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上皮间质转化

IGF-1-induced epithelial –mesenchymal transition in MCF-7cells is mediated by MUC1

Gaoyong Liao a ,b ,c ,d ,1,Mengyu Wang b ,c ,d ,1,Yu Ou a ,?,Yong Zhao b ,c ,d ,?

School of Life Science and Technology,China Pharmaceutical University,Nanjing,China b

Simcere Pharmaceutical Co.,Ltd.,Nanjing,Jiangsu,China c

BioSciKin Co.,Ltd.,Nanjing,Jiangsu,China d

MtC BioPharma Co.,Ltd.,Nanjing,Jiangsu,China

a b s t r a c t

a r t i c l e i n f o Article history:

Received 3June 2014Accepted 18June 2014

Available online 24June 2014Keywords:IGF-1MUC1EMT

Migration PI3K/Akt ERK

Metastases are the major cause of death from cancer.IGF-1signaling pathway has been shown to have strong implication in the epithelial –mesenchymal transition (EMT)process.However,the mechanisms of how IGF-1promotes EMT have not been fully elucidated.Mucin 1(MUC1),a transmembrane glycoprotein,engages in multiple cancer-related signaling pathways and functions as an oncoprotein that contributes to metastases.Here we provide evidence showing that IGF-1upregulates MUC1expression in MCF-7cells in a PI3K/Akt signal-ing pathway-dependent manner.The overexpression of MUC1is critical for IGF-1-induced EMT of MCF-7cells because the knockdown of MUC1prevented the EMT of MCF-7cells as demonstrated by various EMT markers including the expression of E-cadherin,N-cadherin,vimentin,?bronectin and the nuclear translocalization of β-catenin.On the other hand,the knockdown of MUC1had no impact on IGF-1-induced activation of PI3K/Akt or MAPK.In summary,our study demonstrated MUC1as a critical downstream effector that mediates IGF-1-induced EMT of MCF-7cells and suggested that MUC1might be a potential therapeutic target for preventing tumor metastases.

1.Introduction

Metastases are the major cause of death from cancer.Numerous stud-ies have generated insights into the molecular basis of tumor metastases such as local invasion,circulation and the adaptation to distal tissue microenvironments [1].The initiation phase of metastases is known as epithelial –mesenchymal transition (EMT),which involves the break-down of cell –cell junctions responsible for maintaining epithelial cell integrity and the loss of cell polarity,generating individual cells with multiple mesenchymal attributes and invasive capability [1,2].

While multiple signaling pathways are known to contribute to the EMT process [3],IGF-1signaling pathway is of particular interest to us because it has been shown to have strong implication in tumor metasta-sis [4–8].For instance,in breast cancer,IGF-1R overexpression was observed in 44%of breast cancer tissue specimens,and high IGF-1level appears to be closely associated with tumor metastasis and poor prognosis [7].On the other hand,attenuation of IGF-1R signaling negatively regulated tumor metastasis in mouse models [9,10].Recent-ly,it has been suggested that IGF-1regulates matrix metalloproteinase

(MMP)activity via the PI3K and the MAPK pathways in human breast cancer cell line MCF-7.The IGF-1signaling pathway has been found to interact with the TGF-βpathway,contributing to the invasiveness and EMT of MCF-7[6].

In recent years,accumulating evidence show that Mucin 1(MUC1),a heterodimeric transmembrane glycoprotein composed of αand βsub-units,engages in multiple cancer-related signaling pathways [11].MUC1α,also known as cancer antigen 15.3(CA15.3),has been used as a serum biomarker for breast cancer.In tumor cells,MUC1is often aber-rantly highly expressed,underglycosylated,and no longer restricted to apical side of the cell.These changes facilitate the interaction between MUC1αand extracellular matrix component ICAM-1[12,13].The aberrant MUC1expression not only promotes the metastatic spread of the cancer cells by enhancing the adhesion of circulating cancer cells to the blood vessels,but also leads to the activation of Src pathway,which is a key mediator of metastatic progression [14,15].

Interestingly,recent studies have found that the transmembrane βsubunit of MUC1functions as an oncoprotein and plays more important role in tumor progression [16–18].MUC1βis involved in multiple path-ways that are important to metastases [16].Of note,the overexpression of MUC1βin MUC1-C transgenic mice results in the acquisition of an invasive tumorigenic phenotype in the mammary gland [19],while knockdown of MUC1in Wnt-overexpressed mice relieves the tumori-genic phenotype in the mammary gland [20].

Cellular Signalling 26(2014)2131–2137

?Corresponding authors.Fax:+862585305535.

E-mail addresses:ouyu2008@https://www.wendangku.net/doc/bd13687442.html, (Y.Ou),zhaoyong@https://www.wendangku.net/doc/bd13687442.html, (Y.Zhao).1

These authors contributed equally to this

work.https://www.wendangku.net/doc/bd13687442.html,/10.1016/j.cellsig.2014.06.004

0898-6568/?2014Elsevier Inc.All rights

reserved.

