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Reconstitution and regulation of the blood-tissue barrier

血组织屏障的重建和调节

基本信息

批准号：
14570196
负责人：
CHIBA Hideki
金额：
$ 2.37万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570196/
关键词：
Tight junction Claudin Blood-brain barrier Blood-tissue barrier MAP kinase Cyclic AMP Protein kinase A Enodothelial cells Tet-onシステム

项目摘要

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.

环磷酸腺苷（cAMP）和丝裂原活化蛋白激酶（MAPK）调节内皮细胞紧密连接的屏障功能，尽管它们的靶点尚不清楚。我们发现cAMP可以诱导猪血脑屏障内皮细胞中claudin-5的磷酸化和基因表达，分别通过蛋白激酶A（PKA）依赖性和非依赖性途径。沿着这条线，我们在密蛋白-5的胞质内羧基末端结构域中的Thr^附近鉴定了PKA的推定磷酸化位点<207>。为了阐明该位点在调节内皮屏障功能中的生物学意义，我们建立了表达强力霉素诱导的野生型claudin-5及其突变体（Thr^替换为Ala）的大鼠肺内皮（RLE）细胞<207>。使用用强力霉素处理的这些细胞，或与cAMP和/或PKA抑制剂H-89组合，我们表明，野生型claudin-5的诱导足以在渗漏的RLE细胞中重建针对菊粉（5 kDa）而不是甘露醇（182 Da）的细胞旁屏障。我们还证明了cAMP激活的PKA靶向内皮细胞中Thr^残基上的claudin-5<207>。此外，我们发现紧密连接<207>蛋白-5的Thr α对于紧密连接蛋白-5信号沿沿着细胞边界的增强，以及跨内皮阻力的快速降低和基于紧密连接蛋白-5的内皮屏障的尺寸选择性松动都是关键的。我们还在密蛋白-1的Thr^附近识别出MAPK的有效磷酸化位点<203>，并确定了该位点的生物学作用。为此，我们产生了表达多西环素诱导的野生型密蛋白-1和其突变体的RLE细胞，所述突变体具有Thr^<203>替换为Ala。我们的研究结果表明，<203>密蛋白-1的Thr β是增强基于密蛋白-1的内皮屏障所必需的。强力霉素诱导的RLE细胞基因表达系统将有助于研究内皮细胞中各种基因产物的功能。