CXCR4 Mediated Chemotaxis Is Regulated by 5T4 Oncofetal Glycoprotein in Mouse Embryonic Cells Thomas D. Southgate1, Owen J. McGinn1, Fernanda V. Castro1, Andrzej J. Rutkowski1,2, Mariam Al-Muftah 1 , Georgi Marinov1, Graeme J. Smethurst1,2, David Shaw1, Christopher M. Ward1, Crispin J. Miller2, Peter L. Stern1* 1 Immunology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom, 2 Applied Computational Biology and Bioinformatics Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom Abstract 5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co- localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site. Citation: Southgate TD, McGinn OJ, Castro FV, Rutkowski AJ, Al-Muftah M, et al. (2010) CXCR4 Mediated Chemotaxis Is Regulated by 5T4 Oncofetal Glycoprotein in Mouse Embryonic Cells. PLoS ONE 5(4): e9982. doi:10.1371/journal.pone.0009982 Editor: Syed A. Aziz, Health Canada, Canada Received February 11, 2010; Accepted March 12, 2010; Published April 1, 2010 Copyright: 2010 Southgate et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work was funded by Cancer Research UK (www.cancerresearchuk.org) Grant No. R108032Q. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pstern@picr.man.ac.uk Introduction 5T4 oncofetal glycoprotein was discovered while searching for molecules with invasive properties likely to be shared by trophoblast and cancer cells [1]. It is expressed by many different carcinomas while showing only low levels in some normal tissues [2]. 5T4 expression has been shown to influence adhesion, cytoskeletal organization and motility [3,4,5], properties which might account for its association with poorer clinical outcome in some cancers [6,7,8,9]. Its <72 kD transmembrane molecules have a short cytoplasmic region, as well as an N-glycosylated extracellular domain with two leucine rich repeat (LRR) regions separated by a hydrophilic sequence and associated N and C terminal flanking regions [10,11]. LRR are found in proteins with diverse functions and are frequently associated with protein-protein interaction [12]. We have recently shown that upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of human and murine embryonic stem cells [13,14] and forms an integrated component of an epithelial-mesenchymal transition (EMT) [15,16]. EMT occurs during embryonic development and is also believed to be important for the metastatic spread of epithelial tumors [17]. To further study this process we conducted a comparative microarray analysis of undifferentiated (5T4 –ve) and early differentiating (5T4 +ve) murine ES cells [18]. 5T4 is up-regulated at an earlier stage of ES differentiation than the widely used down-regulation of the SSEA-1 marker [13] while cell sorting for surface 5T4 expression provided an additional level of stringency in the definition of ES cell populations compared to stratifications used in some other microarray studies [19,20]. Any transcriptional changes may be important in governing the balance of self-renewal/pluripotency and differentiation in ES cells, or in the regulation of 5T4 cell surface expression. Such properties may also be functionally important in tumor progression. One significant transcriptional change identified was the down-regulation of transcripts for the PLoS ONE | www.plosone.org 1 April 2010 | Volume 5 | Issue 4 | e9982