Contents lists available at ScienceDirect

Cellular Signalling

j o u r n a l h o me p a g e :ww w.e l s e v i e r.c o m /l o c a t e /c e l l s i g

Many studies have been done to explore the molecular mechanisms of how MUC1is upregulated in malignant cells.For example,MUC1can be regulated by transcriptional factor HIF1-αand STATs in response to hypoxia,INFγ,IL-6or EGFR stimulation[21–25].MUC1promoter also contains a consensus androgen receptor(AR)-element,which allows MUC1to be regulated by AR in AR-sensitive cell lines[26].Gene ampli-?cation often contributes to elevated level of MUC1in malignant cells [27].In addition,MUC1expression also appears to be regulated by tumor-related microRNAs both positively and negatively[28,29].

Despite of the above studies,the relationship between IGF-1-induced EMT and MUC1has not been reported.In this study,we inves-tigated the role of MUC1in IGF-1-induced EMT.

2.Material and methods

2.1.Cells and antibodies

MCF-7cell line was purchased from the American Type Culture Collection(Manassas,VA)and cultured in Dulbecco's modi?ed Eagle's medium(DMEM,Hyclone)supplemented with10%fetal bovine serum(FBS,Gibico),100U/ml penicillin,and100μg/ml streptomycin. When indicated,cells were treated with IGF-1(Peprotech),EGF (Peprotech),FGF(Invitrogen),HGF(R&D),cycloheximide(CHX, Sigma),U0126(Santa Cruz)or LY294002(Santa Cruz).

Antibodies used for immunoblot include MUC1-C antibody(Ab5, Neomarkers),anti-p-ERK1/2,anti-p-Akt(Cell Signaling Technology), anti-β-catenin(Santa Cruz),anti-GAPDH(Beyotime),and horseradish peroxidase-conjugated secondary antibodies(Sigma).

2.2.Plasmid construction and cell transfection

shRNA-MUC1-CD(5′-GGATCCCGGTACCATCAATGTCCACGTTGATA TCCGCGTGGACATTGATGGTACCTTTTTTCCAAAAGCTT-3′)was cloned into the site of the BamHI–HindIII site of the U6vector(Genscript). The plasmid was transfected into MCF-7cells using TransIT-BrCa Trans-fection Reagent(Mirus)according to the manufacture's protocol.Brief-ly,MCF-7cells were plated in12-well plates at150,000cells/well24h before transfection.Transfection was carried out by adding a2:1(v/w) of TransIT-BrCa Reagent/DNA mixture in a?nal volume of100μl into the cultured cells.After24h incubation,cells were applied for various experiments.

2.3.Cell migration assay

Cell migration assays were performed using scratch wound healing test or Oris Cell Migration Assay Kit(Platypustech).For scratch wound healing test,MCF-7cells were cultured to90%con?uence in6-well plates.Scratches were made using a200μl pipet tip.The cells were then cultured in serum-starved condition for24h before stimulated with different growth factors and continued culturing for up to72h.Im-ages of the cells were taken at selected time points to evaluate the scratch wound healing condition.For Oris Cell Migration Assays,the cells were cultured under similar condition and the kit manual was followed.Cell images were captured and analyzed by ImageXpress Micro system(Molecular Devices).2.4.Immuno?uorescence assay

Immuno?uorescence staining ofβ-catenin was performed as de-scribed previously[30].In brief,MCF-7cells were?xed in ice-cold 100%ethanol overnight,washed with PBST,blocked with5%milk in PBST,and incubated with rabbit anti-β-catenin monoclonal antibody (Santa Cruz)overnight.After washing,cells were incubated with Alexa Fluor594goat anti-rabbit IgG(H+L)(Invitrogen).Fluorescence signal was measured and analyzed by ImageXpress Micro System.

2.5.Nuclear protein extraction

Whole-cell or nuclear lysates forβ-catenin detection were isolated using nuclear and cytoplasmic protein extraction kit(Beyotime).The isolated proteins were then analyzed using immunoblotting.

3.Results

3.1.IGF-1stimulates MCF-7migration and MUC1expression

MCF-7cells were treated with EGF,FGF,HGF or IGF-1at various con-centrations.All of the growth factors promoted cell migration to a differ-ent extent,with the best stimulative effect seen for IGF-1(Fig.1A).IGF-1 promotes the mobility of MCF-7in a dose-dependent manner and at the concentration of200ng/ml,the cells completely occupied the well, while none of the other growth factors achieved similar effect in the range of concentration tested.The strong migration-promoting activity of IGF-1was also seen in a time-course scratch wound healing assay (Fig.1B).In addition,MCF-7cells treated with IGF-1exhibited striking morphology changes which are expected to be seen in cells undergoing a transition to an invasive status(Fig.1C).

Interestingly,it was noticed that IGF-1treatment led to a strong induction of MUC1expression(Fig.1D,left?gure).This induction is dependent on protein synthesis because CHX treatment completely blocked the MUC1accumulation(Fig.1D,right?gure).In addition,it ap-pears that the ability of the growth factors to induce MCF-7migration is correlated with their abilities to induce MUC1overexpression,because IGF-1which induced the strongest MUC1expression also has the most potent cell migration-promoting effect,while HGF which only slightly induced MUC1expression has the weakest migration-promoting activ-ity.The above results led to the hypothesis that IGF-1-stimulated MCF-7 migration is mechanistically linked to MUC1's function.

