Apoptotic cell extrusion depends on single-cell synthesis of sphingosine-1-phosphate by sphingosine kinase 2

Collecting duct cells are physiologically subject to the hypertonic environment of the kidney. This condition is necessary for kidney maturation and function but represents a stress condition that requires active strategies to ensure epithelial integrity. Madin-Darby Canine Kidney (MDCK) cells develop the differentiated phenotype of collecting duct cells when subject to hypertonicity, serving as a model to study epithelial preservation and homeostasis in this particular environment. The integrity of epithelia is essential to achieve the required functional barrier. One of the mechanisms that ensure integrity is cell extrusion, a process initiated by sphingosine-1-phosphate (S1P) to remove dying or surplus cells while maintaining the epithelium barrier. Both types start with the activation of S1P receptor type 2, located in neighboring cells. In this work, we studied the effect of cell differentiation induced by hypertonicity on cell extrusion in MDCK cells, and we provide new insights into the associated molecular mechanism. We found that the different stages of differentiation influence the rate of apoptotic cell extrusion. Besides, we used a novel methodology to demonstrate that S1P increase in extruding cells of differentiated monolayers. These results show for first time that cell extrusion is triggered by the single-cell synthesis of S1P by sphingosine kinase 2 (SphK2), but not SphK1, of the extruding cell itself. Moreover, the inhibition or knockdown of SphK2 prevents cell extrusion and cell-cell junction protein degradation, but not apoptotic nuclear fragmentation. Thus, we propose SphK2 as the biochemical key to ensure the preservation of the epithelial barrier under hypertonic stress.