Wnt Activation After Inhibition Restores Trabecular Meshwork Cells Toward a Normal Phenotype
Wnt Activation After Inhibition Restores Trabecular Meshwork Cells Toward a Normal Phenotype
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DOI：
10.1167/iovs.61.6.30
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发表时间：
2020-06-01
影响因子：
4.4
通讯作者：
Raghunathan, VijayKrishna
中科院分区：
文献类型：
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作者：
Dhamodaran, Kamesh;Baidouri, Hasna;Raghunathan, VijayKrishna
目的. Wnt是一种时空调节的信号通路，其抑制与青光眼、眼内压升高（TOP）和细胞硬化相关。这种变化是永久性的还是可能逆转，目前尚不清楚。在这里，我们确定抑制后Wnt通路的激活是否逆转了病理表型。将来自非青光眼供体的原代人小梁网（hTM）细胞在不存在或存在以下Wnt调节剂的情况下培养12天：（i）LGK 974（Porcn抑制剂，10 μ M）;（ii）LY 2090314（pGSK β抑制剂，250 nM）;或（iii）LGK 974 9天，随后LY 2090314 3天。Western印迹法检测Wnt的表达，定量PCR法检测细胞外基质（ECM）相关基因的表达。通过免疫荧光和原子力显微镜测定细胞骨架形态和细胞硬度。通过pGSK β（0.3倍; P < 0.01）的下调、AXIN 2（6.7倍; P < 0.001）和LEF 1（3.8倍; P < 0.001）的过表达证实了Wnt激活。Wnt抑制导致F-肌动蛋白的急剧变化，这与随后的Wnt激活解决。同时，Wnt抑制（11.86 kPa; P < 0.01）导致细胞硬度升高，随后Wnt激活（4.195 kPa; P < 0.01）导致细胞硬度降低，并伴有磷酸化雅普（1.8倍; P < 0.001）和TAZ（1.4倍; P < 0.001）的显著过表达。此外，抑制后的Wnt激活显著抑制ECM基因（TIMP 1和CTGF，P < 0.01）、交联基因（LOX和TGM 2，P < 0.05）、基质金属蛋白酶抑制剂（TIMP 1和PAIL，P < 0.001）和MMP 1/9/14的过表达（P < 0.01）。这些数据有力地证明，在正常hTM细胞中，Wnt途径的激活逆转了由Wnt抑制引起的病理表型，因此可能是降低IOP的可行治疗剂。
PURPOSE. Wnt is a spatiotemporally regulated signaling pathway whose inhibition is associated with glaucoma, elevated intraocular pressure (TOP), and cell stiffening. Whether such changes are permanent or may be reversed is unclear. Here, we determine if activation of Wnt pathway after inhibition reverses the pathologic phenotype.METHODS. Primary human trabecular meshwork (hTM) cells from nonglaucomatous donors were cultured for 12 days in the absence or presence of Wnt modulators: (i) LGK974 (Porcn inhibitor, 10 mu M); (ii) LY2090314 (pGSK beta inhibitor, 250 nM); or (iii) 9 days of LGK974 followed by 3 days of LY2090314. Wnt modulation were determined by Western blotting and extracellular matrix (ECM) related genes were evaluated by quantitative PCR. Cytoskeletal morphology was determined by immunofluorescence and cell stiffness by atomic force microscopy.RESULTS. Wnt activation was confirmed by downregulation of pGSK beta (0.3-fold; P < 0.01), overexpression of AXIN2 (6.7-fold; P < 0.001), and LEF1 (3.8-fold; P < 0.001). Wnt inhibition resulted in dramatic changes in F-actin, which were resolved with subsequent Wnt activation. Concurrently, cell stiffness that was elevated with Wnt inhibition (11.86 kPa; P < 0.01) decreased with subsequent Wnt activation (4.195 kPa; P < 0.01) accompanied by significant overexpression of phosphorylated YAP (1.8-fold; P < 0.001) and TAZ (1.4-fold; P < 0.001). Additionally, Wnt activation after inhibition significantly repressed ECM genes (SPARC and CTGF, P < 0.01), cross-linking genes (LOX and TGM2, P < 0.05), inhibitors of matrix metalloproteinases (TIMP1 and PAIL, P < 0.001), and overexpressed MMP 1/9/14 (P < 0.01).CONCLUSIONS. These data strongly demonstrate that, in normal hTM cells, activation of the Wnt pathway reverses the pathological phenotype caused by Wnt inhibition and may thus be a viable therapeutic for lowering IOP.