Caspase activation is not required for villous cytotrophoblast fusion into syncytiotrophoblasts

The villous trophoblast renews itself by fusion of individual stem cells (cytotrophoblasts, CT) with a functional syncytium (syncytiotrophoblast, ST). The literature indicates that fusion occurs with limited activation (proteolytic cleavage) of caspase-8 in CT and is inhibited either by blocking caspase-8 synthesis or inhibiting activation with a caspase-8-specific inhibitor, zIETD. We challenge part of this evidence: inhibition of differentiation with caspase-8 inhibitors. Br-cAMP-stimulated differentiation of isolated CT into multinucleated syncytia in culture is not blocked with three different low molecular weight inhibitors of caspase-8: broad caspase inhibitors zVAD-fmk and qVD-OPh and the caspase-8-specific inhibitor zIETD-fmk. Syncytialization was determined by desmoplakin staining of intracellular boundaries surrounding >2 nuclei and by diffusion within fused cells of long-lived cytoplasmic staining from half of original CT to the unstained half. Differentiation of isolated CT into hCGβ-secreting syncytiotrophoblast was also not blocked by the inhibitors nor was upregulation of hCGβ secretion blocked in ST-stripped and regenerated 5 day explant cultures. The ratio of CT to ST nuclei present was also not changed in explant cultures by caspase inhibitors. The effectiveness of caspase inhibitors was demonstrated by their ability to completely block TNFα-induced apoptosis. We conclude that activation of caspases in general, and caspase-8 in particular, is not required for villous CT differentiation into ST. However, another role of intact caspase-8 (proform) in CT differentiation remains possible.