Ocular Surface Research Group, Singapore Eye Research Institute, 169856, Singapore; Eye-Academic Clinical Programme, Duke-NUS Medical School, 169857, Singapore.
Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos, Singapore, 138673, Singapore.
Ocul Surf. 2023 Apr;28:155-164. doi: 10.1016/j.jtos.2023.04.002. Epub 2023 Apr 8.
Transglutaminase (TG)-2 is a ubiquitous multi-functional protein expressed in all living cells. The purpose of the current study was to investigate the role of TG-2 in corneal barrier function and its potential regulation of epithelial junctional proteins and transcription factors.
Corneal barrier function to ions in TG-2 and TG-2 mice was assessed by Ussing chamber assay. Hypo-osmolar water or FITC-dextran was applied on top of mouse eyes to evaluate the corneal barrier function to water and macromolecules. Western blots, qPCR and immunofluorescent staining were used to investigate the expression of tight junction proteins in TG-2 and TG-2 mouse corneas, and also in TG-2 knockdown human corneal epithelial cells.
Corneal explants from TG-2 mice had a lower trans-epithelial electrical resistance compared to TG-2 mice. When challenged by hypo-osmolar water, the central corneal thickness of TG-2 mice increased faster, and these mice had a faster rise of fluorescence in the anterior chamber after ocular exposure to FITC-dextran, compared to TG-2. Claudin-1 protein and transcript levels were reduced in the cornea of TG-2 mice and in TG-2 knockdown human corneal epithelial cells. Slug which previously reported suppressing Claudin-1 transcription, was increased at both protein and transcript level in TG-2 knockdown cells. TG-2 and Claudin-1 protein levels were unchanged in shRNA and shTG cells after MG132 treatment, while Slug accumulated in treated cells.
TG-2 may positively regulate Claudin-1 through repressing Slug at transcript level, and thus it is critical for normal corneal barrier function.
转谷氨酰胺酶（TG）-2 是一种普遍存在的多功能蛋白，存在于所有活细胞中。本研究的目的是探讨 TG-2 在角膜屏障功能中的作用及其对上皮连接蛋白和转录因子的潜在调节作用。
通过 Ussing 室测定法评估 TG-2 和 TG-2 小鼠的角膜对离子的屏障功能。将低渗盐水或 FITC-葡聚糖施加于小鼠眼睛的顶部，以评估角膜对水和大分子的屏障功能。使用 Western blot、qPCR 和免疫荧光染色来研究 TG-2 和 TG-2 小鼠角膜中紧密连接蛋白的表达，也研究了 TG-2 敲低的人角膜上皮细胞中的表达情况。
与 TG-2 小鼠相比，TG-2 小鼠的角膜外植体具有较低的跨上皮电阻。当受到低渗盐水的挑战时，TG-2 小鼠的中央角膜厚度增加得更快，并且这些小鼠在前房暴露于 FITC-葡聚糖后，荧光在前房的上升速度更快。与 TG-2 小鼠相比，TG-2 小鼠角膜中的 Claudin-1 蛋白和转录本水平降低，并且在 TG-2 敲低的人角膜上皮细胞中也是如此。先前报道的抑制 Claudin-1 转录的 Slug 在 TG-2 敲低细胞中在蛋白和转录本水平上均增加。在用 MG132 处理 shRNA 和 shTG 细胞后，TG-2 和 Claudin-1 蛋白水平保持不变，而 Slug 在处理的细胞中积累。
TG-2 可能通过在转录本水平上抑制 Slug 来正向调节 Claudin-1，因此对正常角膜屏障功能至关重要。
