1、JCB Article The Rockefeller University Press 30 00 J Cell Biol Vol 191 No 3 631 644 www jcb org cgi doi 10 1083 jcb 201006094JCB631 D Li and J Zhou contributed equally to this paper Correspondence to Fei Wang feiwang life uiuc edu Abbreviations used in this paper bFGF basic FGF E cadherin epithelial
2、 cadherin ESC embryonic stem cell hE Fc E cadherin Fc proteins hESC human ESC hiPSC human iPSC iPSC induced pluripotent stem cell MEF mouse embryonic fibroblast mESC mouse ESC mTOR mammalian target of rapamycin Introduction Human embryonic stem cells ESCs hESCs derived from the inner cell mass of bl
3、astocyst stage human embryos can self renew in culture indefinitely and have the remarkable potential to develop into nearly all differentiated cell types Thomson et al 1998 allowing them to be used for bio medical research drug discovery and cell based therapies Daley and Scadden 2008 Rossant 2008
4、hESC long term self renewal requires cell proliferation and survival with continuous repression of differentiation Recent studies have revealed some important molecular elements that support hESC long term undifferentiated growth and survival Extrinsic factors such as basic FGF bFGF Thomson et al 19
5、98 Levenstein et al 2006 TGF Xu et al 2008 and insulin like growth factor 1 Bendall et al 2007 are required for stability of a network of transcription factors including OCT 4 NANOG and SOX2 which function in concert to posi tively regulate target genes necessary for pluripotency and repress a varie
6、ty of lineage specification factors Jaenisch and Young 2008 Receptor tyrosine kinases including ERBB2 and insulin like growth factor 1 receptor Wang et al 2007 are necessary for hESC proliferation and survival The sig nals from the extrinsic factors are integrated by intracellular molecules such as
7、mammalian target of rapamycin mTOR Zhou et al 2009 to repress differentiation activities and or H uman embryonic stem cells ESCs hESCs prolif erate as colonies wherein individual cells are strongly adhered to one another This architecture is linked to hESC self renewal pluripotency and survival and
8、depends on epithelial cadherin E cadherin NMMIIA nonmuscle myosin IIA and p120 catenin E cadherin and p120 catenin work within a positive feedback loop that promotes localized accumulation of E cadherin at intercellular junctions NMMIIA stabilizes p120 catenin protein and controls E cadherin mediate
9、d intercellular adhesion Perturbations of this signaling network disrupt colony formation destabilize the transcriptional regula tory circuitry for pluripotency and impair long term survival of hESCs Furthermore depletion of E cadherin markedly reduces the efficiency of reprogramming of human somati
10、c cells to an ESC like state The feedback regulation and mechanical biochemical integration pro vide mechanistic insights for the regulation of intercellular adhesion and cellular architecture in hESCs during long term self renewal Our findings also contribute to the understanding of microenvironmen
11、tal regulation of hESC identity and somatic reprogramming Integrated biochemical and mechanical signals regulate multifaceted human embryonic stem cell functions Dong Li 1 2 Jiaxi Zhou 1 2 Lu Wang 1 2 Myung Eun Shin 1 2 Pei Su 1 2 Xiaohua Lei 5 Haibin Kuang 5 Weixiang Guo 5 Hong Yang 1 2 Linzhao Che
12、ng 6 Tetsuya S Tanaka 1 3 Deborah E Leckband 4 Albert B Reynolds 7 Enkui Duan 5 and Fei Wang1 2 1Institute for Genomic Biology 2Department of Cell and Developmental Biology 3Department of Animal Sciences and 4Department of Chemical and Molecular Engineering University of Illinois at Urbana Champaign
13、 Urbana IL 61801 5State Key Laboratory of Reproductive Biology Institute of Zoology Chinese Academy of Science Beijing 100101 China 6Stem Cell Program Institute for Cell Engineering School of Medicine Johns Hopkins University Baltimore MD 21205 7Department of Cancer Biology Vanderbilt University Nas
14、hville TN 37232 2010 Li et al This article is distributed under the terms of an Attribution Noncommercial Share Alike No Mirror Sites license for the first six months after the publication date see http www rupress org terms After six months it is available under a Creative Commons License Attributi
15、on Noncommercial Share Alike 3 0 Unported license as described at http creativecommons org licenses by nc sa 3 0 T H E J O U R N A L O F C E L L B I O L O G Y JCB VOLUME 191 NUMBER 3 2010 632 Results Inhibition of NMMII causes profound defects in hESCs To identify the regulatory components of plurip
16、otency in hESCs we screened a collection of pharmacological inhibitors against kinases and other signaling molecules To eliminate indirect effects of feeders on hESCs we used feeder free culture condi tions under which untreated control cells grew as compact and tight colonies with well defined edge
17、s Fig 1 A Xu et al 2001 We passaged hESCs as small cell clusters a condition known to improve cell survival This screening effort led us to identify mTOR as a central regulator of hESC pluripotency Zhou et al 2009 With this screen we also found that treat ment of cells with blebbistatin a highly spe
