Novel peptides for deciphering structural and signalling functions of E-cadherin in mouse embryonic stem cells

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.

Figure 1. Abrogation of E-cadherin in mESCs leads to repression of transcripts associated with naïve pluripotency.

(a) RT-PCR analysis of Tbx3, Nanog, Tet1, Klf4, Nr0b1, Nr5a2, and Esrrb transcripts in wtD3 and E-cadherin−/− mESCs. (b) Phase contrast microscopy images of wtD3 mESCs treated with control vehicle or the E-cadherin binding peptide Epep. (c) qPCR analysis of Nanog, Klf4, Tbx3, Esrrb, Nr0b1, Nr5a2 and Tet1 transcript expression in mESCs treated with vehicle control or Epep. (d) Western blot analysis of phosphorylated STAT3 (pSTAT3), total STAT3 and α-tubulin proteins in wtD3 mESCs treated with control vehicle (C) or Epep. (e) Biacore Surface Plasmon Resonance (SPR) analysis was performed using human recombinant E-cadherin-Fc chimaera protein bound to an HTE chip by high capacity His-tagged protein capture. Capture of the recombinant E-cadherin-Fc chimaera protein on 6 independent HTE chips for high capacity His-tagged protein capture (A1-A6) over time is shown. (f) Biacore SPR equilibrium binding analysis using affinity capture of Epep at 0, 1, 2.5, 5, 10 and 30 μM. (g) Concentration curve of Epep binding to recombinant E-cadherin-Fc chimaera protein at various concentrations to enable K_D calculation. (h) Phase contrast image showing that fluorescent tagged Epep (Epep-F) is able to inhibit cell-cell contact in wtD3 mESCs. (j) Immunofluorescence microscopy analysis of Epep-F binding (green) to Ecad^−/− and wtD3 mESCs following incubation at 500 μm for 15 minutes at 37 °C. Nuclei are stained with DAPI (Blue). (k) Quantification of Epep-F binding to wtD3 and Ecad^−/− mESCs using ImageJ analysis. Data shows the mean fluorescence intensity and standard deviation. Epep - SWELYYPLRANL.

Figure 2. Inhibition of E-cadherin in mESCs using Epep or DECMA-1 results in differential gene transcript expression.

(a) Phase contrast images of mESCs treated with Epep or DECMA-1 neutralising antibody for 24 h. (b) qPCR analysis of Nanog, Klf4, Esrrb, Tbx3, Nr0b1, Nr5a2 and Tet1 transcript expression in mESCs treated with vehicle control (D3v), Epep or DECMA-1. (c) (i) Immunofluorescence microscopy analysis of NANOG protein expression in wtD3 mESCs treated with vehicle control (Control), Epep or DECMA-1 (DECMA). Nuclei are stained with DAPI (Blue). (ii) Quantification of NANOG protein expression in the images shown in (i) using ImageJ analysis. Data shows the mean fluorescence intensity and standard deviation. (d) Western blot analysis of phosphorylated STAT3 (pSTAT3), total STAT3 and α-tubulin proteins in wtD3 mESCs treated with control vehicle (wtD3), Epep or DECMA-1 (DECMA). (e) Phase contrast images of wtD3 mESCs exhibiting restoration of cell-cell contact 24 h post-removal of Epep or DECMA-1 treatment. (f) qPCR analysis of Nanog, Klf4, Esrrb, Tbx3, Nr0b1, Nr5a2 and Tet1 transcript expression in wtD3 mESCs 24 h post-removal of Epep (Epep_off) or DECMA-1 (Decma-1_off) treatment. (g) Apoptosis analysis using Annexin V and propridium iodide fluorescence flow cytometry in wtD3 mESCs treated with vehicle control (Control), DECMA-1 or Epep. The lower left box of the flow cytometry plots shows the viable population, the lower right box the early apoptotic cells and the top right box the late apoptotic or necrotic cells. Epep - SWELYYPLRANL.

Figure 3. Analysis of specific residues required for Epep induced loss of cell-cell contact and the minimum residues required for this function.

(a) Diagrammatic representation of Epep, EpepW2R and Epep∆ctm peptides used in the analysis. (b) Phase contrast images of mESCs treated with Epep, EpepW2R or Epep∆ctm for 24 h. (c) Biacore SPR equilibrium binding analysis of Epep, EpepW2R or Epep∆ctm at 30 μM. (d) Biacore SPR equilibrium binding analysis of Epep∆ctm at 0, 1, 2.5, 5, 10 and 30 μM and (e) binding curve for the calculation of peptide K_D. (f) Western blot analysis of phosphorylated STAT3 (pSTAT3), total STAT3 and α-tubulin (α-tub) proteins in wtD3 mESCs treated with control vehicle (wtD3), Epep, EpepW2R or Epep∆ctm. (g) qPCR analysis of Nanog, Klf4, Esrrb, Tbx3, Nr0b1 and Nr5a2 transcript expression in mESCs treated with vehicle control (D3v), Epep, EpepW2R or Epep∆ctm at 10 μM. (h) Immunofluorescence microscopy analysis of EpepW2R-F binding (green) to Ecad^−/− and wtD3 mESCs following incubation at 500 μm for 15 minutes at 37 °C. Nuclei are stained with DAPI (Blue). (j) Quantification of EpepW2R-F binding to wtD3 and Ecad^−/− mESCs using ImageJ analysis. Data shows the mean fluorescence intensity and standard deviation. Epep – SWELYYPLRANL; EpepW2R – SRELYYPLRANL; Epep∆ctm – SWELYYP.

