Analysis of Wnt signaling β-catenin spatial dynamics in HEK293T cells

BMC Systems Biology

Antony W Burgess²  David W Smith³  Yumiko Hirokawa⁴  Bruce S Gardiner³  Chin Wee Tan¹ 

[1]Department of Infrastructure Engineering, University of Melbourne, Parkville, VIC, Australia

[2]Department of Surgery, University of Melbourne, Parkville, VIC, Australia

[3]School of Computer Science and Software Engineering, The University of Western Australia, Perth, WA, Australia

[4]Ludwig Institute for Cancer Research, Melbourne-Parkville Branch, Parkville, VIC, Australia

关键词: Systems biology;    Confocal microscopy;    Compartmentalization;    Computational model;    β-catenin;    HEK293T;    Wnt signaling;   

Others  :  866504
DOI  :  10.1186/1752-0509-8-44

 received in 2013-11-29, accepted in 2014-03-31,  发布年份 2014

【 摘 要 】

Background

Wnt/β-catenin signaling is involved in different stages of mammalian development and implicated in various cancers (e.g. colorectal cancer). Recent experimental and computational studies have revealed characteristics of the pathway, however a cell-specific spatial perspective is lacking. In this study, a novel 3D confocal quantitation protocol is developed to acquire spatial (two cellular compartments: nucleus and cytosol-membrane) and temporal quantitative data on target protein (e.g. β-catenin) concentrations in Human Epithelial Kidney cells (HEK293T) during perturbation (with either cycloheximide or Wnt3A). Computational models of the Wnt pathway are constructed and interrogated based on this data.

Results

A single compartment Wnt pathway model is compared with a simple β-catenin two compartment model to investigate Wnt3A signaling in HEK293T cells. When protein synthesis is inhibited, β-catenin decreases at the same rate in both cellular compartments, suggesting diffusional transport is fast compared to β-catenin degradation in the cytosol. With Wnt3A stimulation, the total amount of β-catenin rises throughout the cell, however the increase is initially (~first hour) faster in the nuclear compartment. While both models were able to reproduce the whole cell changes in β-catenin, only the compartment model reproduced the Wnt3A induced changes in β-catenin distribution and it was also the best fit for the data obtained when active transport was included alongside passive diffusion transport.

Conclusions

This integrated 3D quantitation imaging protocol and computational modeling approach allowed cell-specific compartment models of the signaling pathways to be constructed and analyzed. The Wnt models constructed in this study are the first for HEK293T and have suggested potential roles of inter-compartment transport to the dynamics of signaling.