3.2.IGF-1induces the epithelial–mesenchymal transitions of MCF-7in a MUC1-dependent manner

IGF-1has been reported to induce EMT of multiple tumor cell lines including MCF-7.This phenomenon was also seen in our study.Treating MCF-7with IGF-1resulted in the changes of several EMT biomarkers, including the elevated expression of vimentin and?bronectin,the loss of E-cadherin(Fig.2A)and nuclear localization ofβ-catenin(Fig.2B and C).Notably,IGF-1-induced MUC1overexpression appeared to precede the changes seen for EMT biomarkers.

To understand the role of MUC1overexpression in IGF-1-induced EMT,we next investigated whether the knockdown of MUC1-C has any impact on the IGF-1-induced EMT.As shown in Fig.3A,transfection of MCF-7with the MUC1siRNA dramatically inhibited cell migration promoted by IGF-1.In line with the migration behavior,MUC1siRNA

Fig.1.Stimulation of MCF-7breast cancer cells with growth factors induces cell migration and MUC1expression.(A)IGF-1exhibited good activity in promoting the migration of MCF-7 cells.The ability of EGF,FGF,HGF and IGF-1in promoting the migration of MCF-7cells is quantitated by using Oris Cell Migration Assay Kit.The cells were treated with the growth factors at indicated concentrations for72h.The vacancy area in each well was measured,normalized and schematically represented to the right.(B)Potent migration-stimulating activity of IGF-1 on MCF-7cells as demonstrated in a time-course scratch wound healing assay.36h post the addition of200ng/ml IGF-1,the scratch mark was completely sealed while little change was seen in the control group.(C)IGF-1induced the morphological changes of MCF-7cells.MCF-7cells were treated with200ng/ml IGF-1and images were taken at indicated time points.S1 and S2show the enlarged images of circled regions.(D)IGF-1induced MUC1expression in MCF-7cells.MCF-7cells were stimulated with200ng/ml EGF,FGF,HGF and IGF-1respectively for an indicated period of time(left?gure).MCF-7cells were stimulated with IGF-1in the presence and absence of10μg/ml CHX for the indicated times(right?gure).Lysates were immunoblotted with anti-MUC1-C and anti-GAPDH.

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signi ?cantly blocked MUC1expression and reversed the IGF-1-induced upregulation of ?bronectin,vimentin and N-cadherin,and the down-regulation of E-cadherin (Fig.3B),suggesting that the IGF-1-induced EMT was blocked upon MUC1silencing.Notably,silencing of MUC1de-creased nuclear location of β-catenin with or without IGF-1(200ng/ml)(Fig.3C).This phenomenon was also supported by immunoblot assay which showed that MUC1siRNA treatment decreased the nuclear portion of β-catenin without affecting the cytoplasmic β-catenin (Fig.3D).Taken together,the IGF-1-induced overexpression of MUC1is associated with the activation of the β-catenin pathway which is crucial to EMT in MCF-7cells.

3.3.IGF-1induces MUC1expression via PI3K/Akt pathway

PI3K/Akt pathway and MAPK pathway are the two major signaling pathways downstream of IGF-1stimulation.To elucidate the potential involvement of PI3K and/or ERK regulation in IGF-1-induced MUC1ex-pression,we treated MCF-7cells with IGF-1in the presence or absence of U0126or LY294002,the MEK-and PI3K-speci ?c inhibitors,respec-tively,and checked MUC1expression.As shown in Fig.4A,IGF-1strong-ly induced MUC1expression,as well as the activation of Akt and ERK as indicated by hyperphosphorylation of the proteins.However,LY294002but not U0126signi ?cantly decreased MUC1level in IGF-1-treated MCF-7cells (Fig.4C).These results suggest that the IGF-1-induced over-expression of MUC1is mediated by PI3K/Akt signaling pathway.

Next,we investigated whether the knockdown of MUC1has any im-pact on the activation of either PI3K/Akt pathway or MAPK pathway upon IGF-1stimulation.Interestingly,silence of MUC1-C oncoprotein could not affect the activation of PI3K/Akt and MEK/ERK signaling in IGF-1-treated MCF-7cells (Fig.4D).This result suggested that MUC1acts downstream of PI3K/Akt signaling pathway to promote IGF-1-induced EMT without apparent feedback effect.4.Discussion

It is well-known that an extensive array of cytokines and growth fac-tors are involved in EMT [3].While numerous studies have revealed the signaling pathways connecting the cell surface stimulation to patholog-ical behavior of EMT,our understanding for the molecular basis of EMT is far from comprehensive.In this study,we focus on the mechanism of IGF-1-induced EMT in human breast cancer.MCF-7cells originate from a low-aggressive form of breast cancer and express a high level of IGF-1R,which makes MCF-7a suitable model for the study.IGF-1stimula-tion increases the invasive potential of MCF-7cells by activating the PI3K and MAPK pathways,as well as the TGF-β1signaling [6].However,the detailed mechanisms that bridge these pathways to the molecular hallmarks of EMT progression upon IGF-1stimulation remain unclear.

Interestingly,we noticed that upon IGF-1treatment,MCF-7cells start to express high levels of MUC1in as short as 1h.In tumor cells,MUC1is aberrantly overexpressed and distributed and has

been

Fig.2.IGF-1-induced overexpression of MUC1precedes the changes of EMT markers in MCF-7cells.(A)IGF-1induced EMT in MCF-7cells.IGF-1treatment led to the decrease in E-cadherin expression and increase in the expression of N-cadherin,vimentin and ?bronectin,which are the markers of EMT.MUC1level started elevating as early as 60min post IGF-1treatment,which was earlier than the expression level changes of other EMT markers.(B)IGF-1induces nuclear localization of β-catenin.Images were taken at 10×magni ?cation.(C)The nuclear translocation of β-catenin after IGF-1treatment was also veri ?ed by Western blot.

