Syntaxin4, P-cadherin, and CCAAT enhancer binding protein β as signaling elements in the novel differentiation pathway for cultured embryonic stem cells

Pluripotent stem cells possess the potential to differentiate into all three germ layers. However, upon removal of the stemness factors, pluripotent stem cells, such as embryonic stem cells (ESCs), exhibit EMT-like cell behavior and lose stemness signatures. This process involves the membrane translocation of the t-SNARE protein syntaxin4 (Stx4) and the expression of the intercellular adhesion molecule P-cadherin. The forced expression of either of these elements induces the emergence of such phenotypes even in the presence of stemness factors. Interestingly, extracellular Stx4, but not P-cadherin, appears to induce a significant upregulation of the gastrulation-related gene brachyury, along with a slight upregulation of the smooth muscle cell-related gene ACTA2 in ESCs. Furthermore, our findings reveal that extracellular Stx4 plays a role in preventing the elimination of CCAAT enhancer binding protein β (C/EBPβ). Notably, the forced overexpression of C/EBPβ led to the downregulation of brachyury and a significant upregulation of ACTA2 in ESCs. These observations suggest that extracellular Stx4 contributes to early mesoderm induction while simultaneously activating an element that alters the differentiation state. The fact that a single differentiation cue can elicit multiple differentiation responses may reflect the challenges associated with achieving sensitive and directed differentiation in cultured stem cells. [Display omitted] •ES cells spontaneously extrude syntaxin4, which disrupts stem cell signatures.•Extracellular syntaxin4 elicits multiple differentiation responses in ES cells.•Extracellular syntaxin4 activates P-cadherin-dependent and -independent pathways.•Extracellular syntaxin4 enables the expression of a CEBPβ gene product, LAP.•LAP protein interferes with the primary effect of extracellular syntaxin4.