and disrupts mTORCl complex
We next investigated how NDGA regulated mTORCl. Our result that deletion of TSC2could not diminish the inhibitory effects NDGA against mTORC1implicates additional mechanisms are involved. We thus examined the effects of NDGA on insulin-and amino acids-stimulated mTORC1, which simulates mTORC1in TSC2-dependent and independent mechanism respectively. Interestingly, both amino acids and insulin-stimulated mTORC1activity were repressed by NDGA, these data implicate NDGA may target mTORC1directly.
Assembling and stability of the mTORC1determines its kinase activity. Surprisingly, NDGA did not stabilize mTOR-Raptor interaction under either presence or absence of amino acids conditions, however, it acted like rapamycin to disrupt the mTOR-Raptor complex and inhibited mTORC1in vitro kinase activity, whereas mTOR-Rictor association remained unchanged. Taken together, these results implicate that disruption of mTOR-Raptor complex by NDGA may contribute to its inhibitory effects against mTORC1and mTORC1may be a direct target of NDGA.
5NDGA inhibits breast tumor growth and targets mTORCl in xenograft
We next asked whether in vivo administration of NDGA inhibits mTORC1activity and prevents breast tumor growth in xenograft. Nude mice bearing MDA-MB-231breast tumor xenografts were dosed with saline or NDGA in a month, the NDGA therapy as single agent markedly inhibited breast tumor growth, most importantly, in vivo administration of NDGA remarkably reduced the levels of mTORC1downstream signals in the breast tumor xenograft. Suggesting that NDGA could inhibit mTORC1and its downstream signaling in vivo. All data were detected and analysed by statistical method, and with significant differences (P=0.000). These results identified mTORC1as a target of NDGA in vivo.
Conclusion
In summary, understanding the mechanisms by which NDGA suppress carcinogenesis and progression of cancer is critical for its clinical application. We made observations regarding effects of NDGA on mTORCl/2signaling in breast cancer cell culture and animal model. Our study suggests that NDGA also functions as direct inhibitor of mTORCl.
These data implicate that NDGA may represent a potential new class of agents for the treatment of breast and other cancers where the mTORC1signaling play a role in the oncogenic process.
一、研究背景
骨的代谢是一个动态过程,并随着机械应力、激素及细胞因子等的改变而维持动态的重建,包括骨形成与骨吸收,如此骨组织才具有正常的形态,发挥应有的功能。在骨代谢中骨细胞的主要作用是接收并传递信号给周围的成骨细胞(OB)和破骨细胞(OC),从而启动骨重建。OC主要负责吸收局部骨组织,于此同时OB则形成新骨以替代被吸收的骨组织。OC是由骨髓造血干细胞中的单核/巨噬细胞系分化而成的多核细胞,其活性受多种细胞因子和激素的调节,但具体的机制不清楚。
骨保护素(osteoprotegerin, OPG)、核因子KB受体活化因子(receptor activator of nuclear factor—kappaB, RANK)及核因子KB受体活化因子配体(receptoractivator of nuclear factor—kappa B ligand, RANKL)系统是自1997年以来发现的一组调控破骨细胞分化激活的细胞因子,三者均属肿瘤坏死因子受体超家族(tumomecrosis factor receptor superfamily, TNFRSF)。成骨细胞及骨髓基质细胞表达RANKL,与破骨细胞前体细胞或破骨细胞表面上的RANK结合后,通过激活调节破骨细胞形成的转录因子,从而促进破骨细胞的形成和分化,并抑制破骨细胞的凋亡。由成骨细胞分泌的OPG可以与RANKL结合,竞争性抑制RANKL与RANK之间的结合,从而抑制破骨细胞的增殖和分化,并最终抑制骨吸收。
越来越多的证据证明许多激素、细胞因子直接或间接地调节RANK/RANKL/OPG系统,从而介导破骨细胞的分化和功能,导致多种骨代谢疾病,包括骨质疏松、糖皮质激素导致的骨丢失、多发性骨髓瘤以及风湿性关节炎等。另外,靶向OPG/RANK/RANKL系统的药物比如外源性的OPG、抗RANKL抗体、RANK—Fc等被证实是有效预防和治疗包括骨质疏松、骨肿瘤的骨溶解疾病。
哺乳动物雷帕霉素靶蛋白(mammalian Target Of Rapamycin, mTOR)是一种保守的丝/苏氨酸蛋白质激酶。在细胞多种生理活动的调控中处于核心地位,接受并整合细胞内外的各种刺激如激素、生长因子、营养、能量、氧气、应激等调控着包括基因转录、蛋白质起始翻译、核糖体生物合成、自噬等过程。人类重大疾病如癌症、心血管疾病、移植排斥及自体免疫紊乱、糖尿病、肥胖、神经系统疾病等均涉及mTOR信号的失调,mTOR信号通路成为近年基础与临床研究的热点。
近年来越来越多的文章报导mTOR信号通路参与骨代谢的调节。mTOR/S6K1通过调节il-6和VEGF的合成,进而调节骨形成。也有文章报导,雷帕霉素(rapamycin)增加成骨样细胞的OPN和OCN的mRNA的表达,通过抑制mTOR活性并激活bmp/smad通路促进人胚胎干细胞的成骨分化。另外,BEZ235作为PI3K和mTOR通路的抑制剂能够促进HMSCS向成骨细胞分化。尽管雷帕霉素对成骨细胞分化的影响仍然存在争论,但目前普遍认为抑制mTORC1信号通路可以促进成骨细胞分化。另外,mTOR的激活是破骨细胞前体以及成熟破骨细胞存活所必需的,有文章指出促红细胞生成素(EPO)能激活mTOR的活性,并分别通过上调NFATcl和下调组织蛋白酶K,增加破骨细胞的数量和降低破骨细胞的活性,而Rapa能够抑制EPO的作用。尽管mTOR在骨代谢中的作用被肯定,但是其具体的机制仍然不明了。
最近发现转录因子CCAAT/enhancer binding protein beta (C/EBPβ)在骨代谢中发挥重要作用。结合转基因小鼠、细胞生物学和药理学方面的研究,发现它不仅可以调控成骨细胞的活性,也可以调节破骨细胞的分化和活性。鉴于RANK/RANKL/OPG系统在骨代谢中的重要作用,特别是对破骨细胞的调控,我们猜想1mTOR是否能通过C/EBPβ调控成骨细胞及其前体释放RANKL/OPG,从而调节破骨细胞的分化和功能。目前,mTOR信号通路对RANKL/OPG的调控仍然存在较大争议,对其中的机制研究尚未见报道。
二、研究结果
1. mTORC1参与去卵巢诱导的破骨细胞形成和骨吸收过程
雌鼠去卵巢可以降低雌激素水平,诱导骨质疏松。首先,将8-12周龄的C57B6小鼠共24只,随机分四组,分别为假手术组(Sham)、去卵巢组(OVX)、假手术+雷帕霉素组、去卵巢+雷帕霉素组。小鼠引颈处死后取材,micro-CT检测分析、骨组织形态计量学分析、骨钙素免疫组化和破骨细胞染色检测表明：在去卵巢组小鼠中,破骨细胞活性增强,骨吸收增加；单纯灌胃雷帕霉素组小鼠破骨细胞活性受抑制,骨吸收减少；此外,雷帕霉素可以逆转去卵巢引起的小鼠骨质疏松。碱性磷酸酶(ALP)和酒石酸酸性磷酸酶(TRAP)分别是成骨细胞分化和破骨细胞分化的特征性外酶,取四组小鼠的血清进行生物化学检测,结果表明抑制mTORC1会增加ALP/TRAP的比例(数据未给出)。
我们进一步检测mTORC1在以上样品中的活性,发现在雷帕霉素组和雷帕霉素+去卵巢组的小鼠标本中,骨吸收的增加都伴随着mTORC1的下游信号分子S6的磷酸化活性降低,说明抑制mTORC1能降低破骨细胞的活性,促进骨吸收过程。