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Fig.3.Silencing of MUC1inhibits the migration and EMT of IGF-1-treated MCF-7cells.(A)Silencing of MUC1counteracted IGF-1-induced migration of MCF-7.MCF-7cells were transfected with MUC1shRNA,followed by IGF-1treatment for 48h.shRNA treatment signi ?cantly reduced IGF-1-stimulated cell migration.(B)The changes of EMT markers induced by IGF-1were also reversed upon MUC1silencing.(C)and (D)The nuclear localization of β-catenin after IGF-1treatment was blocked upon MUC1silencing,as demonstrated by immuno ?uorescence staining (C)and Western blot

(D).

Fig.4.IGF-1upregulates MUC1via PI3K/Akt but not MAKP signaling pathway.IGF-1treatment activates both PI3K/Akt and MAKP signaling pathway in MCF-7cells.The cells were stim-ulated with 200ng/ml IGF-1for the indicated times.The peak of the activation of Akt and ERK is well ahead of MUC1overexpression.Activation of p-ERK or p-Akt was effectively inhibited by the speci ?c inhibitor U0126or LY294002.MCF-7cells were treated with gradient concentrations of inhibitors for 24h to determine the proper concentration to be applied in MCF-7.Inhibition of PI3K/Akt signaling pathway signi ?cantly blocked IGF-1-induced MUC1expression.MCF-7cells were stimulated with 200ng/ml IGF-1in the presence of 10μM U0126or 10μM LY294002for the indicated times.Silencing of MUC1has no obvious effects on IGF-1-mediated activation of PI3K/Akt or MAKP signaling pathway in MCF-7cells.

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associated with high metastasis and poor prognosis [10,11,16].While MUC1is a transmembrane glycoprotein without any kinase activity,it was found in recent years that the cytoplasmic domain of MUC1,MUC1-C,interacts with multiple cancer-related pathways including EGFR pathway,Wnt/β-catenin pathway,PI3K/Akt pathway and MEK/ERK pathway [31].These facts promote us to further investigate the role of MUC1in IGF-1-induced EMT.

Surprisingly,it is noticed that the upregulation of MUC1level ap-pears to be earlier than the changes of hallmarks of EMT progression (E-cadherin,N-cadherin,?bronectin and vimentin).More importantly,the knockdown of MUC1signi ?cantly reduced the expression level changes seen for those EMT markers,and partially inhibited the mobil-ity changes of MCF-7cells after IGF-1treatment.The partial inhibition of the cell mobility maybe due to (1)the incomplete silencing of MUC1and (2)the involvement of other mechanisms that promotes cell mobil-ity upon IGF-1stimulation.All these evidences suggest that MUC1is a downstream effector that is required for IGF-1-induced EMT in MCF-7cells.

This notion is supported by the fact that PI3K antagonist LY294002could also suppress the overexpression of MUC1,while neither the overexpression (data not shown)nor the silencing of MUC1-C has any impact on the phosphorylation of Akt or MAPK (Fig.4D).

The exact mechanism of how MUC1overexpression is linked to IGF-1-induced EMT progression remains to be elucidated.One possible mechanism is through β-catenin,which is well-known for its role in EMT progression [32,33].Our study showed that MUC1-C was required for the nuclear translocation of beta-catenin upon IGF-1stimulation.This result is consistent with the ?nding by other lab.Yamamoto M.et al.reported that the MUC1-C subunit contains a SAGNGGSSLS motif which binds directly to the β-catenin in the cytoplasm.This binding sta-bilizes β-catenin and activates the complex translocation to nuclear [34].The interaction between MUC1-C and β-catenin promotes mam-mary gland tumorigenesis in MUC1-overexpressed transgenic mice [35].In addition,it has been reported that the interaction of MUC1-C with cofactors such as β-catenin,p120-catenin and estrogen receptor

βpromotes their nuclear translocation,and induces expression of EMT genes [16].A hypothetical mechanism of how MUC1is involved in IGF-1-induced EMT and cancer metastasis is schematically presented in Fig.5.

In summary,our study proved a critical role of MUC1in IGF-1-induced EMT of MCF-7cells and suggested that MUC1might be a poten-tial therapeutic target for preventing tumor metastases.While our study uses MCF-7cells as a model,the mechanism revealed here might be applied to other cells because it was found that IGF-1also can increase the expression level of MUC1on MDA-MB-468breast cancer cell line (Supplementary Fig.1)and promotes its metastasis as well [36].Further studies are needed to check whether MUC1also contributes to the metastasis-promoting activity by other growth factors.

