Vorontsova Irene, Lam Leo, Delpire Eric, Lim Julie, Donaldson Paul
Department of Optometry and Vision Science, University of Auckland, New Zealand The New Zealand National Eye Centre, University of Auckland, New Zealand.
Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, Tennessee, United States.
Invest Ophthalmol Vis Sci. 2014 Dec 16;56(1):310-21. doi: 10.1167/iovs.14-15911.
To identify whether the kinases that regulate the activity of cation chloride cotransporters (CCC) in other tissues are also expressed in rat and human lenses.
The expression of with-no-lysine kinase (WNK 1, 3, 4), oxidative stress response kinase 1 (OSR1), and Ste20-like proline alanine rich kinase (SPAK) were determined at either the transcript or protein levels in the rat and human lenses by reverse-transcriptase PCR and/or Western blotting, respectively. Selected kinases were regionally and subcellularly characterized in rat and human lenses. The transparency, wet weight, and tissue morphology of lenses extracted from SPAK knock-out animals was compared with wild-type lenses.
WNK 1, 3, 4, SPAK, and OSR1 were identified at the transcript level in rat lenses and WNK1, 4, SPAK, and OSR1 expression confirmed at the protein level in both rat and human lenses. SPAK and OSR1 were found to associate with membranes as peripheral proteins and exhibited distinct subcellular and region-specific expression profiles throughout the lens. No significant difference in the wet weight of SPAK knock-out lenses was detected relative to wild-type lenses. However, SPAK knock-out lenses showed an increased susceptibility to opacification.
Our results show that the WNK 1, 3, 4, OSR1, and SPAK signaling system known to play a role in regulating the phosphorylation status, and hence activity of the CCCs in other tissues, is also present in the rat and human lenses. The increased susceptibility of SPAK lenses to opacification suggests that disruption of this signaling pathway may compromise the ability of the lens to control its volume, and its ability to maintain its transparency.
确定在其他组织中调节阳离子氯共转运体（CCC）活性的激酶在大鼠和人类晶状体中是否也有表达。
分别通过逆转录聚合酶链反应和/或蛋白质免疫印迹法，在转录水平或蛋白质水平上测定大鼠和人类晶状体中无赖氨酸激酶（WNK 1、3、4）、氧化应激反应激酶1（OSR1）和类Ste20脯氨酸丙氨酸丰富激酶（SPAK）的表达。对大鼠和人类晶状体中选定的激酶进行区域和亚细胞特征分析。将从SPAK基因敲除动物中提取的晶状体的透明度、湿重和组织形态与野生型晶状体进行比较。
在大鼠晶状体中在转录水平鉴定出WNK 1、3、4、SPAK和OSR1，在大鼠和人类晶状体的蛋白质水平均证实了WNK1、4、SPAK和OSR1的表达。发现SPAK和OSR1作为外周蛋白与膜相关联，并在整个晶状体中表现出独特的亚细胞和区域特异性表达模式。相对于野生型晶状体，未检测到SPAK基因敲除晶状体的湿重有显著差异。然而，SPAK基因敲除晶状体显示出对浑浊的易感性增加。
我们的结果表明，已知在调节其他组织中CCC的磷酸化状态进而活性方面起作用的WNK 1、3、4、OSR1和SPAK信号系统在大鼠和人类晶状体中也存在。SPAK基因敲除晶状体对浑浊的易感性增加表明，该信号通路的破坏可能会损害晶状体控制其体积的能力及其维持透明度的能力。
