Cyclic AMP induces phosphorylation of claudin-5 immunoprecipitates and expression of claudin-5 gene in blood-brain-barrier endothelial cells via protein kinase A-dependent and -independent pathways |
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Authors: | Ishizaki Tsutomu Chiba Hideki Kojima Takashi Fujibe Masato Soma Tamotsu Miyajima Hideaki Nagasawa Kunihiko Wada Ikuo Sawada Norimasa |
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Affiliation: | Department of Pathology, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan. |
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Abstract: | Cyclic AMP (cAMP) promotes functions of tight junctions in endothelial cells, although its target remains unknown. We showed here that cAMP increased gene expression of claudin-5 and decreased that of claudin-1 in porcine blood-brain-barrier endothelial cells via protein kinase A (PKA)-independent and -dependent pathways, respectively. cAMP also enhanced immunoreactivity of claudin-5 along cell borders and in the cytoplasm, reorganized actin filaments, and altered signals of claudin-5, occludin, ZO-1, and ZO-2 along cell boundaries from zipperlike to linear patterns. In contrast, claudin-1 was detected only in the cytoplasm in a dotlike pattern, and its immunolabeling was reduced by cAMP. Interestingly, 31- and 62-kDa claudin-5 immunoprecipitates in the NP-40-soluble and -insoluble fractions, respectively, were highly phosphorylated on threonine residue(s) upon cAMP treatment. All these changes induced by cAMP, except for claudin-5 expression and its signals in the cytoplasm, were reversed by an inhibitor of PKA, H-89. We also demonstrated that cAMP elevated the barrier function of tight junctions in porcine blood-brain-barrier endothelial cells in PKA-dependent and -independent manners. These findings indicate that both PKA-induced phosphorylation of claudin-5 immunoprecipitates and cAMP-dependent but PKA-independent induction of claudin-5 expression could be involved in promotion of tight-junction function in endothelial cells. |
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Keywords: | blood– brain barrier cAMP PKA tight junctions claudins claudin-1 claudin-5 occludin phosphorylation |
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