2. mTORCl活性增加下调RANKL的表达
为了进一步验证mTORC1对RANKL表达水平的调节,我们通过体外培养小鼠骨髓基质细胞系OP9、大鼠原代骨髓间充质干细胞BMSC,用mTORC1抑制剂雷帕霉素处理细胞,分别在mRNA水平和蛋白水平检测RANKL的表达,结果表明抑制mTORC1会导致RANKL表达水平的显著下调(P=0.000)。mTORC1的上游抑制基因——结节性硬化症复合物(tuberous sclerosis complex, TSC)由TSC1和TSC2组成,它们二者任一缺失都会导致mTORC1的过度活化,为了进一步验证mTORC1对RANKL的调控作用,我们体外培养TSC2未敲除和TSC2敲除的小鼠胚胎成纤维细胞系MEFTSC2+/+和MEFTSC2-/-,分别在mRNA水平和蛋白水平检测RANKL的表达,发现1mTORC1过度活化可以显著增加RANKL的表达水平(P=0.000)。为了进一步验证以上结果,我们同时用TSC2的siRNA体外敲除OP9细胞系的TSC1(P=0.024)。以上结果均确定,mTORC1对RANKL的调控作用。
3. mTORC1活性增加上调OPG的表达
破骨细胞形成和骨吸收过程受RANKL/OPG系统的调控,除了检测RANKL水平受mTORC1调节的现象,我们同时在体外培养的上述细胞系中分别检测了OPG的mRNA表达水平和蛋白表达水平,结果表明mTORC1活性被雷帕霉素抑制后会导致OPG表达水平的上调(P=0.004,P=0.000),在MEFTSC2+/+和MEFTSC2-/-细胞系和TSC2siRNA干扰实验中均证明OPG的表达受mTORC1负调节(P=0.000,P=0.000)。以上数据均通过统计学检验方法检验,并具有显著性差异。
4. mTORC1调节RANKL/OPG的转录活性
由于mTORC1活性调节了RANKL/OPG的表达水平,我们通过荧光素酶报告系统来进一步验证mTORC1是否调节了RANKL/OPG的转录活性。我们通过体外转染含有荧光素酶报告基因的质粒给OP9细胞,并用mTORC1抑制剂雷帕霉素处理,通过荧光素酶报告基因检测仪检测和分析证实：抑制mTORC1活性会显著抑制RANKL的转录活性(P=0.000),和增强OPG的转录活性(P=0.011)。以上数据均通过统计学检验方法检验,并具有显著性差异。
5. mTORCl通过抑制Akt降低β-catenin的mRNA的稳定性
在上述结果中,我们证实TSC缺失导致的mTORC1过度活化可以引起RANKL/OPG的比例降低,从而抑制骨吸收。
在前期实验中,我们用雷帕霉素体外处理OP9细胞系,通过mRNA芯片筛选出其中表达差异显著的转录因子之一：C/EBPp (p-catenin),介于目前已报导的β-catenin对RANKL/OPG和骨代谢的调节作用,我们推测mTORC1通过对mTORC1/2负反馈途径的下游分子Akt磷酸化的调节来调控β-catenin,进而调节RANKL/OPG的比例。
我们通过体外培养大鼠骨髓间充质干细胞BMSC、小鼠骨髓基质细胞系OP9,用雷帕霉素处理细胞和敲除细胞的TSC基因,得到mRNA水平和蛋白水平检测结果符合我们的预期：抑制mTORC1活性,会引起β-catenin的表达上调(P=0.000),同时伴随1mTORC2下游信号分子Akt的Ser473位点磷酸化的活性增强。为了进一步验证,我们通过用显著性抑制性Akt的腺病毒(Dominant negative Akt Ad virus)感染骨髓间充质干细胞,并用雷帕霉素处理细胞,结果证实：当Akt被抑制时,能完全抵抗mTORC1活性受阻所引起的β-catenin的表达。这说明,1mTORC1很可能是通过Akt来维持β-catenin的表达稳定性的。以上数据均通过统计学检验方法检验,并具有显著性差异。
6. mTORC1通过β-catenin信号通路调节RANKL/OPG
根据1mTORC1对RANKL/OPG的调节作用和β-catenin对骨代谢的调控作用,我们通过体外实验对以上分子机制进行探讨,当抑制β-catenin信号时,mTORC1对RANKL/OPG的影响,从而验证mTORC1能通过β-catenin来调节RANKL/OPG.我们对体外培养的细胞用同种属的β-catenin的siRNA干扰实验来敲除β-catenin的表达,同时用雷帕霉素处理细胞,所得结果证明：当β-catenin被抑制时,mTORC1失去了对RANKL/OPG的调控作用。
三、结论
综上所述,我们的研究揭示了一条新的涉及mTORC1、p-catenin、Akt与RANKL/OPG系统对骨代谢调节的信号转导通路。通过这条通路,mTORC1被抑制,进而通过mTORC1/2负反馈抑制途径激活Akt (Ser473)的磷酸化,激活的磷酸化Akt通过减少β-catenin的降解来改变RANKL/OPG的比例,进而调控破骨细胞的发生和骨吸收过程。mTORC1-Akt-β-catenin-RANKL/OPG途径对骨代谢的调控作用为雷帕霉素治疗骨质疏松病人的分子机制增添了新的内容。目前,我们通过可诱导的全身性敲除TSC1/Raptor和成骨细胞特异性敲除TSC1/Raptor的转基因小鼠,对于mTOR成为治疗骨质疏松的靶点及其治疗效果进行进一步的研究。
第二部分去甲二氢化愈创木酸(NDGA)以mTORC1为靶点抑制乳腺癌的生长
一、研究背景
去甲二氢化愈创木酸Nordihydroguaiaretic acid (NDGA)是一种来源于石炭酸灌木Larrea divaricatta的天然的酚类化合物,目前文献报道它在体外和体内均具有抗肿瘤活性。据许多临床研究报道得知,NDGA(去甲二氢化愈创木酸)的许多化学同型物正被开发为难治性实体瘤的临床药物。虽然NDGA(去甲二氢化愈创木酸)具有很好的临床药用价值,但其发挥抗肿瘤作用的分子机制目前仍然没有得到清晰的解释,这对其能得到广泛应用是一个极大的限制。
哺乳动物雷帕霉素靶蛋白(mammalian Target Of Rapamycin, mTOR)是一种保守的丝／苏氨酸蛋白质激酶。在细胞多种生理活动的调控中处于核心地位,接受并整合细胞内外的各种刺激如激素、生长因子、营养、能量、氧气、应激等调控着包括基因转录、蛋白质起始翻译、核糖体生物合成、自噬等过程。人类重大疾病如癌症、心血管疾病、移植排斥及自体免疫紊乱、糖尿病、肥胖、神经系统疾病等均涉及mTOR信号的失调,mTOR信号通路成为近年基础与临床研究的热点。在本实验室前期的研究成果中发现,不饱和脂肪酸花生四烯酸(Arachidonic acid, AA)及其代谢产物能以mTORC1为靶点促进乳腺癌的发生发展,而花生四烯酸的代谢途径之一脂氧合酶途径在其中发挥了重要作用,NDGA(去甲二氢化愈创木酸)做为一种强抗氧化剂是抑制脂氧合酶途径的重要物质。我们推测mTORC1能作为NDGA(去甲二氢化愈创木酸)具有抗肿瘤和抑制乳腺癌生长作用的分子机制的潜在靶点。
在我们的研究中,我们发现并证明了哺乳动物雷帕霉素靶标复合物1(mTORC1)是NDGA(去甲二氢化愈创木酸)的一个靶点,这个结论在体外培养的乳腺癌细胞和体内移植瘤小鼠身上都得到了验证。
二、研究结果
1. NDGA(去甲二氢化愈创木酸)在乳腺癌细胞中抑制mTORC1而不是mTORC2
我们体外培养三种乳腺癌细胞系,在不同时间点(时间梯度)给细胞加入同一浓度的NDGA(去甲二氢化愈创木酸)和在同一时间点给细胞加入不同浓度(浓度梯度)的NDGA(去甲二氢化愈创木酸),药物处理完成后,我们通过用免疫印迹(western blot)检测mTORC1和mTORC2下游途径信号分子的表达水平和活化水平来验证NDGA(去甲二氢化愈创木酸)的作用。
我们发现NDGA(去甲二氢化愈创木酸)能抑制mTORC1的活性而不是mTORC2的活性,并具有一定的时间梯度和浓度梯度。
说明NDGA(去甲二氢化愈创木酸)能特异性地以mTORC1而不是mTORC2为靶点而发挥作用。
2. NDGA(去甲二氢化愈创木酸)抑制mTORCl下游信号分子和抑制乳腺癌细胞的增殖
细胞周期蛋白D1(cyclinD1),缺氧诱导因子(HIF-α),血管内皮生长因子(VEGF)都是mTORC1下游重要的信号分子,它们的表达水平直接受到mTORCl活性的调节并与由于mTORC1过度活化导致的肿瘤发生发展具有重要关系。我们在体外培养的乳腺癌细胞中证明,NDGA(去甲二氢化愈创木酸)能抑制以上信号分子的表达水平,并在体外培养的条件下,抑制不同的乳腺癌细胞系的增殖水平(P=0.000)。
以上数据同样提示了mTORC1作为NDGA(去甲二氢化愈创木酸)的靶标,对于乳腺癌的治疗具有重要意义。
3. NDGA(去甲二氢化愈创木酸)抑制mTORC1通过部分活化AMPK/TSC2
AMP激活蛋白激酶(AMPK)和结节性硬化症复合物2(TSC2)都是mTORC1上游的抑制基因。能量缺乏和其它多种信号刺激或药物都能引起AMPK的磷酸化从而抑制mTORC1活性；活化的AMPK能直接磷酸化TSC2并抑制mTORC1活性。为了探讨NDGA(去甲二氢化愈创木酸)作用mTORC1的分子机制,我们在体外敲除乳腺癌细胞的AMPK或TSC2发现,NDGA(去甲二氢化愈创木酸)能部分通过AMPK或TSC2途径来抑制：mTORC1的活性。
4. NDGA(去甲二氢化愈创木酸)抑制氨基酸和胰岛素引起的mTORC1活化,并破坏mTOR-Raptor相互作用
胰岛素对mTORC1信号通路的激活是依赖于TSC途径的,而氨基酸对mTORC1信号通路的激活是不依赖于TSC2的。有趣的是,NDGA(去甲二氢化愈创木酸)不但能抑制氨基酸引起的mTORC1活化,它同样能抑制胰岛素引起的mTORC1活性升高。因此,我们对NDGA(去甲二氢化愈创木酸)是否能直接作用mTORC1复合物本身进行验证。
(Rapamycin)一样干扰mTOR-Raptor直接的相互作用并能抑制mTORC1的体外激酶活性。这个结果让我们明确,NDGA(去甲二氢化愈创木酸)抗肿瘤的分子机制,可能是直接以mTORC1为靶标来发挥作用的。
5. NDGA(去甲二氢化愈创木酸)以mTORCl为靶点抑制乳腺癌荷瘤小鼠肿瘤的生长
体外水平的实验结果给了我们很好的提示,因此我们建立了乳腺癌体外移植瘤动物模型来验证NDGA能否以mTORC1为靶点,并抑制乳腺癌的生长。
我们用NDGA(去甲二氢化愈创木酸)腹腔注射体外移植乳腺癌细胞的裸鼠,给药期间测量肿瘤大小,给药一个月后取材,测量,称重,鉴定。结果证明,NDGA(去甲二氢化愈创木酸)在体内水平能有效抑制乳腺癌的生长,并显著下调mTORC1信号通路的下游信号分子的活性。以上数据均通过统计学检验方法检验,并具有显著性差异,P=0.000。