18、cific inhibitor of NMMII Straight et al 2003 induced profound morphological changes in H9 hESCs Blebbistatin treated cells formed poorly developed colonies in which a significant fraction of cells were dissociated from one another and spread out as single cells Fig 1 A several representative cell mo
19、rphologies are shown in Fig S1 A The effect of blebbistatin on colony formation was dose dependent with an inhibitory concentration for 50 inhi bition IC50 disruption of 50 of colonies of 4 M which is close to the reported IC50 value for inhibiting ATPase activity of myosin II 2 M Straight et al 200
20、3 Exposure of cells to 10 M blebbistatin caused the poorly aggregated colonies to increase from 9 for control cells to 82 Table S1 This dose was used for the rest of the experiments Disruption of colony integrity is often associated with al tered expression of pluripotency markers Indeed blebbistati
21、n treatment reduced the activity of AP an ESC marker and the levels of SOX2 NANOG and OCT 4 proteins in H9 cells Fig 1 B and C and Fig S1 B quantification of SOX2 NANOG and OCT 4 levels is shown in Table S1 SOX2 NANOG and OCT 4 are components of the core transcriptional regulatory circuitry for plur
22、ipotency Jaenisch and Young 2008 and have also been used to derive human and mouse iPSCs Takahashi and Yamanaka 2006 Takahashi et al 2007 Yu et al 2007 Park et al 2008 Interestingly the mRNA levels of POU5F1 the gene encoding OCT 4 SOX2 and NANOG decreased only slightly after blebbistatin treatment
23、Fig S1 C indicating that the regulation largely occurred posttranscriptionally Blebbi statin s effect on the pluripotency factors was also observed in H1 hESCs Thomson et al 1998 a human iPSC hiPSC line MMW2 Mali et al 2010 and W4 129 mouse ESCs mESCs Fig S1 D F implying a conserved function of NMMI
24、I in pluripotent stem cells Furthermore although blebbistatin treat ment for 5 d exerted little effect blebbistatin treated cells 7 4 vs control cells 5 3 long term treatment 7 d caused in creased apoptosis in hESCs 17 7 vs control cells on day 8 6 3 P 7 d led to marked apoptosis in hESCs NMMIIA dep
25、leted cells 32 5 non targeting shRNA control cells on day 8 7 3 P 0 001 The elevated apoptosis in NMMIIA depleted cells was preceded by a robust reduction in the c Myc protein see Fig 3 F un published data c Myc is a transcription factor and acts as a key regulator of cell growth and survival In con
26、trast depletion of NMMIIB had little effect on the pluripotency factors colony formation and long term cell survival Fig 1 A and E Table S1 and not depicted The shRNA sequences targeting NMMIIA or NMMIIB effectively depleted their respective targets in H9 cells demonstrating high specificity of shRN
27、As Fig 1 F These results suggest that NMMIIA but not NMMIIB is neces sary for colony formation stability of the OCT 4 NANOG SOX2 circuitry and long term survival of hESCs The requirement of NMMIIA for supporting the ESC state might explain in part the early developmental defects observed in NMMIIA n
28、ull mice NMMIIA modulates E cadherin adhesion and protein expression in hESCs We next assessed NMMIIA s subcellular localization in hESCs Immunofluorescence of NMMIIA was mainly distributed at cell cell junctions colocalizing with the adhesion molecule E cadherin Fig 2 A This raises the possibility
29、that NMMIIA might regulate intercellular junctions in hESCs Consistent with this idea NMMIIA depletion for 4 d markedly impaired the ac cumulation of E cadherin at the junctional sites and caused it to distribute diffusely or in punctuate structures in the cytoplasm Fig 2 B Interestingly although NM
30、MIIB was also localized to cell cell junctions Fig S2 C its depletion failed to alter E cadherin localization not depicted Similar to NMMIIA de pletion blebbistatin treatment caused redistribution of E cadherin immunofluorescence to the cytoplasm Fig 2 B The effect of blebbistatin was rapid altering
31、 E cadherin localization within 24 h of treatment Fig 2 B To further dissect how NMMIIA regulates E cadherin we asked whether NMMIIA was necessary for E cadherin dependent intercellular adhesion in hESCs We used an estab lished adhesion assay Verma et al 2004 to assess the ability of cells to attach
32、 to the immobilized E cadherin Fc proteins hE Fc In this assay short term blebbistatin treatment 2 h robustly reduced the number of cells adhered to the E cadherin substrate Fig 2 C suggesting that NMMII activity is re quired for E cadherin mediated intercellular contact forma tion and adhesion NMMI
33、I dependent cytoskeletal contractility has been im plicated as a key regulator of mechanical signals in a variety of cell types including stem cells McBeath et al 2004 Engler et al 2006 Chowdhury et al 2010 In this study we also showed that NMMII was necessary for E cadherin mediated mechani cal ten
