Protective role of FKBP51 in calcium entry‐induced endothelial barrier disruption

Abstract

Pulmonary artery endothelial cells (PAECs) express a cation current, ISOC (store-operated calcium entry current), which when activated permits calcium entry leading to inter-endothelial cell gap formation. The large molecular weight immunophilin FKBP51 inhibits ISOC but not other calcium entry pathways in PAECs. However, it is unknown whether FKBP51-mediated inhibition of ISOC is sufficient to protect the endothelial barrier from calcium entry-induced disruption. The major objective of this study was to determine whether FKBP51-mediated inhibition of ISOC leads to decreased calcium entry-induced inter-endothelial gap formation and thus preservation of the endothelial barrier. Here, we measured the effects of thapsigargin-induced ISOC on the endothelial barrier in control and FKBP51 overexpressing PAECs. FKBP51 overexpression decreased actin stress fiber and inter-endothelial cell gap formation in addition to attenuating the decrease in resistance observed with control cells using electric cell-substrate impedance sensing. Finally, the thapsigargin-induced increase in dextran flux was abolished in FKBP51 overexpressing PAECs. We then measured endothelial permeability in perfused lungs of FKBP51 knockout (FKBP51–/–) mice and observed increased calcium entry-induced permeability compared to wild-type mice. To begin to dissect the mechanism underlying the FKBP51-mediated inhibition of ISOC, a second goal of this study was to determine the role of the microtubule network. We observed that FKBP51 overexpressing PAECs exhibited increased microtubule polymerization that is critical for inhibition of ISOC by FKBP51. Overall, we have identified FKBP51 as a novel regulator of endothelial barrier integrity, and these findings are significant as they reveal a protective mechanism for endothelium against calcium entry-induced disruption.

S100A6 is a positive regulator of PPP5C‐FKBP51‐dependent regulation of endothelial calcium signaling

“…In doxycycline‐treated cells, with increased FKBP51 expression, I SOC was decreased by ~52% (~14 pA at −80 mV) with no change in reversal potential. Indeed, this result is consistent with our previous observations in rat PAECs . Finally, using doxycycline‐inducible FKBP51 over‐expressing cells, we wanted to determine whether upregulation of FKBP51 in PMVECs protects the endothelial barrier, as it does in PAECs .…”

“…We first observed that upregulation of the PPP5C‐FKBP51 axis inhibits I SOC in PMVECs. As this observation supports our earlier data in PAECs, it suggests that inhibition of I SOC by the PPP5C‐FKBP51 axis is a fundamental mechanism in endothelial cells. Second, we found that S100A6 is required for the PPP5C‐FKBP51‐mediated inhibition of I SOC , working through the PPP5C arm of the axis.…”

“…T231 is part of a flanking region around the microtubule binding repeats which is important for binding to microtubules, and tau T231 phosphorylation is important for tau’s ability to bind and stabilize microtubules . We were particularly interested in phosphorylation of this site not only as a readout of PPP5C activity but also because it may affect microtubule polymerization status, which is a key determinant of FKBP51’s ability to inhibit I SOC …”

“…The TRPC4 subunit in particular is required for channel activity through its interaction with the spectrin membrane skeleton . In addition to interacting with the spectrin membrane skeleton, the channel interacts with a number of accessory proteins, overall comprising what we have termed the ISOC heterocomplex . For instance, we have previously shown that Orai1 associates with and modulates ISOC channel activity .…”

FK506 regulates Ca2+ release evoked by inositol 1,4,5‐trisphosphate independently of FK‐binding protein in endothelial cells

“…Other FKBPs may also be the target of the drugs. FKBP51 and FKBP52 regulate steroid receptor signalling and store operated Ca 2+ entry and some TRP channels (Hamilton et al, 2018;Kadeba et al, 2013;Sinkins, Goel, Estacion, & Schilling, 2004) to modulate cell function. The present findings suggest that rapamycin and FKBP do not modulate Ca 2+ release.…”