Supplementary data to this article can be found online at https://www.wendangku.net/doc/bd13687442.html,/10.1016/j.cellsig.2014.06.004.Acknowledgment

We thank Dr.Stewart Leung and Dr.Jing Jin for proof reading the manuscript.References

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G.Liao et al./Cellular Signalling26(2014)2131–2137

TGFβ1诱导肺泡上皮细胞间质转化

TGF-β1诱导肺泡上皮细胞间质转化作者：黄振杰,郑金旭,莫凯天,苏石芳作者单位：广西北海市人民医院呼吸科;江苏大学附属医院呼吸科;上海市第八人民医院消化科【摘要】目的：通过观察TGF- 1诱导下A549细胞出现的细胞形态学和E-cad表达的变化，探讨上皮细胞-间质细胞转化(epithelial-mesenchymal transition,EMT)过程在肺纤维化发病机制中的作用。方法：体外培养A549细胞，以TGF- 1进行干预，收集不同时段的细胞，应用荧光实时定量PCR(RT-PCR)检测TGF- 1干预前后E-cad的mRNA表达变化;倒置相差显微镜观察细胞形态学的变化;间接免疫荧光观察E-cad蛋白表达的变化。结果：倒置相差显微镜观察到TGF- 1干预后A549细胞由鹅卵石状变为梭形，形态如同肌纤维母细胞。间接免疫荧光显示A549细胞的E-cad表达(红色荧光染色)随时间延长逐渐减少。RT-PCR显示E-cad的mRNA表达下调(P 0.05)。结论：TGF- 1在体外诱导肺泡上皮细胞向间质细胞转化,肺泡上皮细胞间质转化是肺纤维化的重要发病机制之一。【关键词】转化生长因子1,A549细胞,上皮细胞间质转化,肺纤维化 Abstract：ObjectiveBy observing the cellular morphology changes and the expression of E-cad after A549 cells were treated with transforming growth factor 1(TGF- 1)，to investigate the role of epithelial-mesenchymal transition (EMT) in the pathogenesy of pulmonary fibrosis.MethodA549 cells cultured in vitro were treated by TGF- 1,then harvested at different time points to assay mRNA expression of E-cad by real-time PCR (RT-PCR) before and after A549 cells being treated by TGF- 1.Cellular morphology changes were observed by phase-contrast microscope.Protein expression of E-cad by indirect immunofluorescence.ResultsAfter being treated by TGF- 1，A549 cells were observed by inverted phase contrast microscope to turn from pebble shape to fusiform shape，a myofibroblast-like morphology.Indirect immunofluorescence showed protein expression of E-cad reduced as time went by(red stain).The mRNA expression of E-cad was down -regulated in RT-PCR(P 0.05).ConclusionTGF- 1 induced EMT of alveolar epithelial cells in vitro suggests that EMT of alveolar epithelial cells might be one of fundamental mechanisms of pulmonary fibrosis. Key Words:Transforming growth factor A549 cells;Epithelial- mesenchymal transition;Pulmonary fibrosis 特发性肺纤维化(IPF) 是最常见的肺间质疾病,也是肺间质纤维化的主要原因。多数肺间质疾病病因不明,其发病机制目前也未完全阐明。传统观点认为各种损伤因素损伤肺泡上皮细胞和毛细血管内皮细胞，炎症细胞浸润，细胞因子失衡，肺实质损伤后继发肺间质细胞增殖过度修复导致纤维化。国外有文献报道IPF是一种涉及异常创伤愈合的功能紊乱，进行性的上皮损伤和(或)激活可能处于纤维形成和间质细胞增殖的核心位置，这种作用是不依赖于炎症的[1-2]。肺泡上皮细胞(AECs)不仅仅是作为发病的促动因素，它们本身就可以通过一个叫做上皮细胞-间质转化(epithelial-mesenchymal transition，EMT)的过程获得间质细胞表型而作为成纤维细胞和肌纤维母细胞的重要来源。在这个新的模式中，肺泡上皮应该被看作纤维化的关键环节之一，它作为一个多能的干细胞具有相当的可塑性，能参与到交替的途径中：上皮再生修复正常的肺泡结构，凋亡，或者通过EMT形成纤维化。本研究通过用TGF- 1诱导A549从肺泡上皮细胞向间质细胞转化，进一步探讨肺泡上皮细胞在肺纤维化发病中的作用,为阐明肺纤维化发病的分子生物学机制及寻求新的治疗方法提供依据。

