TGFβ2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways.
TGFβ2 Induces the Formation of Cross-Linked Actin Networks (CLANs) in Human Trabecular Meshwork Cells Through the Smad and Non-Smad Dependent Pathways.
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DOI：
10.1167/iovs.16-19672
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发表时间：
2017-02-01
影响因子：
4.4
通讯作者：
Mao W
中科院分区：
文献类型：
--
作者：
Montecchi-Palmer M;Bermudez JY;Webber HC;Patel GC;Clark AF;Mao W
眼内压升高是由于病理变化（包括交联肌动蛋白网络（CLAN）的形成）导致小梁网（TM）处的房水（AH）流出阻力增加所致。转化生长因子β2（TGFβ2）在原发性开角型青光眼（POAG）患者的AH和TM中升高，并诱导POAG相关的TM改变，包括CLAN。我们确定了单个TGFβ2信号通路在CLAN形成中的作用。培养的非青光眼人TM（NTM）细胞用对照或TGFβ2处理，加或不加TGFβ受体抑制剂Smad 3、c-Jun N-末端激酶（JNK）、细胞外信号调节激酶（ERK）、P38或Rho相关蛋白激酶（ROCK）。NTM细胞用TGFβ2加抑制剂共处理10天或先用TGFβ2预处理10天，然后用抑制剂处理1小时。用鬼笔环肽-Alexa-488和4′，6-二脒基-2-苯基吲哚（DAPI）对NTM细胞进行免疫染色。使用单因素ANOVA和Dunnett事后检验分析数据。TGFβ2显着诱导CLAN形成（n = 6至12，P < 0.05），该形成可被TGFβ受体、Smad 3和ERK抑制剂完全抑制，也可被JNK、P38和ROCK抑制剂完全或部分抑制，具体取决于细胞株。ROCK抑制剂作用1小时可完全消除形成的CLANs（P < 0.05），而TGFβ受体、Smad 3抑制剂和ERK抑制剂作用1小时可部分或完全消除CLANs。JNK和P38抑制剂显示部分消退或无消退。在这些抑制剂中，ROCK抑制剂对肌动蛋白应力纤维的破坏性最大，而ERK抑制剂的破坏性最小。使用各种途径抑制剂预防和解决NTM细胞中TGFβ2诱导的CLAN。除了CLAN抑制，这些抑制剂中的一些也有不同的影响肌动蛋白应力纤维。
Increased intraocular pressure results from increased aqueous humor (AH) outflow resistance at the trabecular meshwork (TM) due to pathologic changes including the formation of cross-linked actin networks (CLANs). Transforming growth factor β2 (TGFβ2) is elevated in the AH and TM of primary open angle glaucoma (POAG) patients and induces POAG-associated TM changes, including CLANs. We determined the role of individual TGFβ2 signaling pathways in CLAN formation. Cultured nonglaucomatous human TM (NTM) cells were treated with control or TGFβ2, with or without the inhibitors of TGFβ receptor, Smad3, c-Jun N-terminal kinases (JNK), extracellular signal regulated kinase (ERK), P38, or Rho-associated protein kinase (ROCK). NTM cells were cotreated with TGFβ2 plus inhibitors for 10 days or pretreated with TGFβ2 for 10 days followed by 1-hour inhibitor treatment. NTM cells were immunostained with phalloidin-Alexa-488 and 4′,6-diamidino-2-phenylindole (DAPI). Data were analyzed using 1-way ANOVA and Dunnett's post hoc test. TGFβ2 significantly induced CLAN formation (n = 6 to 12, P < 0.05), which was completely inhibited by TGFβ receptor, Smad3, and ERK inhibitors, as well as completely or partially inhibited by JNK, P38, and ROCK inhibitors, depending on cell strains. One-hour exposure to ROCK inhibitor completely resolved formed CLANs (P < 0.05), whereas TGFβ receptor, Smad3 inhibitor, and ERK inhibitors resulted in partial or complete resolution. The JNK and P38 inhibitors showed partial or no resolution. Among these inhibitors, the ROCK inhibitor was the most disruptive to the actin stress fibers, whereas ERK inhibition showed the least disruption. TGFβ2-induced CLANs in NTM cells were prevented and resolved using various pathway inhibitors. Apart from CLAN inhibition, some of these inhibitors also had different effects on actin stress fibers.