Liu X, Wu Z, Sheibani N, Brandt C R, Polansky J R, Kaufman P L
Department of Ophthalmology and Visual Sciences, University of Wisconsin, F4/328 CSC, 600 Highland Avenue, Madison, WI, USA.
Exp Eye Res. 2003 Aug;77(2):181-8. doi: 10.1016/s0014-4835(03)00118-0.
We determined the effects of a low dose of the actin-disrupting agent latrunculin (LAT)-A on dexamethasone (DEX)-induced changes in actin organization, focal adhesions, and production of extracellular matrix proteins in cultured human trabecular meshwork (HTM) cells. HTM cells were cultured to a highly confluent stage with stable endothelium-like morphology and incubated with 0.1 or 0.2 microM DEX and/or 0.1 microM LAT-A. Changes in the actin cytoskeleton and vinculin-containing focal contacts were evaluated by immunofluorescence microscopy. Expression of thrombospondin-1 (TSP1) and fibronectin (FN) in HTM cells was evaluated by Western blot analysis. The results showed that DEX induced morphological changes and actin reorganization in HTM cells. The cells partly recovered after DEX withdrawal, but the addition of low dose LAT-A hastened the recovery. In addition, DEX failed to induce changes when co-incubated with LAT-A for at least 4 weeks, and for at least 2 weeks when cells were pre-treated with LAT-A for 2 weeks. HTM cells treated with 0.1 microM LAT-A only for 5 days showed mild disorganization of the actin cytoskeleton and focal adhesions, which persisted during the 4 weeks of treatment. DEX stimulated production of FN in HTM cells independent of LAT-A treatment. LAT-A and, to a lesser extent, DEX inhibited production of TSP1 by HTM cells. Although LAT-A is not a DEX receptor antagonist, it is able to prevent the effects of DEX on the actin cytoskeleton in cultured HTM cells at a dose subthreshold for increasing outflow facility in monkeys. This suggests that LAT-A at low doses may be useful in treating steroid and other glaucomas. TSP1 may be an important target of LAT-A in HTM cells and modulation of TSP may influence the actin cytoskeleton of the trabecular meshwork (TM), and consequently, intraocular pressure.
我们确定了低剂量的肌动蛋白破坏剂拉春库林（LAT）-A对培养的人小梁网（HTM）细胞中地塞米松（DEX）诱导的肌动蛋白组织、粘着斑以及细胞外基质蛋白产生变化的影响。将HTM细胞培养至高度汇合阶段，使其具有稳定的内皮样形态，并用0.1或0.2微摩尔/升的DEX和/或0.1微摩尔/升的LAT-A进行孵育。通过免疫荧光显微镜评估肌动蛋白细胞骨架和含纽蛋白的粘着斑的变化。通过蛋白质印迹分析评估HTM细胞中血小板反应蛋白-1（TSP1）和纤连蛋白（FN）的表达。结果表明，DEX诱导HTM细胞发生形态变化和肌动蛋白重组。撤去DEX后细胞部分恢复，但添加低剂量的LAT-A加速了恢复。此外，当与LAT-A共同孵育至少4周时，DEX未能诱导变化，而当细胞用LAT-A预处理2周时，至少2周内DEX未能诱导变化。仅用0.1微摩尔/升的LAT-A处理5天的HTM细胞显示肌动蛋白细胞骨架和粘着斑有轻度紊乱，在处理的4周内持续存在。DEX刺激HTM细胞中FN的产生，与LAT-A处理无关。LAT-A以及在较小程度上DEX抑制HTM细胞中TSP1的产生。虽然LAT-A不是DEX受体拮抗剂，但它能够在低于增加猴子房水流出率阈值的剂量下，预防DEX对培养的HTM细胞中肌动蛋白细胞骨架的影响。这表明低剂量的LAT-A可能对治疗类固醇性青光眼和其他青光眼有用。TSP1可能是LAT-A在HTM细胞中的一个重要靶点，对TSP的调节可能影响小梁网（TM）的肌动蛋白细胞骨架，进而影响眼压。
