Tumminia S J, Mitton K P, Arora J, Zelenka P, Epstein D L, Russell P
Laboratory for Mechanisms of Ocular Diseases, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2735, USA.
Invest Ophthalmol Vis Sci. 1998 Jul;39(8):1361-71.
Human trabecular meshwork (HTM) cells were mechanically stretched in vitro as a potential model for the distension of this tissue that can occur in vivo in response to increased pressure gradients. Cell morphology and certain components of the signal transduction pathways, including the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase (JNK) pathways, were evaluated for stretch-induced alterations.
Primary HTM cells grown in tissue culture were subjected to a mechanical stretch lasting from 10 seconds to 4 days. The actin cytoskeletal network was visualized by phalloidin staining. Proteins phosphorylated on their tyrosine residues were isolated using an immunoaffinity system and were analyzed by gel electrophoresis and immunostaining. Mitogen-activated protein kinase activity was evaluated using an in-gel assay system, and the mRNA levels of c-fos and c-jun were determined by quantitation of competitive reverse transcription-polymerase chain reaction. In addition, the amount of c-Fos protein was estimated by chemiluminescent immunoblot analysis.
On stretching, the HTM cells elongated but regained their normal morphologic characteristics within 24 hours. Unstretched HTM cells displayed a diffuse F-actin microfilament network, whereas stretched cells exhibited complex geodesic patterns. Ten seconds after stretching began, the level of tyrosine phosphorylation on the six major phosphoproteins significantly decreased between 80% and 100%, whereas the level of paxillin tyrosine phosphorylation significantly increased 39%. Stretching caused MAPK activity and the amount of mRNA and protein of the immediate-early gene c-fos to decrease more than 60% within 2 minutes, but within 15 to 30 minutes they increased above or equivalent to normal levels. The level of c-jun mRNA was unchanged by stretching.
In response to a mechanical stretch, major cytoskeletal alterations occur in HTM cells, which involve changes in the levels of tyrosine phosphorylation. Mechanotransduction (signal transduction by mechanical stimulation) through the MAPK signaling pathway was significantly depressed immediately after stretching; however, the JNK pathway appeared to be unaffected. The data suggest that HTM cells adapt to mechanical stress by altering the cytoskeletal network and signaling cascades.
在体外对人小梁网（HTM）细胞进行机械拉伸，以此作为该组织扩张的潜在模型，这种扩张在体内可因压力梯度增加而发生。评估细胞形态以及信号转导通路的某些成分，包括丝裂原活化蛋白激酶（MAPK）和c-Jun氨基末端蛋白激酶（JNK）通路，以检测拉伸诱导的改变。
将在组织培养中生长的原代HTM细胞进行持续10秒至4天的机械拉伸。用鬼笔环肽染色观察肌动蛋白细胞骨架网络。使用免疫亲和系统分离酪氨酸残基磷酸化的蛋白质，并通过凝胶电泳和免疫染色进行分析。使用凝胶内分析系统评估丝裂原活化蛋白激酶活性，通过竞争性逆转录-聚合酶链反应定量测定c-fos和c-jun的mRNA水平。此外，通过化学发光免疫印迹分析估计c-Fos蛋白的量。
拉伸时，HTM细胞伸长，但在24小时内恢复其正常形态特征。未拉伸的HTM细胞显示出弥漫性的F-肌动蛋白微丝网络，而拉伸后的细胞呈现出复杂的测地线模式。拉伸开始10秒后，六种主要磷蛋白的酪氨酸磷酸化水平显著降低80%至100%，而桩蛋白酪氨酸磷酸化水平显著增加39%。拉伸导致MAPK活性以及即刻早期基因c-fos的mRNA和蛋白量在2分钟内降低超过60%，但在15至30分钟内它们增加至高于或等同于正常水平。拉伸未改变c-jun mRNA的水平。
响应机械拉伸，HTM细胞发生主要的细胞骨架改变，这涉及酪氨酸磷酸化水平的变化。拉伸后立即通过MAPK信号通路的机械转导（通过机械刺激进行信号转导）显著受抑；然而，JNK通路似乎未受影响。数据表明HTM细胞通过改变细胞骨架网络和信号级联反应来适应机械应力。