内皮细胞间质转化的信号调节和生物学意义

622 中国循环杂志 2016年6月第31卷第6期（总第216期）Chinese Circulation Journal，June，2016，Vol. 31 No.6（Serial No.216）内皮细胞间质转化的信号调节和生物学意义何光庭、吴洁综述，黄巧冰审校内皮细胞间质转化（Endo-MT）是内皮细胞间连接丢失、细胞形态改变和迁移进入周围组织的生物学过程。Endo-MT 是上皮细胞间质转化（EMT）的一种，是新近发现的一种细胞转化类型，早期研究显示Endo-MT 在胚胎期心内膜的发育中发挥了十分关键的作用[1]。然而，近来研究显示Endo-MT 与EMT 一样，也参与各种疾病的发生发展，如肿瘤及心、肺、肾等重要器官的纤维化。1 Endo-MT 的信号调节 Endo-MT 过程中，内皮细胞失去特异性抗原，如血小板-内皮细胞黏附因子（CD31）和VE-钙黏蛋白（VE-cadherin），获得间质细胞抗原，如ɑ-平滑肌肌动蛋白（ɑ-SMA）、波形蛋白和成纤维细胞特异性蛋白1（FSP1），同时其功能发生明显改变,并获得较强增殖或迁移能力。转化生长因子- β（TGF-β）、Notch 信号、Wnt 信号以及MicroRNAs 等参与对Endo-MT 的调节。1.1 TGF-β信号通路 TGF-β是转化生长因子-β超家族蛋白中的一种，属于多功能蛋白质，可以影响细胞的生长、分化、凋亡等过程。另外，它还参与体内各种疾病如癌症[2]、心血管疾病[3]和组织纤维化[4]的发生。TGF-β包括三个亚型：TGF -β1、TGF -β2和TGF -β3。TGF -β可以结合到细胞表面的TGF -β受体（TGF-βR）从而将其激活，TGF-βR 通过Smad 依赖信号通路调控细胞内复杂的信号反应。 TGF-β和骨形态发生蛋白（BMPs）均是通过具有高亲和力的跨膜I 型和II 型丝氨酸/苏氨酸激酶受体的联合作用介导信号从膜表面传递到细胞核[5]。配体和Ⅱ型受体结合形成异四聚体后，磷酸化I 型受体使其活化。磷酸化的I 型受体继而通过磷酸化细胞质中一组被称为Smads（TGF-β信号通路的主要胞内中介）的蛋白质使信号传递下去。TGF-β I 型受体使Smad2和Smad3磷酸化，而BMP I 型受体则引起Smad1、Smad5和Smad8磷酸化。Smad1,2,3,5,8为受体摘要在内皮细胞间质转化（Endo-MT）过程中，内皮细胞间连接丢失，失去内皮细胞特异性标志物血小板-内皮细胞黏附因子（CD31）和VE-钙黏蛋白（VE-cadherin），获得间质细胞特异性标志物ɑ-平滑肌肌动蛋白(ɑ-SMA)、波形蛋白和成纤维细胞特异性蛋白1( FSP1)等，并获得间质细胞运动和收缩的特性，由原来的鹅卵石样结构转变为细长梭形结构，同时其侵袭力明显增强。转化生长因子-β、Notch 信号、Wnt 信号以及MicroRNAs 等参与对内皮间质转化的调节。Endo-MT 参与了各种疾病，如肿瘤、心、肺、肾等重要器官纤维化的发生、发展。关键词综述；内皮细胞；信号；肿瘤；纤维化调节性Smads，它们磷酸化后与共同Smad 即Smad4发生联系。这些杂聚肽Smad 复合体入核后通过与其他转录因子协同作用来调节特异基因的转录应答，调控Endo-MT 发生[6]。TGF-β通过Smad4依赖的信号途径，上调细胞内鸟嘌呤核苷酸交换因子Arhgef5的表达，参与对Endo-MT 的调控；研究中特异性下调Arhgef5的表达水平，α-SMA 的表达部分减少，但并没有被完全抑制，说明在这个过程中可能还有其他因子参与[7]。α-SMA 启动子含有心肌相关转录因子（MRTF-A）的作用位点CArG 盒[CC（A/T）6 GG]，当TGF-β诱导微血管内皮细胞发生Endo-MT，MRTF-A 与血清应答因子（SRF）形成稳定的三元络合物结合到此位点，诱导α-SMA 表达，间接参与调控Endo-MT；当这个位点发生突变，间质细胞标志物表达亦受到抑制[8] 。体内炎症因子[9]、血流高切应力[10]等也能够触发细胞内TGF-β/Smad 信号反应，调节Endo-MT。除了Ⅰ型和Ⅱ型受体外，参与TGF-β信号通路调控的还有辅助受体，如TGF-βR Ⅲ和内皮糖蛋白（endoglin），它们通过配合或增强配体和信号受体结合来调节TGF-β信号通路，调节信号在细胞内的传递和定位，参与调控Endo-MT。 1.2 Notch 信号通路 Notch 和TGF-β一样，能够诱导体外培养的内皮细胞发生Endo-MT。它通过调节转录因子Snail、Slug 以及ZEB1的表达介导Endo-MT。Notch 上调内皮细胞中Snail 和Slug 蛋白的表达[11]，Snail 和Slug 是已知能抑制内皮细胞黏附分子VE-cadherin 表达的转录因子，VE-cadherin 在维持内皮细胞间的稳定中发挥关键作用，当它的表达受到抑制，内皮细胞间黏附连接被破坏，内皮细胞更容易发生表型转变。有研究表明[12]，在房室管发育中，Notch 信号通路被激活，诱导鸟苷酸环化酶异二聚体亚基Gucy1a3和Gucy1b3转录，Gucy1a3和Gucy1b3构成NO 受体，同时促使内皮细胞分泌整合素A，进而激活磷脂酰肌醇3-激酶/蛋白激酶B（PI3-kinase/ 综述

