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

Exp Cell Res. 2003 Nov 1;290(2):275-88. doi: 10.1016/s0014-4827(03)00354-9.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, CD
  • Blood-Brain Barrier*
  • Cadherins / genetics
  • Cadherins / metabolism
  • Claudin-1
  • Claudin-5
  • Claudins
  • Cyclic AMP / pharmacology*
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Endothelium, Vascular / metabolism*
  • Gene Expression
  • Immunoenzyme Techniques
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism*
  • Occludin
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Precipitin Tests
  • RNA, Messenger / metabolism
  • Rabbits
  • Reverse Transcriptase Polymerase Chain Reaction
  • Swine
  • Zonula Occludens-1 Protein
  • Zonula Occludens-2 Protein


  • Antigens, CD
  • CLDN1 protein, human
  • CLDN2 protein, human
  • Cadherins
  • Claudin-1
  • Claudin-5
  • Claudins
  • Cldn1 protein, mouse
  • Cldn5 protein, mouse
  • Membrane Proteins
  • OCLN protein, human
  • Occludin
  • Ocln protein, mouse
  • Phosphoproteins
  • RNA, Messenger
  • TJP1 protein, human
  • TJP2 protein, human
  • Tjp1 protein, mouse
  • Tjp2 protein, mouse
  • Zonula Occludens-1 Protein
  • Zonula Occludens-2 Protein
  • cadherin 5
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases