The Role of S100A6 in Calcium-Induced Endothelial Barrier Disruption

Background During inflammation, mediators increase calcium influx into endothelial cells through the store‐operated calcium entry (SOCE) channels. Increasing calcium influx through the SOCE current Isoc promotes endothelial barrier disruption through formation of inter‐endothelial cell gaps. Inter‐endothelial cell gap formation leads to increased endothelial permeability, a vascular event contributing to pathophysiological states such as acute respiratory distress syndrome. Identifying mechanisms of Isoc inhibition will aid in development of therapeutic strategies against endothelial permeability‐associated vascular inflammation. We have shown that FK506‐binding protein 51 (FKBP51), along with protein phosphatase 5 (PP5C), inhibits Isoc and calcium‐induced inter‐endothelial cell gap formation. Phosphatase activity of PP5C is required for the FKBP51‐mediated inhibition of Isoc . However, in the basal state PP5C has very low activity and to exert its effect, PP5C needs an activator. S100 proteins are potent activators of PP5C. S100 proteins are calcium‐dependent proteins and specifically S100A6 shows calcium‐induced translocation from cytosol to the plasma membrane, where the ISOC channel is located. However, it is unknown whether S100A6 contributes to the PP5C‐FKBP51‐mediated inhibition of inter‐endothelial cell gap formation. We previously showed that S100A6 associates with the ISOC channel and PP5C in a calcium‐dependent manner in pulmonary microvascular endothelial cells (PMVECs). Here we provide direct evidence that S100A6 activates PP5C in PMVECs and contributes to the inhibition of PP5C‐FKBP51‐mediated inhibition of Isoc and inter‐endothelial cell gap formation . Methods To determine whether S100A6 activates PP5C we analyzed the phosphorylation status of tau as a read‐out of PP5C activity in PMVECs. Whole cell patch clamp electrophysiology was used to measure Isoc in PMVECs. Global SOCE was determined by measuring intracellular calcium using the fluorophore Fura 2‐AM in PMVECs. Next, we performed electric cell‐substrate impedance sensing (ECIS) to measure inter‐endothelial cell gap formation in PMVECs. Results Phosphorylation of tau was dramatically increased in FKBP51 over‐expressing PMVECs following knock‐down of S100A6 and was significantly decreased in PMVECs over‐expressing S100A6, overall indicating that S100A6 activates PP5C. Over‐expression of S100A6 nearly abolished Isoc . Further, upon knock‐down of S100A6, FKBP51‐mediated Isoc inhibition was diminished. Global SOCE was reduced in PMVECs over‐expressing either FKBP51 or S100A6. And knock‐down of S100A6 abolished the FKBP51‐mediated decrease in SOCE. In either FKBP51 or S100A6 over‐expressing PMVECs inter‐endothelial cell gap formation was reduced. However, upon S100A6 knock‐down, FKBP51‐mediated decreased gap formation was abrogated. Conclusion Together these findings suggest that S100A6 activates PP5C leading to the PP5C‐FKBP51‐mediated inhibition of Isoc and inter‐endothelial cell gap formation. Support or Funding Information NIH R56HL107778 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .