Thr(207) of claudin-5 is involved in size-selective loosening of the endothelial barrier by cyclic AMP.
We have recently shown that cyclic AMP (cAMP) increases claudin-5 immunoreactivity along cell boundaries and could promote phosphorylation of claudin-5 on threonine residues in porcine blood-brain barrier (BBB) endothelial cells via a protein kinase A (PKA)-dependent pathway (Exp. Cell Res. 290 [2003] 275). Along this line, we identified a putative phosphorylation site for PKA at Thr(207) in the intracytoplasmic carboxyl terminal domain of claudin-5. To clarify the biological significance of this site in regulation of endothelial barrier functions, we established rat lung endothelial (RLE) cells expressing doxycycline (Dox)-inducible wild-type claudin-5 and a mutant with a substitution of Ala for Thr(207) (CL5T207A). We show that induction of wild-type claudin-5 is sufficient to reconstitute the paracellular barrier against inulin (5 kDa), but not mannitol (182 Da), in leaky RLE cells. By contrast, the barrier against both molecules was induced in the mutant cells. We also demonstrate that, upon cAMP treatment, Thr(207) of claudin-5 is involved in enhancement of claudin-5 immunoreactive signals along cell borders, rapid reduction in transendothelial electrical resistance (TER), and loosening of the claudin-5-based endothelial barrier against mannitol, but not inulin. cAMP decreased the claudin-5-based endothelial barrier, strongly suggesting that other tight-junction molecule(s) are required to elevate endothelial barrier functions in response to cAMP.
Soma
Tamotsu
T
Department of Pathology, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
Chiba
Hideki
H
Kato-Mori
Yuko
Y
Wada
Takuro
T
Yamashita
Toshihiko
T
Kojima
Takashi
T
Sawada
Norimasa
N
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D062507:Claudin-5; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004318:Doxycycline / Q000494:pharmacology; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D007150:Immunohistochemistry; D007444:Inulin / Q000378:metabolism / Q000493:pharmacokinetics; D008353:Mannitol / Q000378:metabolism / Q000493:pharmacokinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D051381:Rats; D012995:Solubility; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D062507:Claudin-5; C491510:Cldn5 protein, rat; D008565:Membrane Proteins; D008353:Mannitol; D007444:Inulin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D004318:Doxycycline; D000409:Alanine