Abstract
We have previously shown that E-cadherin regulates the naive pluripotent state of mouse embryonic stem cells (mESCs) by enabling LIF-dependent STAT3 phosphorylation, with E-cadherin null mESCs exhibiting over 3000 gene transcript alterations and a switch to Activin/Nodal-dependent pluripotency. However, elucidation of the exact mechanisms associated with E-cadherin function in mESCs is compounded by the difficulty in delineating the structural and signalling functions of this protein. Here we show that mESCs treated with the E-cadherin neutralising antibody DECMA-1 or the E-cadherin binding peptide H-SWELYYPLRANL-NH2 (Epep) exhibit discrete profiles for pluripotent transcripts and NANOG protein expression, demonstrating that the type of E-cadherin inhibitor employed dictates the cellular phenotype of mESCs. Alanine scanning mutation of Epep revealed residues critical for Tbx3, Klf4 and Esrrb transcript repression, cell-cell contact abrogation, cell survival in suspension, STAT3 phosphorylation and water solubility. STAT3 phosphorylation was found to be independent of loss of cell-cell contact and Activin/Nodal-dependent pluripotency and a peptide is described that enhances STAT3 phosphorylation and Nanog transcript and protein expression in mESCs. These peptides represent a useful resource for deciphering the structural and signalling functions of E-cadherin and demonstrate that complete absence of E-cadherin protein is likely required for hierarchical signalling pathway alterations in mESCs.
Highlights
Abrogation of E-cadherin in mESCs leads to a more polarized actin cytoskeleton organisation[13] which is associated with Ecad−/−mESCs switching from LIF/BMP- to Activin/Nodal-dependent pluripotency[14]
Abrogation of E-cadherin mediated cell-cell contacts in mESCs using Epep leads to repression of pluripotency associated transcripts and STAT3 phosphorylation
To further confirm the specific binding of Epep to E-cadherin a fluorescein-5(6)-isothiocyanate label was conjugated to the NH2-terminus of the peptide (Epep-F) separated by a Nova linker molecule to prevent the fluorescent label interfering with the peptide binding function
Summary
Abrogation of E-cadherin in mESCs leads to a more polarized actin cytoskeleton organisation[13] which is associated with Ecad−/−mESCs switching from LIF/BMP- to Activin/Nodal-dependent pluripotency[14]. The exact mechanism associated with this switch is not clear: it may reflect altered E-cadherin signalling via STAT3 phosphorylation[15] which directly influences the pluripotent phenotype, or it may be an indirect effect due to the altered actin cytoskeleton activating/inhibiting unknown proteins/pathways. At present it remains unknown whether the transcriptional and post-translational modifications associated with loss of E-cadherin are a result of direct or indirect (or both) regulation via E-cadherin. Our data demonstrates that the structural and signalling functions of E-cadherin can be demarcated using a range of peptides based on the Epep sequence, which will allow further analysis of the function of this protein in mESC pluripotency to be investigated
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