Figure 4. Epep treatment of mESCs does not induce maintenance of pluripotency via the Activin/Nodal signalling pathways.

(a) Immunofluorescence microscopy analysis of NANOG protein expression in wtD3 mESCs treated with vehicle control (Control), EpepW2R or Epep∆ctm. Nuclei are stained with DAPI (Blue). (b) Quantification of NANOG protein expression in the images shown in (a) using ImageJ analysis. Data shows the mean fluorescence intensity and standard deviation. (c) Apoptosis analysis using Annexin V and propridium iodide fluorescence flow cytometry in wtD3 mESCs treated with EpepW2R or Epep∆ctm. The lower left box of the flow cytometry plots shows the viable population, the lower right box the early apoptotic cells and the top right box the late apoptotic or necrotic cells. (d) Immunofluorescence microscopy analysis of OCT4 protein expression in wtD3 mESCs treated with the Activin/Nodal inhibitor SB431542 or the FGFR1 inhibitor SU5402 and (i) vehicle control, (ii) Epep, (iii) EpepΔCtm and (iv) EpepW2R. (v) Relative fluorescence intensity of OCT4 protein expression in (i)-(iv) assessed using ImageJ analysis. (e) (i) Western blot analysis of phosphorylated ERK1/2 (pERK1/2), total ERK1/2 and α-tubulin proteins in wtD3 mESCs treated with control vehicle (D3), Epep or EpepW2R. (ii) Graph showing the ratio of pERK1/2 and α-tubulin protein levels from the western blot in e(i) using ImageJ densitometry analysis. Epep – SWELYYPLRANL; EpepW2R – SRELYYPLRANL; Epep∆ctm – SWELYYP.

Figure 5. Alanine scanning mutational analysis of Epep reveals discrete residues associated with transcript repression and solubility.

(a) Biacore SPR equilibrium binding analysis using affinity capture of EpepP7R at 0.3, 1, 3, 5, 10 μM. (b) Concentration curve of EpepP7R binding to recombinant E-cadherin-Fc chimaera protein at various concentrations to enable K_D calculation. (c) Phase contrast images of wtD3 mESCs treated with vehicle control (10% v/v DMSO in H₂0), Epep and EpepP7R. (d) The alanine scanning mutations synthesised based on the sequence of Epep. (e) Biacore SPR equilibrium binding analysis using affinity capture of EpepS1A, EpepW2A, EpepE3A, EpepL4A, EpepY5A and EpepY6A. (f) K_D values for EpepS1A, EpepW2A, EpepE3A, EpepL4A, EpepY5A and EpepY6A calculated using Biacore SPR equilibrium affinity capture analysis. (g) Phase contrast images of wtD3 mESCs treated with vehicle control (wtD3), Epep, EpepS1A, EpepW2A, EpepE3A, EpepL4A, EpepY5A and EpepY6A at 10 μM. (h) qPCR analysis of Nanog, Klf4, Tbx3, Esrrb, Nr0b1 and Nr5a2 transcript expression in mESCs treated with vehicle control (D3v), Epep, EpepS1A, EpepW2A, EpepE3A, EpepL4A, EpepY5A and EpepY6A at 10 μM. Epep - SWELYYPLRANL; EpepS1A – AWELYYPLRANL; EpepW2A – SAELYYPLRANL; EpepE3A – SWALYYPLRANL; EpepL4A – SWEAYYPLRANL; EpepY5A – SWELAYPLRANL; EpepY6A – SWELYAPLRANL; EpepP7R – SWELYYRLRANL.

Figure 6. The function of specific residues in Epep related to transcript expression, solubility, cell-cell contact and STAT3 phosphorylation.

(a) Effect of different peptides and DECMA-1 neutralising antibody on cell-cell contact, STAT3 phosphorylation and transcripts expression in mESCs. (b) Western blot analysis of phosphorylated STAT3 (pSTAT3), total STAT3 and α-tubulin proteins in wtD3 mESCs treated with Epep, EpepE3A or EpepY6A. (c) Immunofluorescence microscopy analysis of NANOG protein expression in wtD3 mESCs treated with vehicle control (Control), EpepE3A or EpepY6A. Nuclei are stained with DAPI (Blue). (d) Summary of the residues within Epep and their function in mESCs ascertained from the mutational peptide analysis. Epep - SWELYYPLRANL; EpepS1A – AWELYYPLRANL; EpepW2R – SRELYYPLRANL; EpepW2A – SAELYYPLRANL; EpepE3A – SWALYYPLRANL; EpepL4A – SWEAYYPLRANL; EpepY5A – SWELAYPLRANL; EpepY6A – SWELYAPLRANL; EpepP7R – SWELYYRLRANL.