The goal of the proposed research is to determine the mechanisms by which the large, molecular weight immunophilins, FKBP51 and FKBP52, regulate store-operated calcium (SOC) entry in pulmonary endothelial cells. This project is of clinical significance because activation of a particular SOC entry channel, the ISOC channel, is a key step leading to the formation of inter-endothelial cell gaps and increased permeability across the endothelial barrier. Increased permeability is a vascular event that occurs in both acute and chronic inflammation and is a contributing factor to morbidity and mortality in Acute Lung Injury. Understanding regulatory mechanisms of SOC entry will aid in development of new anti-inflammatory strategies, however regulation is poorly understood. It has been observed that FKBP51 and FKBP52 regulate store-operated calcium entry in opposing manners. FKBP51 is of specific interest because it inhibits store-operated calcium entry and may lead to reduced endothelial permeability. Further, as FKBP51 is preferentially expressed in microvascular endothelial cells, it may be the key determinant of this cell type's resistance to barrier disruptin by inflammatory agonists. Finally, FKBP51 is the most highly glucocorticoid-inducible gene in human lung, and its expression and activity can be pharmacologically manipulated, potentially leading to development of beneficial therapeutic interventions. Specifically, in vitro and in situ experimental models will be used to determine whether these immunophilins are part of the ISOC channel heterocomplex (Specific Aim 1), whether specific domains are important for facilitating or inhibiting channel function (Specific Aims 2-3), and whether relative immunophilin expression is a key determinant of inter-endothelial cell gap formation (Specific Aim 4). Specific domain mutants of FKBP51 and FKBP52 are readily available. Techniques to be used include: differential fractionations, immunoprecipitation and Homogenous Time-Resolved Fluorescence to determine protein-protein interactions; fluorescence microscopy and patch-clamp electrophysiology to measure calcium channel function; nuclear magnetic resonance spectroscopy to measure proline- containing peptide bond isomerization; video microscopy, permeability measurements and resistance measurements to measure inter-endothelial cell gap formation in vitro; and the isolated, perfused lung model measuring filtration coefficient (Kf), wet/dry lung weight ratio, alveolar fluid volume fraction and albumin permeability (Evan's Blue dye in bronchoalveolar lavage fluid) in situ.

Public Health Relevance

The goal of the proposed research is to determine mechanisms by which certain calcium ion channels (SOC channels) are regulated. This project is of clinical significance because SOC channels play a role in the formation of inter-endothelial cell gaps and increased permeability across the endothelial barrier. Increased permeability is a vascular event that occurs in both acute and chronic inflammation. Thus, understanding regulation of SOC channels may lead to the development of novel and beneficial therapeutic interventions.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
4R01HL107778-04
Application #
9000733
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Xiao, Lei
Project Start
2013-02-01
Project End
2017-01-31
Budget Start
2016-02-01
Budget End
2017-01-31
Support Year
4
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of South Alabama
Department
Biochemistry
Type
Schools of Medicine
DUNS #
172750234
City
Mobile
State
AL
Country
United States
Zip Code
36688
Hamilton, Caleb L; Kadeba, Pierre I; Vasauskas, Audrey A et al. (2018) Protective role of FKBP51 in calcium entry-induced endothelial barrier disruption. Pulm Circ 8:2045893217749987
Hamilton, Caleb L; Abney, Kevin A; Vasauskas, Audrey A et al. (2018) Serine/threonine phosphatase 5 (PP5C/PPP5C) regulates the ISOC channel through a PP5C-FKBP51 axis. Pulm Circ 8:2045893217753156
Xu, Ningyong; Cioffi, Donna L; Alexeyev, Mikhail et al. (2015) Sodium entry through endothelial store-operated calcium entry channels: regulation by Orai1. Am J Physiol Cell Physiol 308:C277-88
Vasauskas, Audrey A; Chen, Hairu; Wu, Songwei et al. (2014) The serine-threonine phosphatase calcineurin is a regulator of endothelial store-operated calcium entry. Pulm Circ 4:116-27
Xu, Ningyong; Francis, Michael; Cioffi, Donna L et al. (2014) Studies on the resolution of subcellular free calcium concentrations: a technological advance. Focus on ""detection of differentially regulated subsarcolemmal calcium signals activated by vasoactive agonists in rat pulmonary artery smooth muscle cells"". Am J Physiol Cell Physiol 306:C636-8
Kadeba, Pierre I; Vasauskas, Audrey A; Chen, Hairu et al. (2013) Regulation of store-operated calcium entry by FK506-binding immunophilins. Cell Calcium 53:275-85