三、结论
综上所述,清楚NDGA(去甲二氢化愈创木酸)抑制肿瘤发生发展过程的分子机制对临床应用具有重要的意义。我们的研究揭示了一条新的NDGA(去甲二氢化愈创木酸)以(?)mTORC1而不是mTORC2为靶标的治疗乳腺癌发展的信号转导通路。通过这条通路,NDGA(去甲二氢化愈创木酸)能抑制mTORC1活性和乳腺癌细胞的增殖；能部分通过AMPK/TSC2发挥抑制作用；NDGA(去甲二氢化愈创木酸)能打破mTOR和Raptor的相互作用并抑制(?)mTORC1的体外激酶反应；NDGA(去甲二氢化愈创木酸)能以mTORC1为靶点并显著抑制乳腺癌体外移植瘤小鼠的乳腺癌的发展。这为NDGA(去甲二氢化愈创木酸)治疗乳腺癌,发挥抗肿瘤作用的分子机制增添了新的内容。
PART ONE
mTORCl regulates RANKL/OPG and osteoclastogenesis through β-catenin signaling pathway
Background
Bone remodeling is in a dynamic process where bone resorption and bone formation take responsibilities. Under internal or external stimuli such as mechanical strength, hormones, cytokines, the variational activities of osteoblasts and osteoclasts would maintain coordinated balance in healthy adults.
The osteoclast, which takes responsibilities for local bone resorption is derived from hematopoietic stem cells through myeloid progenitor cells to mono/macrophage and into osteoclast lineage. The molecular mechanism of the activation of osteoclast activity is not clear. Osteoclast differentiation and activation are considered to be under the regulation of RANK/RANKL/OPG system since1997. Receptor activator of nuclear factor-kappaB (RANK), receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG) is belongs to tumornecrosis factor receptor super-family (TNFRSF). Osteoblast and bone marrow stromal cells both express RANKL, while they bind to RANK which express on the surface of osteoclast progenitors and mature osteoclast, they will promote the formation and differiation of osteoclast and inhibit the apoptosis through activating the transcriptional factor. OPG is secreted by osteoblast and able to bind to RANKL, which competitively inhibit the combining of RANKL and RANK, as a result of the suppression of osteoclast proliferation, differentiation and bone resorption.
At present, several evidences demonstrate that multiple hormone, cytokines can regulate RANK/RANKL/OPG system directly or indirectly, lead to many bone metabolism diseases, including osteoporosis, bone loss induced by glucocorticoids, multiple myeloma, rheumatoid arthritis and so on. Beside these, which target OPG/RANK/RANKL system such as exogenetic OPG, anti-RANKL antibody and RANK-Fc have been confirmed as potent and effectual medicines to prevent and treat osteolytic diseases.
Mammalian target of rapamycin (mTOR), is a protein Ser-Thr kinase that functions as a central element in a signaling pathway involved in the control of cell growth and proliferation. Disregulation of mTOR signaling associated to human major diseases, for instance, cancer, cardiovascular diseases, graft rejection and autoimmune diseases, diabetes, adiposity, nerve system diseases. mTOR signaling pathway have been become to a certain target in recent years.
mTOR signaling pathway take part in the regulation of bone metaboslism is definitely confirmed:mTOR/S6K1regulates il-6and VEGF to affect bone formation; rapamycin promote mRNA expression of OPN and OCN in osteoblast-like cells, and promote osteogenic differentiation of human embryonic stem cell through inhibit mTOR activity and activate bmp/smad pathways. What's more, BEZ235as an inhibitor of PI3K/mTOR pathway, is able to stimulate human bone marrow mesenchymal stem cells (hMSC) differentiate to osteoblast. Rapamycin promote osteoblast differentiation is still in argument, but most consideration about mTORC1signaling pathway inhibition can promote osteoblast formation were supported. On the other hand, mTOR activation is necessary for the survival of osteoclast progenitor cells and mature osteoclast. Someone pointed out erythropoletin hematopoietin (EPO) can activate mTOR and up-or down-regulate NFATcl or cathepsin k to enhance osteoclast formation and decrease osteoclast activity, however rapamycin can reverse these. Despite mTOR has been considered as a regulator in bone metabolism, the concrete mechanism is still unclear.
Recently published articles have found that the transcriptional factor CCAAT/enhancer binding protein beta (C/EBPβ) plays a key role in bone metabolism. C/EBPβ not only regulates osteoblast activity, but also regulates osteoclast differentiation and activity under the investigation on transgenic mice, cell biology and pharmacology. In view of the importance of RANK/RANKL/OPG system, especially the regulation of osteoclast, we considered that wether mTOR can control osteoblast and its progenitors to secrete RANKL/OPG via C/EBPβ, and modulate osteoclast differentiation and function.
