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.
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.
Department of Pathology, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
Tsutomu
T
Ishizaki
Hideki
H
Chiba
Takashi
T
Kojima
Masato
M
Fujibe
Tamotsu
T
Soma
Hideaki
H
Miyajima
Kunihiko
K
Nagasawa
Ikuo
I
Wada
Norimasa
N
Sawada
D000818:Animals; D015703:Antigens, CD; D001812:Blood-Brain Barrier; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D062445:Claudin-1; D062507:Claudin-5; D057167:Claudins; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression; D007124:Immunoenzyme Techniques; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D062793:Occludin; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D062445:Claudin-1; D062507:Claudin-5; D057167:Claudins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D008565:Membrane Proteins; D062793:Occludin; D010750:Phosphoproteins; D012333:RNA, Messenger; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D000099152:Cadherin 5; C568108:CLDN1 protein, human; C491875:CLDN2 protein, human; C568113:Cldn1 protein, mouse; C491511:Cldn5 protein, mouse; C568985:OCLN protein, human; C568986:Ocln protein, mouse; C569042:TJP1 protein, human; C569046:TJP2 protein, human; C569043:Tjp1 protein, mouse; C569047:Tjp2 protein, mouse