Calcium‐Dependent Localization of S100A6 in Pulmonary Microvascular Endothelial Cells

Abstract

During inflammation, mediators increase calcium influx into endothelial cells through 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 permeability. Identifying mechanisms of ISOC inhibition may aid in the development of therapeutic strategies against endothelial permeability‐associated vascular inflammation. Our studies indicate FK506‐binding protein 51 (FKBP51), in conjunction with protein phosphatase 5 (PPP5C), inhibit ISOC and calcium entry‐induced inter‐endothelial cell gap formation. Further, we determined that the calcium‐binding protein S100A6 is necessary for the PPP5C‐FKBP51‐mediated inhibition of ISOC in pulmonary microvascular endothelial cells (PMVECs). In PMVECs, S100A6 interacts with the ISOC channel in a calcium‐dependent manner, i.e., following an increase in cytosolic calcium. However, we do not know whether calcium entry specifically through the ISOC channel or through other SOCE channels that provides the calcium source to promote S100A6 interaction with the ISOC channel.MethodsPMVECs were used for co‐precipitation and immunocytochemistry‐based studies. When cells are treated with rolipram (R) plus thapsigargin (Tg), ISOC and other SOCE channels are activated. However, when cells are treated with Tg alone, ISOC is not activated, but other SOCE channels are activated. The TRPC4 subunit of the ISOC channel was immunoprecipitated and probed for co‐precipitation of S100A6.ResultsFollowing R/Tg treatment, robust co‐precipitation of S100A6 with TRPC4 was observed, whereas following Tg alone only minor co‐precipitation was seen. Immunocytochemistry revealed that in untreated cells S100A6 localization is principally cytosolic. Following R/Tg treatment, but not Tg alone, a distinct membrane‐delimited pool of S100A6 was observed, particularly at sites of cell‐cell contact. Membrane localization was confirmed by co‐localization with wheat germ agglutinin (WGA).ConclusionOverall, our data reveal that calcium entry specifically through the ISOC channel is an important determinant of S100A6 translocation to the plasma membrane promoting interaction of S100A6 with the ISOC channel.Support or Funding InformationNIH HL R56HL107778