Results
1mTORCl is involved in OVX-induced osteoclastogenesis and bone resorption
Ovariectomized (OVX) female mice will keep low estrogen level and lead to osteoporosis. Firstly, we randomly grouped24C57B6mice into four, respectively, including Sham, OVX, Sham+rapamycin, OVX+rapamycin. Mice were executed then detected and analysed bone samples for bone mineral density (BMD), micro-CT, bone histomorphometry, osteocalcin immunohistochemistry and osteoclast tartaric acid and acid phosphatase (TRAP) staining. These data demonstrated that, osteoclast activity and bore resorption enhanced in OVX mice, and osteoclast activity and bone resorption decreased in rapamycin-treated mice, besides this, rapamycin can dominantly rescure osteoporosis in OVX mice.
Alkaline phosphatase (ALP) and tartaric acid and acid phosphatase (TRAP) are respectively the characterized exoenzymes of osteoblast and osteoclast differentiation. We collected serum of four groups mice for biochemical detection and found that mTORC1inhibition will increase ALP/TRAP (data not shown). We further assay mTORC1activity in above samples, as a result of activated mTORC1lead to activated bone resorption progression.
2RANKL expression is positively regulated by mTORCl activity
To confirm mTORC1regulate RANKL expression level, we in vitro culture mouse bone marrow stromal cell line OP9, rat primary bone marrow stem cell BMSC and treated cells with mTORCl inhibitor rapamycin, then detected mRNA and protein expression levels of RANKL, the data showed us mTORC1inhibition dominantly down-regulate RANKL expression level(P=0.000).
mTORCl upstream suppressor------Tuberous Sclerosis Complex (TSC) is organized with TSC1and TSC2, either of them lost will over-activate mTORCl. In orter to confirm mTORC1take part in RANKL regulation, we cultured mouse embryo fibroblast cell lines MEFTSC2+/+and MEFTSC2-/-, and use TSC2siRNA to knockdown TSC1in OP9cell line, then detected RANKL mRNA and protein expression levels(P=0.000, P=0.024). All data were detected and analysed by statistical method, and with significant differences.
3OPG expression is negatively regulated by mTORCl activity
Osteoclastogenesis and bone resorption is regulated by RANKL/OPG system.
Except for measuring mTORCl regulates RANKL, we have also detected OPG expression on mRNA and protenin level in above cell lines. Data showed us mTORC1activity is suppressed by rapamycin while up-regulating OPG expression(P=0.004, P=0.000), and in embryo fibroblast cell lines or TSC2knockdown OP9cell line, OPG expression is denifitely negatively regulated by mTORC1(P=0.000, P=0.000). All data were detected and analysed by statistical method, and with significant differences.
4mTORCl regulates transcriptional activity of RANKL/OPG
We next investigated how mTORC1regulated the expression of RANKL/OPG. Luciferase reporter gene system is a useful system for detecting transcriptional factor activity. To determine the role of mTORC1activity in RANKL/OPG regulation, we use luciferase reporter gene plasmids which have been constructed with RANKL or OPG genes, and companied by rapamycin treatment in OP9cell line. These results suggest that mTORC1activity is required for RANKL and OPG transcriptional activity. All data were detected and analysed by statistical method, and with significant differences (P=0.000, P=0.011).
5mTORC1decreases mRNA stability of beta-catenin via inhibition of Akt
In previous experiments, a screening test via mRNA chip to find a signifinantly variable transcriptional factor:C/EBPβ (β-catenin), in rapamycin-treated OP9cell line. β-catenin regulated RANKL/OPG has been shown recently, we supposed mTORC1is able to regulate RANKL/OPG through β-catenin signaling pathway while affacting the phosphorylation level of Akt (Ser473), which is a downstream signal of mTORC2.
Indeed, we found that mTORC1activity inhibition raised an induction of β-catenin expression, meanwhile, mTORC2downstream signal Akt (Ser473) was phosphorylated enhanced in rapamycin-treated or TSC knockdowning cell lines.
To further investigate mTORC1/Akt/β-catenin signaling pathway, we use dominant negtice Akt adenovirus to knockdown Akt activity, then treat cells with rapamycin. The data show us when Akt was totally blocked, β-catenin expression is not affected by mTORCl activity. We proposed that mTORC1may decreases mRNA stability of β-catenin via Akt inhibition. All data were detected and analysed by statistical method, and with significant differences.(P=0.000)
6mTORCl regulates RANKL/OPG through beta-catenin signaling
As we known, mTORC1and β-catenin mainly in the regulation of RANKL/OPG system, we performed in vitro measuring the molecular mechanism. Interestingly, β-catenin siRNA knockdown interrupted the regulation of mTORC1to RANKL/OPG expression level.
Conclusion
In summary, our study reveals a novel regulatory pathway that involves RANKL/OPG system and bone resorption progress. Through this novel pathway, the key regulator Akt drives β-catenin to regulate RANKL/OPG balance dependent of mTORC1. The activated Akt in turn increases mRNA stability of β-catenin through a mechanism involved mTORC1activation to reduce RANKL secretion and induce OPG expression, finally regulate osteoclastogenesis and bone resorption.
mTORCl-Akt-catenin-RANKL/OPG pathway is meaningful to provide a new molecular mechanism to treat osteoporosis patient with rapamycin. At present, we determine the further investigation using inducible systemic-specific knockout TSC1/Raptor transgenic mice and osteoblast-specific knockout TSC1/Raptor mice, to confirm mTORC1as a key regulator in osteoclastogenesis and bone metabolism.
PART TWO
mTORCl is a target of nordihydroguaiaretic acid to prevent breast tumor growth in vitro and in vivo
Background
Nordihydroguaiaretic acid (NDGA) is a natural phenolic compound isolated from the creosote bush Larrea divaricatta that has anti-tumor activities both in vitro and in vivo. Its analogues are in clinical development for use in refractory solid tumors. But the mechanisms underlying the anti-cancer effect of NDGA are not fully understood. In this study, we identified mammalian target of rapamycin complex1(mTORCl) as a target of NDGA both in cultured breast cancer cells and in xenograft models.
Mammalian target of rapamycin (mTOR), is a protein Ser-Thr kinase that functions as a central element in a signaling pathway involved in the control of cell growth and proliferation. Disregulation of mTOR signaling associated to human major diseases, for instance, cancer, cardiovascular diseases, graft rejection and autoimmune diseases, diabetes, adiposity, nerve system diseases. mTOR signaling pathway have been become to a certain target in recent years. In our previous research data we have found that unsaturated fatty acid arachidonic acid (AA) and its metabolites can target mTORC1and promote breast cancers. Lipoxygenase pathway is a critical way in arachidodic acid metabolism, NDGA as a strong antioxidant against to lipoxygenase and dominantly inhibits arachidonic acid production. We suppose that mTORC1can be a potent target of NDGA in prevention of breast cancer growth and carcinogenesis.
Together our data provide a novel mechanism for NDGA activity which could help explain its anti-cancer activity. NDGA repressed breast tumor growth and targeted mTORC1and its downstream signaling in vitro and in vivo.
Results
1NDGA inhibits mTORCl in breast cance cells
To investigate the potential effect of NDGA on mTOR signaling in breast cancer cells, we cultured several breast cancer cell lines in vitro and treated them with NDGA, we found that NDGA effectively suppressed basal levels of phosphorylation of S6(S235/236) and4E-BP1(downstream target of S6K1and mTORC1) in a dose-and time-dependent manner in all tested cells. NDGA can particularly target mTORC1but not mTORC2.
2NDGA inhibits mTORCl downstream signaling and suppresses proliferation in breast cancer cells
Cyclin D1, HIF-α and VEGF are three key mTORC1downstream signaling molecules whose expression are positively regulated by mTORC1and responsible for mTORCl overactivation-related carcinogenesis and progression of cancer. we next examine the effect of NDGA on Cyclin D1, HIF-α and VEGF protein levels in MCF-7cells.
Taken together, these results further confirmed mTORC1signaling is a target of NDGA and is involved in prevention of breast cancer proliferation. All data were detected and analysed by statistical method, and with significant differences.(P=0.000)
3NDGA inhibits mTORCl in part through activation of AMPK/TSC2
AMP-activated protein kinase (AMPK) and tuberous sclerosis complex1/2(TSC1/2) are upstream signals that negatively regulate mTORCl. Energy starvation and various stimuli/drugs have been shown to induce phosphorylation and activation of AMPK, activated AMPK phosphorylates TSC directly and inhibit mTORC1. To understand the mechanism through which NDGA inhibits mTORCl, the roles of AMPK/TSC in this process were examined. Interestingly, NDGA increased phosphorylation of AMPK, and knockdown of AMPK or TSC2by siRNA, partially repressed NDGA-induced mTORC1activity decreased.