上皮-间质细胞转化的分子机制及其在肿瘤转移中的作用

龙源期刊网 https://www.wendangku.net/doc/bd13687442.html, 上皮-间质细胞转化的分子机制及其在肿瘤转移中的作用作者：笪正来源：《中外医学研究》2011年第29期【摘要】上皮细胞间质转化(epithelial-mesenchymal transition，EMT)是具有极性的上皮细胞转换成为具有移行能力的间质细胞并获得侵袭和迁移能力的过程，它存在于人体多个生理和病理过程中。上皮-间质转化(EMT)在恶性肿瘤的侵袭转移过程中起着关键的作用，研究EMT 的始发因素及其下游通路在肿瘤生长、侵袭、转移中的作用，阻断这一机制的发生发展，对恶性肿瘤的侵袭转移前的早期诊断、早期治疗有着非常重要的意义。【关键词】上皮-间质转化(EMT)；肿瘤侵袭；肿瘤转移；分子机制 EMT在医学中是一类生理组织变化状况，其一般是上皮细胞在特殊的情况下发生向间质细胞转化的形式，这种转化最大的特点在于失去上皮细胞表型、获得间质细胞特性等。从医学发展历史看，对于EMT的研究发现最早在发育生物学中，研究人员通过细胞实验总结出了相关的结论。经过长期实验发现，EMT对恶性肿瘤侵袭、转移、变化的影响较大，针对这一点，本文主要研究了EMT的发生机制以及其在肿瘤侵袭转移中的相关影响。 1EMT的概念在生物学研究工作深入开展的同时，人们对于各种生物学理念的认识更加充分。1982 年，Garry Greenburg［1］和Hay等［2］通过体外细胞实验获得了巨大的收获，发现晶状体上皮细胞在胶原凝胶中产生成伪足而出现出间质细胞的状态，EMT概念由此被提出来。若上皮细胞产生EMT之后，形态上由立方形上皮细胞则转化为梭形的间充质细胞的形态。同时，还观察到上皮细胞标志物的表达下调或者缺失，包括：E-钙黏蛋白(E-Cad)、黏蛋白、角蛋白、桥粒斑蛋白等；间质细胞标记物的表达上调，包括：波形蛋白、N-钙黏蛋白、纤连蛋白、表达上调。 2EMT的形成及肿瘤转移导致EMT产生的因素是多个方面的，其包括：蛋白分子、转录调节因子、MicroRNA等方面的变化。这些都会给患者的身体组织造成不利影响，容易使得肿瘤细胞被袭击而出现转移，由此增加了医生治疗的难度。 2.1E-钙黏蛋白(E-cad)钙连接素属于上皮组织中的细胞间跨膜黏连糖蛋白分子，其往往要借助于Ca2+才能发挥作用。钙连接素均参与了细胞间的连接，包括：E-cad、N-cad、P-cad三

上皮间质转化的分子标志物

四综述四 D O I :10.3760/c m a .j .i s s n .1673-436X.2012.017.019作者单位:200433同济大学附属上海市肺科医院呼吸内科通信作者:李惠萍,E m a i l :l i w 2013@126.c o m 上皮间质转化的分子标志物张霞李惠萍 ?摘要? 上皮间质转化(e p i t h e l i a lm e s e n c h y m a l t r a n s i t i o n ,E M T )参与胚胎发生与器官发育二组织修复与器官纤维化二肿瘤转移等多种生理病理过程,体现了上皮细胞的可塑性三用于E M T 过程标识的分子标志物多种多样,主要包括细胞表面标志物二细胞支架标志物二细胞外基质蛋白和转录因子这四类三对其中一些常见分子标志物进行检测,其表达在E M T 过程中或升或降;而明确分子标志物在不同类型 E M T 过程中的变化规律, 对E M T 相关疾病的研究具有重要意义三?关键词? 上皮间质转化; 分子标志物;器官纤维化;肿瘤B i o m a r k e r s o f e p i t h e l i a lm e s e n c h y m a l t r a n s i t i o n Z HA N GX i a ,L IH u i -p i n g .D e p a r t m e n t o f R e s p i r a t o r y M e d i c i n e ,t h e S h a n g h a iP u l m o n a r y H o s p i t a l ,T o n g j i U n i v e r s i t y ,S h a n g h a i 200433,C h i n a C o r r e s p o n d i n g a u t h o r :L IH u i -p i n g , E m a i l :l i w 2013@126.c o m ?A b s t r a c t ? E p i t h e l i a lm e s e n c h y m a l t r a n s i t i o n (E M T ),w h i c he x h i b i tt h e p l a s t i c i t y o fe p i t h e l i a l c e l l s ,p l a y s a ni m p o r t a n tr o l ei n e m b r y o n i c d e v e l o p m e n t ,t i s s u e r e p a i r ,o r g a n f i b r o s i s ,a n d t u m o r m e t a s t a s i s .Av a s t v a r i e t y o f b i o m a r k e r s h a v e b e e nu s e d t od e m o n s t r a t e a l l s u b t y p e s o fE M T ,a n dm a i n l y i n v o l v ec e l l -s u r f a c e m a r k e r s ,c y t o s k e l e t a l m a r k e r s ,e x t r a c e l l u l a r p r o t e i n s ,t r a n s c r i p t i o n f a c t o r s .T o e x a m i n e a f e wo f t h em o r e c o mm o nm a r k e r s ,s o m e o fw h i c h a r e a c q u i r e d a n d s o m e o fw h i c h a r e a t t e n u a t e d d u r i n g t r a n s i t i o n ,a n d t o c l e a r h o wb i o m a r k e r s c h a n g e i n d i f f e r e n t s u b t y p e s o f E M Tc a n p r o v i d e a v a l u a b l e t h e r a p e u t i c s t r a t e g y f o r t h e d i s e a s e s .?K e y w o r d s ? E p i t h e l i a lm e s e n c h y m a l t r a n s i t i o n ;B i o m a r k e r ;O r g a n f i b r o s i s ;T u m o r 上皮间质转化(e p i t h e l i a l m e s e n c h y m a l t r a n s i t i o n ,E MT )参与机体多个生理病理过程,尤其是在肝二肺二肾等器官纤维化二肿瘤侵袭与转移相关的E MT 成为了研究热点三体现了上皮细胞的可塑性三关于E MT 的研究多数是在某种特定条件刺激下进行的,主要判断依据为形态观察以及细胞标志物的检测,种类繁多而又缺乏可重复的特异性指标三那么这些不同条件刺激产生的E MT 之间有何异同,又有何内在联系?是否拥有共同的基础机制呢? 想要解答这些问题,了解各型E MT 的分子标志物的变化情况,对于E MT 相关疾病的研究具有重要意义三 1 E M T 概述 E MT 是指上皮细胞通过特定程序向间质细胞转化的生物学过程三是由G r e e n b e r g 和H a y 在 1982年首先发现的, 他们在凝胶中培养的晶状体上皮细胞失去了极性,转变为具有伪足的间质样细胞, 从而提出了E MT 的概念三随后的研究发现E MT 现象存在于人体多个生理和病理过程中,并依据E MT 的特定发生生物学环境二功能的不同及其对机体的不同影响将其大致分为3种类型[1 ]:Ⅰ型E MT 与胚胎发生二器官发育相关,一方面原始上皮细胞转化为间质细胞参与原肠胚形成和神经胚形成,另一方面通过与E MT 相反的间质上皮转化(m e s e n c h y a l e p i t h e l i a l t r a n s i t i o n ,M E T )过程产生次级上皮细胞,实现胚胎形成过程中细胞类型的多样化;Ⅱ型E MT 与创伤愈合二组织再生和器官纤维化相关,主要生物学作用是机体对创伤和炎症的应答,通过次级上皮细胞向成纤维细胞转化来修复组织损伤,一旦刺激消失,E MT 过程也随之停止,如果刺激持续存在,这一过程也将持续存在,最终导致器官纤维化;Ⅲ型E MT 与肿瘤侵袭和转移相关,是指与上皮细胞恶性肿瘤相关的表型转化,这类E MT 发生于部分恶性肿瘤细胞中,肿瘤上皮细胞通过E MT 获得了很强侵袭和转移能力,随血流转移至不同部位,进一步通过M E T 过程形成上皮细胞的肿瘤转移灶三至今对3种类型E MT 的研究中所涉四 8531四国际呼吸杂志2012年9月第32卷第17期 I n t JR e s p i r ,S e p t e m b e r 2012,V o l .32,N o .17

