Reconstitution and regulation of the blood-tissue barrier

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可通过蛋白激酶A(PKA)依赖和非依赖途径诱导猪血脑屏障内皮细胞中claudin-5的磷酸化和基因表达。沿着这条线，我们在claudin-5的胞质内羧基末端结构域的大约207&gt；处发现了一个假定的PKA的磷酸化位点。为了阐明该位点在调节内皮屏障功能中的生物学意义，我们建立了表达多西环素诱导的野生型Claudin-5及其突变体的大鼠肺内皮细胞(RLE)，并将其替换为Ala。用多西环素处理这些细胞，或联合cAMP和/或PKA抑制剂H-89，我们发现野生型claudin-5的诱导足以重建泄漏的RLE细胞对菊糖(5 KDa)的细胞旁屏障，但不能重建甘露醇(182 KDa)的细胞旁屏障。我们还证明了cAMP激活的PKA靶向内皮细胞中Thr207&gt；残基上的claudin-5。此外，我们发现claudin-5的Thr^和207是沿细胞边界增强claudin-5信号的关键，以及跨内皮细胞阻力的快速降低和基于claudin-5的内皮屏障的大小选择性松动。我们还在Claudin-1的203&gt；附近发现了MAPK的一个有效的磷酸化位点，并确定了该位点的生物学作用。为此，我们建立了表达多西环素诱导的野生型claudin-1及其突变体的RLE细胞，并将Thr^&lt；203&gt；替换为Ala。我们的研究结果表明，Claudin-1的苏氨酸是增强基于Claudin-1的内皮屏障所必需的。RLE细胞中多西环素诱导的基因表达系统将有助于研究各种基因产物在血管内皮细胞中的功能。

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.