4NDGA suppresses amino acid-and insulin-stimulated mTORCl activity and disrupts mTORCl complex
We next investigated how NDGA regulated mTORCl. Our result that deletion of TSC2could not diminish the inhibitory effects NDGA against mTORC1implicates additional mechanisms are involved. We thus examined the effects of NDGA on insulin-and amino acids-stimulated mTORC1, which simulates mTORC1in TSC2-dependent and independent mechanism respectively. Interestingly, both amino acids and insulin-stimulated mTORC1activity were repressed by NDGA, these data implicate NDGA may target mTORC1directly.
Assembling and stability of the mTORC1determines its kinase activity. Surprisingly, NDGA did not stabilize mTOR-Raptor interaction under either presence or absence of amino acids conditions, however, it acted like rapamycin to disrupt the mTOR-Raptor complex and inhibited mTORC1in vitro kinase activity, whereas mTOR-Rictor association remained unchanged. Taken together, these results implicate that disruption of mTOR-Raptor complex by NDGA may contribute to its inhibitory effects against mTORC1and mTORC1may be a direct target of NDGA.
5NDGA inhibits breast tumor growth and targets mTORCl in xenograft
We next asked whether in vivo administration of NDGA inhibits mTORC1activity and prevents breast tumor growth in xenograft. Nude mice bearing MDA-MB-231breast tumor xenografts were dosed with saline or NDGA in a month, the NDGA therapy as single agent markedly inhibited breast tumor growth, most importantly, in vivo administration of NDGA remarkably reduced the levels of mTORC1downstream signals in the breast tumor xenograft. Suggesting that NDGA could inhibit mTORC1and its downstream signaling in vivo. All data were detected and analysed by statistical method, and with significant differences (P=0.000). These results identified mTORC1as a target of NDGA in vivo.
Conclusion
In summary, understanding the mechanisms by which NDGA suppress carcinogenesis and progression of cancer is critical for its clinical application. We made observations regarding effects of NDGA on mTORCl/2signaling in breast cancer cell culture and animal model. Our study suggests that NDGA also functions as direct inhibitor of mTORCl.
These data implicate that NDGA may represent a potential new class of agents for the treatment of breast and other cancers where the mTORC1signaling play a role in the oncogenic process.
引文
[1]. Manolagas, S.C. and R.L. Jilka, Bone marrow, cytokines, and bone remodeling. Emerging insights into the pathophysiology of osteoporosis. Mechanism of Disease,1995.5(332):p.305-311.
[2]. von Tirpitz, C., et al., Effect of systemic glucocorticoid therapy on bone metabolism and the osteoprotegerin system in patients with active Crohn's disease. European Journal of Gastroenterology & Hepatology,2003.11(15):p. 1165-1170.
[3]. Pearse, R.N., et al., Multiple myeloma disrupts the TRANCE osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression. PNAS,2001.20(98).
[4]. Hussein, O., et al., Rapamycin inhibits osteolysis and improves survival in a model of experimental bone metastases. Cancer Lett,2011(314):p.176-184.
[5]. Smink, J.J., P. Tunn and A. Leutz, Rapamycin inhibits osteoclast formation in giant cell tumor of bone through the CEBPβ-MafB axis. J Mol Med, (90):p. 25-30.
[6]. Turner, C.H., et al., Do bone cells behave like a neuronal network. Calcif Tissue Int,2002(70):p.435-442.
[7]. Harada, S. and G.A. Rodan, Control of osteoblast function and regulation of bone mass. Nature,2003(423):p.349-355.
[8]. Simonet, W.S., et al., Osteoprotegerin a novel secreted protein involved in the regulation of bone density. Cell,1997(89):p.309-319.
[9]. Armstrong, A.P., et al., A RANK TRAF6-dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function. The Journal of Biological Chemistry,2002.46(277):p.44347-44356.
[10]. Min, H., et al., Osteoprotegerin Reverses Osteoporosis by Inhibiting Endosteal Osteoclasts and Prevents Vascular Calcification by Blocking a Process Resembling Osteoclastogenesis. J. Exp. Med.,2000.4(192):p.463-474.
[11]. Yano, K., et al., Immunological characterization of circulating osteoprotegerin osteoclastogenesis inhibitory factor increased serum concentrations in postmenopausal women with osteoporosis. Journal of Bone and mineral Research,1999.4(14):p.518-527.
[12]. Bucay, N., et al., osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes & Development,1998(12):p. 1260-1268.
[13]. Berry, M.A., et al., Evidence of a role of tumor necrosis factor alpha in refractory asthma. N Engl J Med,2006(354):p.697-708.
[14]. Yasusa and Hisataka, [Bone and bone related biochemical examinations. Bone and collagen related metabolites. Receptor activator of NF-kappaB ligand (RANKL)]. Clinical Calcium,2006.6(16):p.964-70.
[15]. Hsu, H., D.L. Lacey and W.J. Boyle, Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl. Acad. Sci.,1999(96):p.3540-3545.
[16]. Hofbauer, L.C., et al., The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res,2000.15(1): p.2-12.
[17]. Jimi, E., et al., Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function. The Journal of Immunology,1999(163):p.434-442.
[18]. Bekker, P.J., D. Holloway and E. Al., The effect of a single dose of osteoprotegerin in postmenopausal women. Journal of Bone AND Mineral Research,2001.2(16):p.348-360.
[19]. McClung, M.R., E.M. Lewiecki and E. Al., Denosumab in postmenopausal women with low bone mineral density. N Engl J Med,2006(354):p.821-31.
[20]. Body, J., P. Greipp and E. Al., A phase I study of AMGN-0007, a recombinant osteoprotegerin construct, in patients with multiple myeloma or breast carcinoma related bone metastases. American Cancer Society,2003(97):p. 887-92.
[21]. Sengupta, S., T.R. Peterson and D.M. Sabatini, Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell,2010. 2(40):p.310-22.
[22]. Luoki, K., T. Zhu and K. Guan, TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival. Cell,2003(115):p.577-90.
[23]. Luoki, K., et al., Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes & Development,2003(17):p.1829-34.
[24]. Gingras, A., B. Raught and N. Sonenberg, Regulation of translation initiation by FRAP mTOR. Genes & Development,2001(15):p.807-26.
[25]. Jefferies, H.B.J., S. Fumagalli and E. Al., Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. The EMBO Journal,1997.12(16):p. 3693-704.
[26]. Sarbassov, D.D. and E. Al., Phosphorylation and regulation of Akt PKB by the rictor-mTOR complex. Science,2005(307):p.1098-101.
[27]. Inoki, K., M.N. Corradetti and K. Guan, Dysregulation of the TSC-mTOR pathway in human disease. Nature Genetics,2005.1(37):p.19-24.
[28]. Romero, D.F., et al., Rapamycin:a bone sparing immunosuppressant? J Bone Miner Res,1995.10(5):p.760-8.
[29]. Morales, J.M., J.M. Campistol and E. Al., Sirolimus-based therapy with or without cyclosporine long-term follow-up in renal transplant patients. Transplantation Proceedings,2005(37):p.693-6.
[30]. R, S., S. J and E. Al., Replacing calcineurin inhibitors with mTOR inhibitors in children. Pediatr Transplantation,2005(9):p.391-97.
[31]. Flechner, S.M., D. Goldfarb and E. Al., Kidney transplantation without calcineurin inhibitor drugs a prospective, randomized trial of sirolimus versus cyclosporine. Transplantation,2002.8(74):p.1070-6.
[32]. Alvarez-Garcia, O., et al., Rapamycin retards growth and causes marked alterations in the growth plate of young rats. Pediatric Nephrology,2007. 22(7):p.954-961.
[33]. J., S.G., et al., Wnt signalling in osteoblasts regulates expression of the receptor activator of NF kappa B ligand and inhibits osteoclastogenesis in vitro. Journal of Cell Science,2006.7(119):p.1283-96.
[34]. Sanchez, C.P. and Y.Z. He, Bone growth during rapamycin therapy in young rats. BMC Pediatr,2009.9:p.3.
