Dexamethasone Induces Cross-Linked Actin Networks in Trabecular Meshwork Cells Through Noncanonical Wnt Signaling
Dexamethasone Induces Cross-Linked Actin Networks in Trabecular Meshwork Cells Through Noncanonical Wnt Signaling
复制标题
DOI：
10.1167/iovs.13-12447
复制
发表时间：
2013-10-01
影响因子：
4.4
通讯作者：
Kao, Winston W. -Y.
中科院分区：
文献类型：
--
作者：
Yuan, Yong;Call, Mindy K.;Kao, Winston W. -Y.
目的.地塞米松（DEX）通过诱导小梁网（TM）细胞中细胞骨架重组形成交联肌动蛋白网络（CLANs）来调节房水流出。Rho相关蛋白激酶（ROCK）已被证明在这一过程中具有重要作用，但导致其激活的上游组分仍然难以捉摸。本研究的目的是证明非经典Wnt信号通路介导DEX诱导的TM细胞CLAN形成。将TM细胞在低血清培养基中用100 nM DEX处理7天以上。每3天更换培养基。收获细胞并对Wnt配体的表达进行分子分析。鬼笔环肽染色显示应力纤维结构。使用针对非经典Wnt受体（Ror 2）的基于慢病毒的shRNA来确定非经典Wnt信号传导在DEX诱导的CLAN形成中的作用。DEX诱导TM细胞应力纤维重排。通过Wnt配体简并PCR、实时定量PCR（qRT-PCR）和Western印迹证实，DEX上调非经典Wnt配体（Wnt 5a）。敲低非经典Wnt信号的受体Ror 2可消除DEX对TM细胞的作用。我们的数据表明，DEX诱导非经典Wnt配体Wnt 5a的上调。重组WNT 5a蛋白通过非经典Wnt受体ROR 2/RhoA/ROCK信号传导轴诱导CLAN形成。鉴于DEX诱导的高眼压和原发性开角型青光眼之间的相似性，我们的研究结果提供了应用ROCK抑制剂治疗原发性开角型青光眼的作用机制。
PURPOSE. Dexamethasone (DEX) regulates aqueous humor outflow by inducing a reorganization of the cytoskeleton to form cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells. Rho-associated protein kinase (ROCK) has been demonstrated to have an important role in this process, but the upstream components leading to its activation remain elusive. The purpose of the study is to demonstrate that noncanonical Wnt signaling mediates the DEX-induced CLAN formation in TM cells.METHODS. The TM cells were treated with 100 nM DEX in low serum medium for over 7 days. The medium was changed every 3 days. The cells were harvested and subjected to molecular analysis for the expression of Wnt ligands. Stress fiber structures were revealed by Phalloidin staining. Lentivirus-based shRNA against noncanonical Wnt receptor (Ror2) was used to determine the role of noncanonical Wnt signaling in DEX-induced CLAN formation.RESULTS. The DEX induced stress fiber rearrangement in TM cells. A noncanonical Wnt ligand (Wnt5a) was upregulated by DEX as demonstrated by Wnt ligand degenerate PCR, real-time quantitative PCR (qRT-PCR), and Western blotting. Knocking-down Ror2, the receptor of noncanonical Wnt signaling, abolished the effects of DEX on the TM cells.CONCLUSIONS. Our data suggest that DEX induces the upregulation of noncanonical Wnt ligand Wnt5a. Recombinant WNT5a protein induces CLAN formation through the noncanonical Wnt receptor ROR2/RhoA/ROCK signaling axis. Given the similarities between DEX-induced ocular hypertension and primary open-angle glaucoma, our results provide a mechanism of action for applying ROCK inhibitor to treat primary open-angle glaucoma.