【 授权许可】

   
2014 Tan et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Harris TJC, Peifer M: Decisions, decisions: β-catenin chooses between adhesion and transcription. Trends Cell Biol 2005, 15:234-237.
• [2]Logan CY, Nusse R: The Wnt Signaling Pathway in Development and Disease. Annu Rev Cell Dev Biol 2004, 20:781-810.
• [3]Cadigan KM, Peifer M: Wnt Signaling from Development to Disease: Insights from Model Systems. Cold Spring Harb Perspect Biol 2009, 1:a002881.
• [4]Polakis P: Wnt signaling and cancer. Genes Dev 1837–1851, 2000:14.
• [5]Clevers H: Wnt/β-catenin signaling in development and disease. Cell 2006, 127:469-480.
• [6]Kishida S, Yamamoto H, Ikeda S, Kishida M, Sakamoto I, Koyama S, Kikuchi A: Axin, a Negative Regulator of the Wnt Signaling Pathway, Directly Interacts with Adenomatous Polyposis Coli and Regulates the Stabilization of β-Catenin. J Biol Chem 1998, 273:10823-10826.
• [7]Behrens J, Jerchow B-A, Würtele M, Grimm J, Asbrand C, Wirtz R, Kühl M, Wedlich D, Birchmeier W: Functional Interaction of an Axin Homolog, Conductin, with β-Catenin, APC, and GSK3β. Science 1998, 280:596-599.
• [8]Liu C, Li Y, Semenov M, Han C, Baeg G-H, Tan Y, Zhang Z, Lin X, He X: Control of β-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 2002, 108:837-847.
• [9]Aberle H, Bauer A, Stappert J, Kispert A, Kemler R: β-catenin is a target for the ubiquitin-proteosome pathway. EMBO J 1997, 16:3797-3804.
• [10]Amit S, Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y, Alkalay I: Axin-mediated CKI phosphorylation of β-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 2002, 16:1066-1076.
• [11]Yanagawa S-i, Matsuda Y, Lee J-S, Matsubayashi H, Sese S, Kadowaki T, Ishimoto A: Casein kinase I phosphorylates the Armadillo protein and induces its degradation in Drosophila. EMBO J 2002, 21:1733-1742.
• [12]Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W: Functional interaction of β-catenin with the transcription factor LEF-1. Nature 1996, 382:638-642.
• [13]Peifer M, Polakis P: Wnt Signaling in Oncogenesis and Embryogenesis–a Look Outside the Nucleus. Science 2000, 287:1606-1609.
• [14]Faux MC, Coates JL, Catimel B, Cody S, Clayton AHA, Layton MJ, Burgess AW: Recruitment of adenomatous polyposis coli and β-catenin to axin-puncta. Oncogene 2008, 27:5808-5820.
• [15]Xiao J-H, Ghosn C, Hinchman C, Forbes C, Wang J, Snider N, Cordrey A, Zhao Y, Chandraratna RAS: Adenomatous Polyposis Coli (APC)-independent Regulation of β-Catenin Degradation via a Retinoid X Receptor-mediated Pathway. J Biol Chem 2003, 278:29954-29962.
• [16]Seo E, E-h J: Axin-independent phosphorylation of APC controls β-catenin signaling via cytoplasmic retention of β-catenin. Biochem Biophys Res Commun 2007, 357:81-86.
• [17]Faux MC, Coates JL, Kershaw NJ, Layton MJ, Burgess AW: Independent Interactions of Phosphorylated β-Catenin with E-Cadherin at Cell-Cell Contacts and APC at Cell Protrusions. PLoS One 2010, 5:e14127.
• [18]Layton MJ, Faux MC, Church NL, Catimel B, Kershaw NJ, Kapp EA, Nowell C, Coates JL, Burgess AW, Simpson RJ: Identification of a Wnt-induced protein complex by affinity proteomics using an antibody that recognizes a sub-population of β-catenin. Biochim Biophys Acta (BBA) - Proteins and Proteomics 2012, 1824:925-937.
• [19]Li VSW, Ng SS, Boersema PJ, Low TY, Karthaus WR, Gerlach JP, Mohammed S, Heck AJR, Maurice MM, Li VSW, Ng SS, Boersema PJ, Low TY, Karthaus WR, Gerlach JP, Mohammed S, Heck AJR, Maurice MM, Mahmoudi T, Clevers H: Wnt Signaling through Inhibition of β-Catenin Degradation in an Intact Axin1 Complex. Cell 2012, 149:1245-1256.
• [20]Lee E, Salic A, Kruger R, Heinrich R, Kirschner MW: The Roles of APC and Axin Derived from Experimental and Theoretical Analysis of the Wnt Pathway. PLoS Biol 2003, 1:E10.
• [21]Kim D, Rath O, Kolch W, Cho K: A hidden oncogenic positive feedback loop caused by crosstalk between Wnt and ERK Pathways. Oncogene 2007, 26:4571-4579.
• [22]Sun Y-C: Examination of effects of GSK3β phosphorylation, β-catenin phosphorylation, and β-catenin degradation on kinetics of Wnt signaling pathway using computational method. Theor Biol Med Model 2009, 6:13. BioMed Central Full Text
• [23]Wawra C, Kuhl M, Kestler HA: Extended analyses of the Wnt/β-catenin pathway: Robustness and oscillatory behaviour. FEBS Lett 2007, 581:4043-4048.
• [24]Cho K-H, Baek S, Sung M-H: Wnt pathway mutations selected by optimal β-catenin signaling for tumorigenesis. FEBS Lett 2006, 580:3665-3670.
• [25]Lloyd Lewis B, Fletcher A, Dale T, Byrne H: Toward a quantitative understanding of the Wnt/β-catenin pathway through simulation and experiment. Wiley Interdiscip Rev Syst Biol Med 2013, 5:391-407.
• [26]Kofahl B, Wolf J: Mathematical modelling of Wnt/β-catenin signalling. Biochem Soc Trans 2010, 38:1281.
• [27]Tan CW, Gardiner BS, Hirokawa Y, Layton MJ, Smith DW, Burgess AW: Wnt Signalling Pathway Parameters for Mammalian Cells. PLoS One 2012, 7:e31882.