[35]. Holstein, J., M. Klein and E. Al., Rapamycin affects early fracture healing in mice. British Journal of Pharmacology,2008(154):p.1055-62.
[36]. Singha, U.K., Y. Jiang and E. Al., Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells. Journal of Cellular Biochemistry,2008(103):p.434-46.
[37]. Isomoto, S., et al., Rapamycin as an inhibitor of osteogenic differentiation in bone marrow-derived mesenchymal stem cells. Journal of Orthopaedic Science,2007.12(1):p.83-88.
[38]. Ogawa, T., et al., Osteoblastic Differentiation Is Enhanced by Rapamycin in Rat Osteoblast-like Osteosarcoma (ROS 17/2.8) Cells. Biochemical and Biophysical Research Communications,1998.249(1):p.226-230.
[39]. Lee, et al., Rapamycin promotes the osteoblastic differentiation of human embryonic stem cells by blocking the mTOR pathway and stimulating the BMP/Smad pathway. Stem cells and Development,2010.4(19):p.557-68.
[40]. Martin, S.K., et al., NVP-BEZ235, A Dual Pan Class I PI3 Kinase and mTOR Inhibitor, Promotes Osteogenic Differentiation in Human Mesenchymal Stromal Cells. Journal of Bone and Mineral Research,2010.10(25):p. 2126-37.
[41]. Sugatani, T. and K.A. Hruska, Aktl/Akt2 and mammalian target of rapamycin/bim play critical roles in osteoclast differentiation and survival, respectively, whereas Akt is dispensable for cell survival in isolated osteoclast precursors. The Journal of Biological Chemistry,2005.5(280):p.3583-9.
[42]. Glantschnig, H., J.E. Fisher and G. Wesolowski, M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase. Cell Death And Differentiation,2003.10(10):p.1165-77.
[43]. Junrong, M., et al., Hyperactivation of mTOR critically regulates abnormal osteoclastogenesis in neurofibromatosis type 1. Journal of Orthopaedic Research,2012.1(30):p.144-52.
[44]. A, O.S.A.L. and E.GA. Pez, Rapamycin induces growth retardation by disrupting angiogenesis in the growth plate. Kidney International,2010(78):p. 561-8.
[45]. Shui, C., B.L. Riggs and S. Khosla, The immunosuppressant rapamycin, alone or with transforming growth factor-beta, enhances osteoclast differentiation of RAW264.7 monocyte-macrophage cells in the presence of RANK-ligand. Calcified Tissue Int,2002.5(71):p.437-46.
[46]. Hofbauer, L.C., et al., Effects of Immunosuppressants on Receptor Activator of NF-κB Ligand and Osteoprotegerin Production by Human Osteoblastic and Coronary Artery Smooth Muscle Cells. Biochemical and Biophysical Research Communications,2001.280(1):p.334-339.
[47]. Mogi, M. and A. Kondo, Down-regulation of mTOR leads to up-regulation of osteoprotegerin in bone marrow cells. Biochemical and Biophysical Research Communications,2009.384(1):p.82-86.
[48]. Okui, et al., Antitumor Effect of Temsirolimus against Oral Squamous Cell Carcinoma Associated with Bone Destruction. Molecular Cancer Therapeutics, 2010.11(9):p.2960-69.
[49]. Smink, J.J., et al., Transcription factor C/EBPbeta isoform ratio regulates osteoclastogenesis through MafB. EMBO J,2009.28(12):p.1769-81.
[50]. Smink, J.J. and A. Leutz, Rapamycin and the transcription factor C/EBPβ as a switch in osteoclast differentiation:implications for lytic bone diseases Journal of Molecular Medicine,2010.88(3):p.227-233.
[51]. Ng, et al., CCAAT/Enhancer Binding Protein Beta Is Up-Regulated in Giant Cell Tumor of Bone and Regulates RANKL Expression. Journal of Cellular Biochemistry,2010.2(110):p.438-46.
[52]. Byon, C.H., Y. Sun and E. Al., Runx2-Upregulated Receptor Activator of Nuclear Factor κB Ligand in Calcifying Smooth Muscle Cells Promotes Migration and Osteoclastic Differentiation of Macrophages. Arterioscl Throm Vas,2011.6(31):p.1387-96.
[53]. Enomoto, H., et al., Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2-/-mice by RANKL transgene. J Biol Chem,2003.278(26):p.23971-7.
[54]. De Toni, et al., OPG is regulated by beta-catenin and mediates resistance to TRAIL-induced apoptosis in colon cancer. Clinical cancer Research,2008. 15(14):p.4713-8.
[55]. Suda, T., T. Nagasawa and E. Al., Regulatory roles of b-catenin and AP-1 on osteoprotegerin production in interleukin-la-stimulated periodontal ligament cells.2009. p.384-9.
[1]Silvia Arteaga, A.A.R.E., Larrea tridentata (Creosote bush), an abundant plant of Mexican and US-American deserts and its metabolite nordihydroguaiaretic acid. Journal of Ethnopharmacology,2005(98):p.231-239.
[2]Sengupta, S., T.R. Peterson and D.M. Sabatini, Regulation of the mTOR Complex 1 Pathway by Nutrients, Growth Factors, and Stress, molecular cell, 2010(40):p.310-322.
[3]Inoki, K., et al., Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes & Development,2003(17):p.1829-1834.
[4]Inoki, K., T. Zhu and K. Guan, TSC2 mediates cellular energy response to control cell growth and survival. Cell,2003(115):p.577-590.
[5]Ma, X.M. and J. Blenis, Molecular mechanisms of mTOR-mediated translational control. Molecular Cell Biology,2009(10):p.307-318.
[6]Inoki, K., M.N. Corradetti and G. Kun-Liang, Dysregulation of the TSC-mTOR pathway in human disease. Nature Genetics,2005.1(37):p.19-24.
[7]Sarbassov, D.D., et al., Phosphorylation and Regulation of AktPKB by the Rictor-mTOR Complex 1. Science,2005(307):p.1098-1101.
[8]A., D., Guertin and D.M. Sabatini, Defining the role of mtor in cancer. Cancer Cell,2007(12):p.9-22.
[9]Soulard, A. and M.N. Hall, SnapShot mTOR signaling. Cell,2007(129):p.434.
[10]Tsang, C.K., et al., Targeting mammalian target of rapamycin (mTOR) for health and diseases. Drug Discov Today,2007(12):p.112-124.
[11]Sancak, Y., et al., The Rag GTPases Bind Raptor and Mediate Amino Acid Signaling to mTORCl. Science,2008(320):p.1496-1501.
[12]Kim, D., et al., mTOR Interacts with Raptor to Form a Nutrient-Sensitive Complex that Signals to the Cell Growth Machinery. Cell,2002(110):p. 163-175.
[13]Meyer, A.N., C.W. McAndrew and D.J. Donoghue, Nordihydroguaiaretic Acid Inhibits an Activated Fibroblast Growth Factor Receptor 3 Mutant and Blocks Downstream Signaling in Multiple Myeloma Cell. Cancer Res,2008(68):p. 7361-7370.
[14]Fujimoto, N., et al., Estrogenic activity of an antioxidant, nordihydroguaiaretic acid (NDGA).lIFE Sci,2004(74):p.1417-1425.
[15]Li, F., et al., Nordihydroguaiaretic acid inhibits transforming growth factor beta type 1 receptor activity and downstream signaling. European Journal Of Pharmacology,2009(616):p.31-37.
[16]Lee, C., et al., Nordihydroguaiaretic acid, an antioxidant, inhibits transforming growth factor-beta activity through the inhibition of Smad signaling pathway. Experimental Cell Research,2003(289):p.335-341.
[17]Park, S. and J. Lee, Inhibitory effect of nordihydroguaiaretic acid on β-catenin. Cell Biochem Funct,2011(29):p.22-29.
[18]Efeyan, A. and D.M. Sabatini, mTOR and cancer,many loops in one pathway. Current Opinion In Cell Biology,2010(22):p.169-176.
[19]Dowling, R.J.O., et al., Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res, 2007(67):p.10804-10812.
[20]Sarbassov, D.D., et al., prolonged rapamycin treatment inhibits mTOR2 assembly and AktPKB. Molecular Cell,2006(22):p.159-168.
[21]Sarbassov, D.D., et al., Phosphorylation and Regulation of Akt PKB by the Rictor-mTOR Complex. Science,2005(307):p.1098-1101.