上皮间质转化的过渡形态

新闻性、★★☆☆创新性、★★★☆科学性★★★☆应用性★★★☆ 胚胎发育与癌症发展中的细胞可塑性变化有着惊人的相似性，而这种可塑性变化受到上皮间质转化epithelial- mesenchymal transition (EMT)过程的调节。胚胎发育时期，上皮状态和间充质状态的细胞能够自由转化。上皮间质转化（EMT）使得细胞具备转移和浸润特性。其反向过程，间质上皮转化mesenchymal-epithelial transition (MET)赋予了细胞极性变化并失去移动能力。EMT会促发癌细胞从病灶分离，转移到其它部位，而MET导致癌细胞停留，并在停留处引起新的肿瘤。长期以来,研究者们将EMT过程中的细胞分为上皮细胞和间质细胞，而忽视了这两种细胞转换过渡的形态。cell 杂志上近期一篇关于EMT的综述文章对上皮细胞和间质细胞之间的过渡形态进行了描述。文中在上皮形态和间充质形态转化过程中增加了EM1，EM2和EM3三种形态（图1），这样既解释了是否具有上皮形态和间质形态共表达标志物的疑虑，又解释了如器官纤维化这种既不是上皮细胞形态又不是间质细胞形态的情况。这些过渡形态反映了EMT转录过程的微妙平衡，而表观遗传和运动能力的改变不可避免的会导致这种平衡的改变。 EMT过程有着复杂的调节网络：表观遗传的调节转录调节选择性剪接蛋白质的稳定亚细胞定位这些调节方式多样性使得EMT的调节往往不是线性的（图2）。EMT调节过程中关于E-cadherin的转录，EMT 转录因子SNAI1, SNAI2, ZEB1,ZEB1和TWIST1等研究较多。文中指出，一些miRNA，如miR-200 和miR-34，能与ZEB1、SNAI1相互作用，形成一个健康状况下的的负反馈网络，其下游标志物主要为E-cadherin，claudins 和occludins。上皮特异性调节蛋白ESRP1和ESRP2能够通过指导特异性剪接，并调节转录过程，影响到其靶标FGFR2、CD44、p120-catenin和Mena等，使细胞保持上皮形态。与之相反的，QKI，SRSF2和RBFOX2能指导剪接，导致其对应靶标circRNAs，RonTK，Cortactin，Dynamin发生变化，引起上皮间质转化。此外，组蛋白的甲基化，一些蛋白的泛素化等都能够影响到EMT的发生。此文丰富了EMT的过程，使得今后对EMT的变化研究能准确到特定时期；另一方面，此文较为完整的阐述了EMT过程中的通路及因子关系，为后续相关研究奠定了基础。