| Project/Area Number |
14570196
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Experimental pathology
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| Research Institution | Sapporo Medical University |
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Principal Investigator |
CHIBA Hideki Sapporo Medical University, School of Medicine, Assistant Professor, 医学部, 講師 (00295346)
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| Co-Investigator(Kenkyū-buntansha) |
SAWADA Norimasa Sapporo Medical University, School of Medicine, Professor, 医学部, 教授 (30154149)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | Tight junction / Claudin / Blood-brain barrier / Blood-tissue barrier / MAP kinase / Cyclic AMP / Protein kinase A / Enodothelial cells / Tet-onシステム |
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| Research Abstract |
Cyclic AMP(cAMP) and mitogen-activated protein kinase(MAPK) modulate the barrier function of tight junctions in endothelial cells, although their targets remain unknown. We showed that cAMP could induce phosphorylation and gene expression of claudin-5 in porcine blood-brain barrier endothelial cells via protein kinase A(PKA)-dependent and -independent pathways, respectively. Along this line, we identified a putative phosphorylation site for PKA at around 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-inducible wild-type claudin-5 and its mutant with substitution of Thr^<207> to Ala. Using these cells treated with doxycycline, or in combination with cAMP and/or the PKA inhibitor H-89, we showed that induction of wild-type claudin-5 was sufficient to reconstitute the paracellular barrier against inulin(5kDa), but not mannitol(182Da), in leaky RLE cells. We also demonstrated that cAMP-activated PKA targeted claudin-5 on the Thr^<207> residue in endothelial cells. Furthermore, we found that Thr^<207> of claudin-5 was critical for enhancement of claudin-5 signals along cell borders, as well as both rapid reduction in transendothelial resistance and size-selective loosening of the claudin-5-based endothelial barrier. We also recognized a potent phosphorylation site for MAPK at around Thr^<203> of claudin-1, and determined the biological role of the site. To this end, we generated RLE cells expressing doxycycline-inducible wild-type claudin-1 and its mutant with substitution of Thr^<203> to Ala. Our findings indicated that Thr^<203> of claudin-1 was required to enhance the claudin-1-based endothelial barrier. The doxycycline-inducible gene expression system in RLE cells will be useful for studying functions of various gene products in endothelial cells.
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