[22]Julien, L. and P.P. Roux, mTOR, the mammalian target of rapamycin. Medicine Science,2010(26):p.1056-1060.
[23]Thoreen, C.C. and D.M. Sabatini, Rapamycin inhibits mTORC1, but not completely. Autophagy,2009(5):p.725-726.
[24]Sarbassov, D.D. and D. Sabatini, Redox Regulation of the Nutrient-sensitive Raptor-mTOR. The Journal of Biological Chemistry,2005(280):p. 39505-39509.
[25]Neve, R.M., A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell,2006(10):p.515-527.
[26]Wang, W. and K.L. Guan, AMP-activated protein kinase and cancer. Acta Physiologica,2009(196):p.55-63.
[27]Thomson, D.M., C.A. Fick and S.E. Gordon, AMPK Activation Attenuates S6K1,4E-BP1, and eEF2 Signaling Responses to High-frequency Electrically Stimulated Skeletal Muscle Contractions. J Appl Physiol,2008.
[28]Li, Y., et al., TSC2 filling the GAP in the mTOR. TRENDS Biochem Sci, 2004(29):p.32-38.
[2]. von Tirpitz, C., et al., Effect of systemic glucocorticoid therapy on bone metabolism and the osteoprotegerin system in patients with active Crohn's disease. European Journal of Gastroenterology & Hepatology,2003.11(15):p. 1165-1170.
[3]. Pearse, R.N., et al., Multiple myeloma disrupts the TRANCE osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression. PNAS,2001.20(98).
[4]. Hussein, O., et al., Rapamycin inhibits osteolysis and improves survival in a model of experimental bone metastases. Cancer Lett,2011(314):p.176-184.
[5]. Smink, J.J., P. Tunn and A. Leutz, Rapamycin inhibits osteoclast formation in giant cell tumor of bone through the CEBPβ-MafB axis. J Mol Med, (90):p. 25-30.
[6]. Turner, C.H., et al., Do bone cells behave like a neuronal network. Calcif Tissue Int,2002(70):p.435-442.
[7]. Harada, S. and G.A. Rodan, Control of osteoblast function and regulation of bone mass. Nature,2003(423):p.349-355.
[8]. Simonet, W.S., et al., Osteoprotegerin a novel secreted protein involved in the regulation of bone density. Cell,1997(89):p.309-319.
[9]. Armstrong, A.P., et al., A RANK TRAF6-dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function. The Journal of Biological Chemistry,2002.46(277):p.44347-44356.
[10]. Min, H., et al., Osteoprotegerin Reverses Osteoporosis by Inhibiting Endosteal Osteoclasts and Prevents Vascular Calcification by Blocking a Process Resembling Osteoclastogenesis. J. Exp. Med.,2000.4(192):p.463-474.
[11]. Yano, K., et al., Immunological characterization of circulating osteoprotegerin osteoclastogenesis inhibitory factor increased serum concentrations in postmenopausal women with osteoporosis. Journal of Bone and mineral Research,1999.4(14):p.518-527.
[12]. Bucay, N., et al., osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes & Development,1998(12):p. 1260-1268.
[13]. Berry, M.A., et al., Evidence of a role of tumor necrosis factor alpha in refractory asthma. N Engl J Med,2006(354):p.697-708.
[14]. Yasusa and Hisataka, [Bone and bone related biochemical examinations. Bone and collagen related metabolites. Receptor activator of NF-kappaB ligand (RANKL)]. Clinical Calcium,2006.6(16):p.964-70.
[15]. Hsu, H., D.L. Lacey and W.J. Boyle, Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl. Acad. Sci.,1999(96):p.3540-3545.
[16]. Hofbauer, L.C., et al., The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res,2000.15(1): p.2-12.
[17]. Jimi, E., et al., Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function. The Journal of Immunology,1999(163):p.434-442.
[18]. Bekker, P.J., D. Holloway and E. Al., The effect of a single dose of osteoprotegerin in postmenopausal women. Journal of Bone AND Mineral Research,2001.2(16):p.348-360.
[19]. McClung, M.R., E.M. Lewiecki and E. Al., Denosumab in postmenopausal women with low bone mineral density. N Engl J Med,2006(354):p.821-31.
[20]. Body, J., P. Greipp and E. Al., A phase I study of AMGN-0007, a recombinant osteoprotegerin construct, in patients with multiple myeloma or breast carcinoma related bone metastases. American Cancer Society,2003(97):p. 887-92.
[21]. Sengupta, S., T.R. Peterson and D.M. Sabatini, Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol Cell,2010. 2(40):p.310-22.
[22]. Luoki, K., T. Zhu and K. Guan, TSC2 Mediates Cellular Energy Response to Control Cell Growth and Survival. Cell,2003(115):p.577-90.
[23]. Luoki, K., et al., Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes & Development,2003(17):p.1829-34.
[24]. Gingras, A., B. Raught and N. Sonenberg, Regulation of translation initiation by FRAP mTOR. Genes & Development,2001(15):p.807-26.
[25]. Jefferies, H.B.J., S. Fumagalli and E. Al., Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70s6k. The EMBO Journal,1997.12(16):p. 3693-704.
[26]. Sarbassov, D.D. and E. Al., Phosphorylation and regulation of Akt PKB by the rictor-mTOR complex. Science,2005(307):p.1098-101.
[27]. Inoki, K., M.N. Corradetti and K. Guan, Dysregulation of the TSC-mTOR pathway in human disease. Nature Genetics,2005.1(37):p.19-24.
[28]. Romero, D.F., et al., Rapamycin:a bone sparing immunosuppressant? J Bone Miner Res,1995.10(5):p.760-8.
[29]. Morales, J.M., J.M. Campistol and E. Al., Sirolimus-based therapy with or without cyclosporine long-term follow-up in renal transplant patients. Transplantation Proceedings,2005(37):p.693-6.
[30]. R, S., S. J and E. Al., Replacing calcineurin inhibitors with mTOR inhibitors in children. Pediatr Transplantation,2005(9):p.391-97.
[31]. Flechner, S.M., D. Goldfarb and E. Al., Kidney transplantation without calcineurin inhibitor drugs a prospective, randomized trial of sirolimus versus cyclosporine. Transplantation,2002.8(74):p.1070-6.
[32]. Alvarez-Garcia, O., et al., Rapamycin retards growth and causes marked alterations in the growth plate of young rats. Pediatric Nephrology,2007. 22(7):p.954-961.
[33]. J., S.G., et al., Wnt signalling in osteoblasts regulates expression of the receptor activator of NF kappa B ligand and inhibits osteoclastogenesis in vitro. Journal of Cell Science,2006.7(119):p.1283-96.
[34]. Sanchez, C.P. and Y.Z. He, Bone growth during rapamycin therapy in young rats. BMC Pediatr,2009.9:p.3.
[35]. Holstein, J., M. Klein and E. Al., Rapamycin affects early fracture healing in mice. British Journal of Pharmacology,2008(154):p.1055-62.
[36]. Singha, U.K., Y. Jiang and E. Al., Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3-E1 cells and primary mouse bone marrow stromal cells. Journal of Cellular Biochemistry,2008(103):p.434-46.
[37]. Isomoto, S., et al., Rapamycin as an inhibitor of osteogenic differentiation in bone marrow-derived mesenchymal stem cells. Journal of Orthopaedic Science,2007.12(1):p.83-88.
[38]. Ogawa, T., et al., Osteoblastic Differentiation Is Enhanced by Rapamycin in Rat Osteoblast-like Osteosarcoma (ROS 17/2.8) Cells. Biochemical and Biophysical Research Communications,1998.249(1):p.226-230.
[39]. Lee, et al., Rapamycin promotes the osteoblastic differentiation of human embryonic stem cells by blocking the mTOR pathway and stimulating the BMP/Smad pathway. Stem cells and Development,2010.4(19):p.557-68.
[40]. Martin, S.K., et al., NVP-BEZ235, A Dual Pan Class I PI3 Kinase and mTOR Inhibitor, Promotes Osteogenic Differentiation in Human Mesenchymal Stromal Cells. Journal of Bone and Mineral Research,2010.10(25):p. 2126-37.