• [28]van Leeuwen IMM, Byrne HM, Jensen OE, King JR: Elucidating the interactions between the adhesive and transcriptional functions of β-catenin in normal and cancerous cells. J Theor Biol 2007, 247:77-102.
• [29]Schmitz Y, Wolkenhauer O, Rateitschak K: Nucleo-cytoplasmic shuttling of APC can maximize β‒catenin/TCF concentration. J Theor Biol 2011, 279:132-142.
• [30]Schmitz Y, Rateitschak K, Wolkenhauer O: Analysing the impact of nucleo-cytoplasmic shuttling of β-catenin and its antagonists APC, Axin and GSK3 on Wnt/β-catenin signalling. Cell Signal 2013, 25:2210-2221.
• [31]Basan M, Idema T, Lenz M, Joanny J-F, Risler T: A Reaction–diffusion Model of the Cadherin-Catenin System: A Possible Mechanism for Contact Inhibition and Implications for Tumorigenesis. Biophys J 2010, 98:2770-2779.
• [32]Stephens DJ, Allan VJ: Light Microscopy Techniques for Live Cell Imaging. Science 2003, 300:82-86.
• [33]Graham FL, Smiley J, Russell WC, Nairn R: Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5. J Gen Virol 1977, 36:59-72.
• [34]Brembeck FH, Schwarz-Romond T, Bakkers J, Wilhelm S, Hammerschmidt M, Birchmeier W: Essential role of BCL9-2 in the switch between β-catenin’s adhesive and transcriptional functions. Genes Dev 2004, 18:2225-2230.
• [35]Cao Y, Liu R, Jiang X, Lu J, Jiang J, Zhang C, Li X, Ning G: Nuclear-Cytoplasmic Shuttling of Menin Regulates Nuclear Translocation of β-Catenin. Mol Cell Biol 2009, 29:5477-5487.
• [36]Fagotto F, Glück U, Gumbiner BM: Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of β-catenin. Curr Biol 1998, 8:181-190.
• [37]Franca-Koh J, Yeo M, Fraser E, Young N, Dale TC: The Regulation of Glycogen Synthase Kinase-3 Nuclear Export by Frat/GBP. J Biol Chem 2002, 277:43844-43848.
• [38]Henderson BR: Nuclear-cytoplasmic shuttling of APC regulates [beta]-catenin subcellular localization and turnover. Nat Cell Biol 2000, 2:653-660.
• [39]Rosin‒Arbesfeld R, Cliffe A, Brabletz T, Bienz M: Nuclear export of the APC tumour suppressor controls β‒catenin function in transcription. EMBO J 2003, 22:1101-1113.
• [40]Townsley FM, Cliffe A, Bienz M: Pygopus and Legless target Armadillo/β-catenin to the nucleus to enable its transcriptional co-activator function. Nat Cell Biol 2004, 6:626-633.
• [41]Yokoya F, Imamoto N, Tachibana T, Yoneda Y: β-Catenin Can Be Transported into the Nucleus in a Ran-unassisted Manner. Mol Biol Cell 1999, 10:1119-1131.
• [42]Wiechens N, Heinle K, Englmeier L, Schohl A, Fagotto F: Nucleo-cytoplasmic Shuttling of Axin, a Negative Regulator of the Wnt-β-Catenin Pathway. J Biol Chem 2004, 279:5263-5267.
• [43]Caspi M, Zilberberg A, Eldar-Finkelman H, Rosin-Arbesfeld R: Nuclear GSK-3[beta] inhibits the canonical Wnt signalling pathway in a [beta]-catenin phosphorylation-independent manner. Oncogene 2008, 27:3546-3555.
• [44]Krieghoff E, Behrens J, Mayr B: Nucleo-cytoplasmic distribution of β-catenin is regulated by retention. J Cell Sci 2006, 119:1453-1463.
• [45]Sharma M, Jamieson C, Johnson M, Molloy MP, Henderson BR: Specific Armadillo Repeat Sequences Facilitate β-Catenin Nuclear Transport in Live Cells via Direct Binding to Nucleoporins Nup62, Nup153, and RanBP2/Nup358. J Biol Chem 2012, 287:819-831.
• [46]Gibson MA, Bruck J: Efficient Exact Stochastic Simulation of Chemical Systems with Many Species and Many Channels. J Phys Chem A 2000, 104:1876-1889.
• [47]Colvin J, Monine MI, Faeder JR, Hlavacek WS, Von Hoff DD, Posner RG: Simulation of large-scale rule-based models. Bioinformatics 2009, 25:910-917.
• [48]Hlavacek WS, Faeder JR, Blinov ML, Posner RG, Hucka M, Fontana W: Rules for Modeling Signal-Transduction Systems. Sci STKE 2006, 2006:re6.
• [49]Nagafuchi A, Ishihara S, Tsukita S: The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-α-catenin fusion molecules. J Cell Biol 1994, 127:235-245.
• [50]Ben-Ze’ev A, Geiger B: Differential molecular interactions of β-catenin and plakoglobin in adhesion, signaling and cancer. Curr Opin Cell Biol 1998, 10:629-639.
• [51]Yokoyama N, Yin D, Malbon C: Abundance, complexation, and trafficking of Wnt/beta-catenin signaling elements in response to Wnt3a. J Mol Signal 2007, 2:11. BioMed Central Full Text
• [52]Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR, Nusse R: Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003, 423:448-452.
• [53]Kishida S, Yamamoto H, Kikuchi A: Wnt-3a and Dvl Induce Neurite Retraction by Activating Rho-Associated Kinase. Mol Cell Biol 2004, 24:4487-4501.
• [54]Obrig TG, Culp WJ, McKeehan WL, Hardesty B: The Mechanism by which Cycloheximide and Related Glutarimide Antibiotics Inhibit Peptide Synthesis on Reticulocyte Ribosomes. J Biol Chem 1971, 246:174-181.
• [55]Nakamura T, Hamada F, Ishidate T, Anai K-I, Kawahara K, Toyoshima K, Akiyama T: Axin, an inhibitor of the Wnt signalling pathway, interacts with β-catenin, GSK-3β and APC and reduces the β-catenin level. Genes Cells 1998, 3:395-403.
• [56]Shampine LF, Reichelt MW: The matlab ode suite. SIAM J Sci Comput 1997, 18:1.