E-cadherin is a cell surface glycoprotein involved in calcium-dependent cell adhesion. E-cadherin expression is essential during embryo development as E-cadherin null (Ecad-/-) embryos fail to develop beyond the blastocyst stage. Furthermore, E-cadherin is down-regulated during epithelial to mesenchymal transition (EMT), a cellular remodelling event which is essential during embryo development and is associated with metastatic tumour progression. Expression of E-cadherin in wild type (WT) embryonic stem (ES) cells is involved in the establishment of cell-cell adhesions, regulation of motility and proliferation and the response of the cells to external growth factors. Accordingly, Ecad-/- ES cells do not form cell-cell adhesions, exhibit increased proliferation and motility, and utilise the Activin/Nodal signalling pathway instead of Lif for the maintenance of pluripotency. The results of microarray analysis comparing WT and Ecad-/- ES cell transcript expression undertaken to investigate the mechanisms underlying these cellular alterations, revealed a 235-fold down-regulation of Junctional adhesion molecule 2 (Jam2), a cell surface protein belonging to the Immunoglobulin superfamily. In this study, we investigate the E-cadherin/Jam2 relationship and demonstrate that Jam2 transcript expression is positively regulated by E-cadherin. We show that although the full length E-cadherin protein encoding the terminal 71 amino acids of the cytoplasmic domain is required for expression of Jam2 in ES cells, -catenin signalling is unlikely to be involved in the regulation of Jam2 by E-cadherin as -cat-/- ES cells also do not express Jam2. Through use of promoter/luciferase constructs we show that the Jam2 promoter is subject to altered regulatory events in Ecad-/- ES cells compared to WT ES cells. Analysis of the Jam2 promoter region using ECR Browser software leads us to hypothesise the involvement of E-boxes, Sp1, Ahr/Arnt, and Zeb1 transcription factors in the regulation of Jam2 in ES cells. Finally, we show that despite the regulation of Jam2 by E-cadherin, and the high level of expression of Jam2 in WT ES cells, that absence of Jam2 from ES cells does not affect the EMT undergone by the cells during spontaneous differentiation. Jam2-/- ES cells undergoing spontaneous differentiation down-regulate E-cadherin cell-surface expression, up-regulate N-cadherin cell-surface expression, show increased transcripts for EMT-associated transcription factors and protease activity, all comparable to that seen in WT ES cells. Furthermore, we confirm that Jam2-/- ES cells up-regulate transcripts representative of the three primary germ layers during spontaneous differentiation, as seen in WT ES cells. Overall, our results show that although Jam2 appears to be dispensable in ES cell biology, further investigation into the regulation of Jam2 by E-cadherin could provide novel insights into the events that take place following loss of E-cadherin in epithelial cells, thereby advancing our knowledge of events during embryo development and metastatic tumour progression.
|Date of Award||1 Aug 2011|
- The University of Manchester
|Supervisor||Christopher Ward (Supervisor)|