[41]. Sugatani, T. and K.A. Hruska, Aktl/Akt2 and mammalian target of rapamycin/bim play critical roles in osteoclast differentiation and survival, respectively, whereas Akt is dispensable for cell survival in isolated osteoclast precursors. The Journal of Biological Chemistry,2005.5(280):p.3583-9.
[42]. Glantschnig, H., J.E. Fisher and G. Wesolowski, M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase. Cell Death And Differentiation,2003.10(10):p.1165-77.
[43]. Junrong, M., et al., Hyperactivation of mTOR critically regulates abnormal osteoclastogenesis in neurofibromatosis type 1. Journal of Orthopaedic Research,2012.1(30):p.144-52.
[44]. A, O.S.A.L. and E.GA. Pez, Rapamycin induces growth retardation by disrupting angiogenesis in the growth plate. Kidney International,2010(78):p. 561-8.
[45]. Shui, C., B.L. Riggs and S. Khosla, The immunosuppressant rapamycin, alone or with transforming growth factor-beta, enhances osteoclast differentiation of RAW264.7 monocyte-macrophage cells in the presence of RANK-ligand. Calcified Tissue Int,2002.5(71):p.437-46.
[46]. Hofbauer, L.C., et al., Effects of Immunosuppressants on Receptor Activator of NF-κB Ligand and Osteoprotegerin Production by Human Osteoblastic and Coronary Artery Smooth Muscle Cells. Biochemical and Biophysical Research Communications,2001.280(1):p.334-339.
[47]. Mogi, M. and A. Kondo, Down-regulation of mTOR leads to up-regulation of osteoprotegerin in bone marrow cells. Biochemical and Biophysical Research Communications,2009.384(1):p.82-86.
[48]. Okui, et al., Antitumor Effect of Temsirolimus against Oral Squamous Cell Carcinoma Associated with Bone Destruction. Molecular Cancer Therapeutics, 2010.11(9):p.2960-69.
[49]. Smink, J.J., et al., Transcription factor C/EBPbeta isoform ratio regulates osteoclastogenesis through MafB. EMBO J,2009.28(12):p.1769-81.
[50]. Smink, J.J. and A. Leutz, Rapamycin and the transcription factor C/EBPβ as a switch in osteoclast differentiation:implications for lytic bone diseases Journal of Molecular Medicine,2010.88(3):p.227-233.
[51]. Ng, et al., CCAAT/Enhancer Binding Protein Beta Is Up-Regulated in Giant Cell Tumor of Bone and Regulates RANKL Expression. Journal of Cellular Biochemistry,2010.2(110):p.438-46.
[52]. Byon, C.H., Y. Sun and E. Al., Runx2-Upregulated Receptor Activator of Nuclear Factor κB Ligand in Calcifying Smooth Muscle Cells Promotes Migration and Osteoclastic Differentiation of Macrophages. Arterioscl Throm Vas,2011.6(31):p.1387-96.
[53]. Enomoto, H., et al., Induction of osteoclast differentiation by Runx2 through receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin regulation and partial rescue of osteoclastogenesis in Runx2-/-mice by RANKL transgene. J Biol Chem,2003.278(26):p.23971-7.
[54]. De Toni, et al., OPG is regulated by beta-catenin and mediates resistance to TRAIL-induced apoptosis in colon cancer. Clinical cancer Research,2008. 15(14):p.4713-8.
[55]. Suda, T., T. Nagasawa and E. Al., Regulatory roles of b-catenin and AP-1 on osteoprotegerin production in interleukin-la-stimulated periodontal ligament cells.2009. p.384-9.
[1]Silvia Arteaga, A.A.R.E., Larrea tridentata (Creosote bush), an abundant plant of Mexican and US-American deserts and its metabolite nordihydroguaiaretic acid. Journal of Ethnopharmacology,2005(98):p.231-239.
[2]Sengupta, S., T.R. Peterson and D.M. Sabatini, Regulation of the mTOR Complex 1 Pathway by Nutrients, Growth Factors, and Stress, molecular cell, 2010(40):p.310-322.
[3]Inoki, K., et al., Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes & Development,2003(17):p.1829-1834.
[4]Inoki, K., T. Zhu and K. Guan, TSC2 mediates cellular energy response to control cell growth and survival. Cell,2003(115):p.577-590.
[5]Ma, X.M. and J. Blenis, Molecular mechanisms of mTOR-mediated translational control. Molecular Cell Biology,2009(10):p.307-318.
[6]Inoki, K., M.N. Corradetti and G. Kun-Liang, Dysregulation of the TSC-mTOR pathway in human disease. Nature Genetics,2005.1(37):p.19-24.
[7]Sarbassov, D.D., et al., Phosphorylation and Regulation of AktPKB by the Rictor-mTOR Complex 1. Science,2005(307):p.1098-1101.
[8]A., D., Guertin and D.M. Sabatini, Defining the role of mtor in cancer. Cancer Cell,2007(12):p.9-22.
[9]Soulard, A. and M.N. Hall, SnapShot mTOR signaling. Cell,2007(129):p.434.
[10]Tsang, C.K., et al., Targeting mammalian target of rapamycin (mTOR) for health and diseases. Drug Discov Today,2007(12):p.112-124.
[11]Sancak, Y., et al., The Rag GTPases Bind Raptor and Mediate Amino Acid Signaling to mTORCl. Science,2008(320):p.1496-1501.
[12]Kim, D., et al., mTOR Interacts with Raptor to Form a Nutrient-Sensitive Complex that Signals to the Cell Growth Machinery. Cell,2002(110):p. 163-175.
[13]Meyer, A.N., C.W. McAndrew and D.J. Donoghue, Nordihydroguaiaretic Acid Inhibits an Activated Fibroblast Growth Factor Receptor 3 Mutant and Blocks Downstream Signaling in Multiple Myeloma Cell. Cancer Res,2008(68):p. 7361-7370.
[14]Fujimoto, N., et al., Estrogenic activity of an antioxidant, nordihydroguaiaretic acid (NDGA).lIFE Sci,2004(74):p.1417-1425.
[15]Li, F., et al., Nordihydroguaiaretic acid inhibits transforming growth factor beta type 1 receptor activity and downstream signaling. European Journal Of Pharmacology,2009(616):p.31-37.
[16]Lee, C., et al., Nordihydroguaiaretic acid, an antioxidant, inhibits transforming growth factor-beta activity through the inhibition of Smad signaling pathway. Experimental Cell Research,2003(289):p.335-341.
[17]Park, S. and J. Lee, Inhibitory effect of nordihydroguaiaretic acid on β-catenin. Cell Biochem Funct,2011(29):p.22-29.
[18]Efeyan, A. and D.M. Sabatini, mTOR and cancer,many loops in one pathway. Current Opinion In Cell Biology,2010(22):p.169-176.
[19]Dowling, R.J.O., et al., Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res, 2007(67):p.10804-10812.
[20]Sarbassov, D.D., et al., prolonged rapamycin treatment inhibits mTOR2 assembly and AktPKB. Molecular Cell,2006(22):p.159-168.
[21]Sarbassov, D.D., et al., Phosphorylation and Regulation of Akt PKB by the Rictor-mTOR Complex. Science,2005(307):p.1098-1101.
[22]Julien, L. and P.P. Roux, mTOR, the mammalian target of rapamycin. Medicine Science,2010(26):p.1056-1060.
[23]Thoreen, C.C. and D.M. Sabatini, Rapamycin inhibits mTORC1, but not completely. Autophagy,2009(5):p.725-726.
[24]Sarbassov, D.D. and D. Sabatini, Redox Regulation of the Nutrient-sensitive Raptor-mTOR. The Journal of Biological Chemistry,2005(280):p. 39505-39509.
[25]Neve, R.M., A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell,2006(10):p.515-527.
[26]Wang, W. and K.L. Guan, AMP-activated protein kinase and cancer. Acta Physiologica,2009(196):p.55-63.
[27]Thomson, D.M., C.A. Fick and S.E. Gordon, AMPK Activation Attenuates S6K1,4E-BP1, and eEF2 Signaling Responses to High-frequency Electrically Stimulated Skeletal Muscle Contractions. J Appl Physiol,2008.
[28]Li, Y., et al., TSC2 filling the GAP in the mTOR. TRENDS Biochem Sci, 2004(29):p.32-38.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |