ILK and cytoskeletal architecture: an important determinant of AQP2 recycling and subsequent entry into the exocytotic pathway.
ILK and cytoskeletal architecture: an important determinant of AQP2 recycling and subsequent entry into the exocytotic pathway.
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DOI：
10.1152/ajprenal.00336.2016
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发表时间：
2016-10
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通讯作者：
Fahmy A. Mamuya;José Luis Cano-Peñalver;Wei Li;D. Rodríguez Puyol;M. Rodriguez Puyol;Dennis Brown;S. de Frutos;H. Lu
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作者：
Fahmy A. Mamuya;José Luis Cano-Peñalver;Wei Li;D. Rodríguez Puyol;M. Rodriguez Puyol;Dennis Brown;S. de Frutos;H. Lu
在过去的十年中，人们为了解水通道蛋白 2 (AQP2) 水通道运输和循环背后的机制付出了巨大的努力，为尿崩症的有效治疗开辟了道路。最近的一项研究表明，整合素连接激酶 (ILK) 条件敲低小鼠会出现多尿症，同时 AQP2 表达下降。为了了解 ILK 是否也调节肾小管细胞中的 AQP2 运输，我们使用稳定表达大鼠 AQP2 的 LLCPK1 细胞（LLC-AQP2 细胞）进行了体外分析。用 ILK 抑制剂 cpd22 和 ILK-siRNA 处理 LLC-AQP2 细胞后，我们观察到 AQP2 在核周区域的积累增加，但胞吞速率没有任何显着增加。这种核周积累并未发生在表达丝氨酸-256-天冬氨酸突变（将 AQP2 保留在质膜中）的细胞中。然后，我们使用罗丹明缀合的转铁蛋白抑制 ILK 后检查了网格蛋白介导的内吞作用。尽管总体转铁蛋白内吞作用没有差异，但内吞的转铁蛋白也在核周区域积累，与 AQP2 共定位。这些积累的囊泡还含有回收内体标记 Rab11。与此同时，ILK 抑制后，通常的加压素诱导的 AQP2 膜积聚被阻止；然而，ILK 抑制并没有显着影响 AQP2 在丝氨酸 256 处的磷酸化或其在丝氨酸 261 处的去磷酸化。相反，我们发现抑制 ILK 会增加 F-肌动蛋白聚合。当F-肌动蛋白用latrunculin解聚时，位于核周的AQP2分散。我们得出的结论是，ILK 在 AQP2 回收过程中协调动态细胞骨架结构中发挥着重要作用，这对于其随后进入胞吐途径是必需的。
Within the past decade tremendous efforts have been made to understand the mechanism behind aquaporin-2 (AQP2) water channel trafficking and recycling, to open a path toward effective diabetes insipidus therapeutics. A recent study has shown that integrin-linked kinase (ILK) conditional-knockdown mice developed polyuria along with decreased AQP2 expression. To understand whether ILK also regulates AQP2 trafficking in kidney tubular cells, we performed in vitro analysis using LLCPK1 cells stably expressing rat AQP2 (LLC-AQP2 cells). Upon treatment of LLC-AQP2 cells with ILK inhibitor cpd22 and ILK-siRNA, we observed increased accumulation of AQP2 in the perinuclear region, without any significant increase in the rate of endocytosis. This perinuclear accumulation did not occur in cells expressing a serine-256-aspartic acid mutation that retains AQP2 in the plasma membrane. We then examined clathrin-mediated endocytosis after ILK inhibition using rhodamine-conjugated transferrin. Despite no differences in overall transferrin endocytosis, the endocytosed transferrin also accumulated in the perinuclear region where it colocalized with AQP2. These accumulated vesicles also contained the recycling endosome marker Rab11. In parallel, the usual vasopressin-induced AQP2 membrane accumulation was prevented after ILK inhibition; however, ILK inhibition did not measurably affect AQP2 phosphorylation at serine-256 or its dephosphorylation at serine-261. Instead, we found that inhibition of ILK increased F-actin polymerization. When F-actin was depolymerized with latrunculin, the perinuclear located AQP2 dispersed. We conclude that ILK is important in orchestrating dynamic cytoskeletal architecture during recycling of AQP2, which is necessary for its subsequent entry into the exocytotic pathway.