Existence of the Canonical Wnt Signaling Pathway in the Human Trabecular Meshwork
Existence of the Canonical Wnt Signaling Pathway in the Human Trabecular Meshwork
复制标题
DOI：
10.1167/iovs.12-9664
复制
发表时间：
2012-10-01
影响因子：
4.4
通讯作者：
Clark, Abbot F.
中科院分区：
文献类型：
--
作者：
Mao, Weiming;Millar, J. Cameron;Clark, Abbot F.
目的。我们之前在青光眼小梁网（GTM）中发现Wnt信号通路抑制剂分泌卷曲相关蛋白1 （sFRP1）水平升高，并发现关键的典型Wnt信号通路基因在小梁网（TM）中表达。本研究的目的是确定人类TM （HTM）中是否存在功能性的典型Wnt信号通路。Western免疫印迹和/或免疫荧光显微镜研究转化和原代HTM细胞中的β -连环蛋白易位和肌动蛋白细胞骨架。采用TCF/LEF荧光素酶法研究功能性典型Wnt信号，并通过定量PCR进一步证实了wnt3a诱导通路靶基因AXIN2的表达。通过玻璃体内注射表达dickkopf相关蛋白-1 （DKK1）的Ad5腺病毒，研究了典型Wnt信号在小鼠IOP中的作用。WNT3a诱导HTM中的β -连环蛋白易位，通过与sFRP1共处理可阻断该易位。同样，WNT3a在TCF/LEF荧光素酶检测中也增强了荧光素酶水平，这也被sFRP1阻断。此外，WNT3a显著提高了AXIN2的表达。然而，WNT3a和sFRP1都不影响肌动蛋白细胞骨架的组织，理论上，这可能是由HTM细胞中的非典型Wnt信号调节的。外源性DKK1（一种典型Wnt信号通路的特异性抑制剂）或sFRP1可将小鼠IOP升高至同等水平。在TM中存在一个典型的Wnt信号通路，这个典型的Wnt信号通路调节IOP，而不是非典型的Wnt信号通路。（Invest Ophthalmol Vis Sci. 2012;53:7043-7051） DOI:10.1167/iovs.12-9664
PURPOSE. We previously discovered elevated levels of secreted frizzled-related protein 1 (sFRP1), the Wnt signaling pathway inhibitor, in the glaucomatous trabecular meshwork (GTM), and found that key canonical Wnt signaling pathway genes are expressed in the trabecular meshwork (TM). The purpose of our study was to determine whether a functional canonical Wnt signaling pathway exists in the human TM (HTM).METHODS. Western immunoblotting and/or immunofluorescent microscopy were used to study beta-catenin translocation as well as the actin cytoskeleton in transformed and primary HTM cells. A TCF/LEF luciferase assay was used to study functional canonical Wnt signaling, which was confirmed further by WNT3a-induced expression of a pathway target gene, AXIN2, via quantitative PCR. Intravitreal injection of an Ad5 adenovirus expressing Dickkopf-related protein-1 (DKK1) was used to study the in vivo effect of canonical Wnt signaling on IOP in mice.RESULTS. WNT3a induced beta-catenin translocation in the HTM, which was blocked by co-treatment with sFRP1. Similarly, WNT3a enhanced luciferase levels in TCF/LEF luciferase assays, which also were blocked by sFRP1. Furthermore, AXIN2 expression was elevated significantly by WNT3a. However, neither WNT3a nor sFRP1 affected actin cytoskeleton organization, which theoretically could be regulated by noncanonical Wnt signaling in HTM cells. Exogenous DKK1, a specific inhibitor for the canonical Wnt signaling pathway, or sFRP1 elevated mouse IOP to equivalent levels.CONCLUSIONS. There is a canonical Wnt signaling pathway in the TM, and this canonical Wnt pathway, but not the noncanonical Wnt signaling pathway, regulates IOP. (Invest Ophthalmol Vis Sci. 2012;53:7043-7051) DOI:10.1167/iovs.12-9664