34、sion in hESCs We applied traction force microscopy which assesses the mechanical interactions between cells and the substrate to determine the traction stress in cells adhered to hE Fc coated substrates with an elastic modulus of 8 5 kPa This stiffness is in the range of normal tissues 1 10 kPa as d
35、ocumented previously Janmey and McCulloch 2007 Single H9 cells were found to exert a mean traction stress of 650 Pa on the substrates Fig 2 D and E whereas blebbistatin treatment G Quantification of the results shown in F The y axis represents relative intensities measured with ImageJ National Insti
36、tutes of Health with values normalized to the signal 100 at 0 h Results from four separate experiments are shown H Western blot analysis of E cadherin protein in H9 cells with or without NMMIIA depletion Asterisks indicate that the value for cells treated with blebbistatin differs statistically from
37、 the control P 0 05 P 0 01 P 0 0001 Error bars indicate mean SEM JCB VOLUME 191 NUMBER 3 2010 636 only slightly decreased Fig S2 D Therefore inhibiting E cadherin activity or reducing E cadherin expression in hESCs impaired cellular association colony formation and stability of the transcription fac
38、tors Furthermore E cadherin depletion mildly up regulated markers for the three germ layers and trophoectoderm Fig S2 D and not depicted The defects were highly reminiscent of those caused by NMMII inhibition or NMMIIA depletion Finally overexpression of E cadherin in hESCs nearly completely rescued
39、 the defects induced by NMMIIA depletion Ectopic expression of E cadherin caused H9 cells to maintain a high level of E cadherin protein even with NMMIIA depleted Sustained E cadherin expression in NMMIIA depleted cells prevented colony disruption and the down regulation of OCT 4 NANOG SOX2 and c My
40、c Fig 3 F Fig S2 E and not de picted Thus E cadherin was capable of supporting colony for mation and stability of the circuitry for pluripotency although NMMIA expression was largely absent Together our results confirm and extend previous evidence Conti et al 2004 Shewan et al 2005 Smutny et al 2010
41、 suggesting that E cadherin serves as a bona fide target of NMMIIA in hESCs to control intercellular adhesion and the pluripotency circuitry NMMIIA controls E cadherin expression via p120 catenin p120 catenin is the prototypic member of a subfamily of Arma dillo domain proteins It binds to the juxta
42、membrane region of cadherin intracellular domains Thoreson et al 2000 p120 catenin ablation in mice and other vertebrates led to embryonic lethality for review see McCrea and Park 2007 demonstrat ing a critical role in early development Earlier studies showed that p120 catenin regulates E cadherin e
43、xpression levels at the cell surface and modulates its signaling behavior Davis et al 2003 Xiao et al 2003 Wildenberg et al 2006 Thus we tested the possibility that p120 catenin might be involved in the regulation of E cadherin expression by NMMIIA Human cells express multiple isoforms of p120 caten
44、in Keirsebilck et al 1998 Isoforms 1 4 differ from each other by the start codon used whereas additional combinations are based on the alternative use of exons A C Western blot analysis of protein lysates from hESCs with two monoclonal antibodies one against isoforms 1 and 2 6H11 Wu et al 1998 and t
45、he other against all isoforms 15D2 Wu et al 1998 recognized one and two major bands respectively Fig 4 A top A side by side analysis of lysates from IMR 90 a human fibroblast cell line indicated that the band of higher molecular mass might be iso form 1 Fig 4 A bottom Mesenchymal cells such as fibro
46、 blasts express p120 catenin isoform 1 Mo and Reynolds 1996 Keirsebilck et al 1998 RT PCR analysis of alternative splicing events at the 5 region confirmed that hESCs expressed mRNA of isoforms 1 3 and at a much lower level 4 Fig S3 A Based on the results from Western blot and RT PCR analyses we inf
47、erred that hESCs mainly express isoforms 1 and 3 In addition RT PCR analysis of alternative splicing at the 3 re gion suggested that hESCs expressed transcripts of isoforms A and C in high abundance and to a much lesser degree iso form B Fig S3 B markedly reduced the level of tractions by 57 to 280
48、Pa Fig 2 D and E Prolonged inhibition of NMMII in hESCs reduced the levels of E cadherin protein Blebbistatin treatment for 72 h caused both H9 and H1 cells to markedly down regulate E cadherin expression which continued to decrease with longer treat ments Fig 2 F and G and Fig S1 D The reduced E ca
49、dherin expression was also observed in MMW2 hiPSCs Fig S1 E indicating conserved modulation in human pluripotent stem cells Interestingly down regulation of E cadherin was either concomitant with or preceded the down regulation of the pluripo tency proteins Fig 2 F and G and Fig S1 D In keeping with
50、 the effect of blebbistatin depletion of NMMIIA but not NMMIIB reduced E cadherin levels Fig 2 H and not depicted Thus NMMIIA is required for E cadherin dependent intercel lular adhesion and mechanical tension and modulates the levels of E cadherin protein in hESCs E cadherin serves as a bona fide t