Poly-l-arginine enhances paracellular permeability via serine/threonine phosphorylation of ZO-1 and tyrosine dephosphorylation of occludin in rabbit nasal epithelium
Poly-l-arginine enhances paracellular permeability via serine/threonine phosphorylation of ZO-1 and tyrosine dephosphorylation of occludin in rabbit nasal epithelium
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DOI：
10.1023/b:pham.0000003383.86238.d1
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发表时间：
2003-11-01
影响因子：
3.7
通讯作者：
Morimoto, Y
中科院分区：
文献类型：
--
作者：
Ohtake, K;Maeno, T;Morimoto, Y
目的。本研究的目的是探讨多聚L-精氨酸(聚L-精氨酸)诱导的异硫氰酸荧光素标记的葡聚糖(MW4.4 kDa，FD-4)紧密连接通透性增加是否与钙依赖的信号转导有关，并且发生在TJ蛋白的磷酸化/去磷酸化之后。将兔离体鼻上皮固定在Ussing型小室中，在不同的抑制剂存在下，测量FD-4的转运和膜电导(GT)，这些抑制剂参与了TJ蛋白的钙依赖途径和磷酸化/去磷酸化。用激光共聚焦扫描显微镜(CLSM)观察TJ蛋白在免疫染色中的分布。0.2 mg/mlPoly-L-Arg引起的FD-4细胞通透性增加不能被钙动员途径的抑制剂所改变，提示Poly-L-Arg的促通透性作用不依赖于Ca~(2+)相关信号。另一方面，蛋白激酶C和酪氨酸磷酸酶抑制剂抑制了多聚L-精氨酸对TJ通透性的增加，提示可能发生了通过非钙依赖的蛋白激酶C的丝氨酸/苏氨酸磷酸化和连接蛋白的酪氨酸去磷酸化。此外，对TJ相关蛋白ZO-1和定位于TJ的整合膜蛋白occludin的免疫荧光监测表明，ZO-1和occludin分别通过PKC活化的丝氨酸/苏氨酸磷酸化和酪氨酸去磷酸化的方式内化TJ，从而提供TJ的解离。我们的结论是，多聚L-精氨酸至少通过ZO-1的丝氨酸/苏氨酸磷酸化和阻塞素的酪氨酸去磷酸化增强FD-4的细胞旁通透性(即大分子)。
Purpose. The purpose of the present study is to explore whether a poly-L-arginine (poly-L-Arg)-induced increase in tight junctions (TJ) permeability of fluorescein isothiocyanate-labeled dextran (MW 4.4 kDa, FD-4) is associated with the Ca2+-dependent signaling and occurs following the phosphorylation/dephosphorylation of TJ proteins.Methods. Excised rabbit nasal epithelium was mounted in an Ussing-type chamber for measurement of FD-4 transport and membrane conductance (Gt) in the presence of various inhibitors that are involved in the Ca2+-dependent pathway and the phosphorylation/ dephosphorylation of TJ proteins. The resultant distribution of TJ proteins was observed using confocal laser scanning microscopy (CLSM) in an immunostaining.Results. The increase in TJ permeability of FD-4 induced by 0.2 mg/ml poly-L-Arg was not altered by treatment with inhibitors of possible Ca2+ mobilization pathways followed by exposure of poly-L- Arg, suggesting that the promoting effect of poly-L- Arg is independent of Ca2+-related signaling. On the other hand, the protein kinase C (PKC) and tyrosine phosphatase inhibitors suppress the increase in TJ permeability by poly-L- Arg, indicating that serine/threonine phosphorylation by way of Ca2+-independent PKC and tyrosine dephosphorylation of junction proteins may have occurred. Furthermore, immunofluorescent monitoring of ZO-1, a TJ associated protein, and occludin, an integral membrane protein localizing at TJ, after preincubation with PKC and tyrosine phosphatase inhibitors followed by poly-L- Arg treatment has shown that the internalization of ZO-1 and occludin occurred by way of serine/threonine phosphorylation by PKC activation and by way of tyrosine dephosphorylation, respectively, providing TJ disassembly.Conclusions. We conclude that poly-L- Arg enhances the paracellular permeability of FD-4 (i.e., macromolecules), at least, by way of both serine/threonine phosphorylation of ZO-1 and tyrosine dephosphorylation of occludin in rabbit nasal epithelium.