The Role of S100A6 in the PP5C‐FKBP51‐Mediated Inhibition of Endothelial Isoc

Abstract

BackgroundDuring 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 the formation of inter‐endothelial cell gaps. Inter‐endothelial cell gap formation leads to increased endothelial permeability, a vascular event contributing to pathophysiological states including inflammation and acute respiratory distress syndrome (ARDS). Identifying mechanisms of Isoc inhibition will aid in development of therapeutic strategies against endothelial permeability‐associated inflammation and ARDS. However, mechanisms of Isoc inhibition are poorly understood. We have shown that the immunophilin FKBP51 inhibits Isoc and contributes to the inhibition of calcium entry‐induced inter‐endothelial cell gap formation. We also determined that protein phosphatase 5 (PP5C) is required for the FKBP51‐mediated inhibition of Isoc and inter‐endothelial cell gap formation. However, PP5C has very low activity in the basal state. To exert its effect PP5C needs an activator. S100 proteins are potent activators of PP5C. S100 proteins are calcium‐dependent proteins and specifically, S100A6 can interact with tetratricopeptide repeat domain containing proteins such as PP5C and FKBP51. Further, 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 Isoc. The goal of this study was to determine whether S100A6 is critical in increasing the phosphatase activity of PP5C in a calcium‐dependent manner in endothelial cells to promote the PP5C‐FKBP51 axis‐mediated inhibition of Isoc.MethodsWe utilized quantitative RT‐PCR and immunoblot to determine whether S100 proteins are expressed in pulmonary endothelial cells. We performed co‐immunoprecipitation experiments to determine if S100A6 associates with the ISOC channel and PP5C protein in pulmonary microvascular endothelial cells (PMVECs) in presence and absence of calcium (2mM). To determine whether S100A6 activates PP5C in PMVECs we analyzed the phosphorylation status of tau protein with or without calcium (2mM), where decreased tau phosphorylation suggests increased PP5C activity. Whole cell patch clamp electrophysiology was used to measure Isoc in cells.ResultsWe showed that S100A6 is highly expressed in pulmonary endothelial cells. S100A6 co‐precipitated with the TRPC4 subunit of the ISOC channel as well as with PP5C in a calcium‐dependent manner. Upon S100A6 knock‐down in PMVECs, phosphorylation of tau increased suggesting that PP5C activity was deceased. In FKBP51 over‐expressed cells, Isoc was inhibited, but this effect was reduced by S100A6 knock‐down.ConclusionTogether these findings suggest that S100A6 activates PP5C leading to the FKBP51‐PP5C‐mediated inhibition of Isoc.Support or Funding InformationSupported by NIH R01HL107778.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.