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ISOFORM‐SPECIFIC SUBCELLULAR DISTRIBUTION AND ACTIVATION OF PKC MODULATE ENDOTHELIAL BARRIER INTEGRITY
Author(s) -
Gaudreault Nathalie,
Perrin Rachel M,
Guo Michael,
Clanton Chase,
Wu Mack H,
Yuan Sarah Y
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.964.14
Subject(s) - protein kinase c , microbiology and biotechnology , gene isoform , endothelium , tight junction , barrier function , immunofluorescence , chemistry , subcellular localization , endothelial stem cell , cytoplasm , biology , signal transduction , in vitro , biochemistry , endocrinology , immunology , antibody , gene
PKC is known to contribute to microvascular injury in diabetes. To determine which PKC isoforms are involved in the regulation of endothelial barrier integrity, we first examined their en face expression in the endothelium of coronary microvessels from diabetic Zucker rats. Immunofluorescence microscopy revealed a significant increase in PKC βII and a decrease in PKC δ expression. To test whether changes in PKCs expression alter microvascular barrier properties, we measured the transendothelial electrical resistance in human coronary microvascular endothelial cell and found that overexpression of PKC βII and inhibition of PKC δ both increased endothelial cell permeability. Using 3D fluorescence microscopy, we observed a distinct translocation of RFP‐tagged PKC βII to the cellular junction upon activation while the δ isoform remained confined to the perinuclear area. Consistently, FRET analysis of PKC activity at distinct subcellular locations demonstrated that PKC βII activity was higher at the cell‐to‐cell junction and plasma membrane surface, whereas PKC δ activity was greater in perinuclear plasma membrane vesicles. Taken together, the data suggest that PKC isoforms target specific subcellular sites leading to differential regulation of endothelial barrier function. Supported by NIH RO1 HL084542 and HL073324.