Department of Ophthalmology, Rostock University Medical Center, 18057 Rostock, Germany.
Department of Medical Biochemistry, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Malá hora 4D, 03601 Martin, Slovakia.
Int J Mol Sci. 2018 Feb 4;19(2):464. doi: 10.3390/ijms19020464.
Following injury, corneal stromal keratocytes transform into repair-phenotype of activated stromal fibroblasts (SFs) and participate in wound repair. Simultaneously, ongoing bi-directional communications between corneal stromal-epithelial cells also play a vital role in mediating the process of wound healing. Factors produced by stromal cells are known to induce proliferation, differentiation, and motility of corneal epithelial cells, which are also subsequently the main processes that occur during wound healing. In this context, the present study aims to investigate the effect of SFs conditioned medium (SFCM) on corneal epithelial cell function along with substance P (SP). Antibody microarrays were employed to profile differentially expressed cell surface markers and cytokines in the presence of SFCM and SP. Antibody microarray data revealed enhanced expression of the ITGB1 in corneal epithelial cells following stimulation with SP whereas SFCM induced abundant expression of IL-8, ITGB1, PD1L1, PECA1, IL-15, BDNF, ICAM1, CD8A, CD44 and NTF4. All these proteins have either direct or indirect roles in epithelial cell growth, movement and adhesion related signaling cascades during tissue regeneration. We also observed activation of MAPK signaling pathway along with increased expression of focal adhesion kinase (FAK), paxillin, vimentin, β-catenin and vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Additionally, epithelial-to-mesenchymal transition (EMT) regulating transcription factors Slug and ZEB1 expression were enhanced in the presence of SFCM. SP enriched the expression of integrin subunits α4, α5, αV, β1 and β3 whereas SFCM increased α4, α5, αV, β1 and β5 integrin subunits. We also observed increased expression of Serpin E1 following SP and SFCM treatment. Wound healing scratch assay revealed enhanced migration of epithelial cells following the addition of SFCM. Taken together, we conclude that SFCM-mediated sustained activation of ZEB1, Slug in combination with upregulated migration-associated integrins and ERK (Extracellular signal-regulated kinase)-FAK-paxillin axis, may lead to induce type 2 EMT-like changes during corneal epithelial wound healing.
受伤后，角膜基质中的角膜基质成纤维细胞（SFs）转变为修复表型的激活的基质成纤维细胞，并参与伤口修复。同时，角膜基质-上皮细胞之间持续的双向通讯也在介导伤口愈合过程中起着至关重要的作用。已知基质细胞产生的因子可诱导角膜上皮细胞的增殖、分化和迁移，这也是伤口愈合过程中主要发生的过程。在这种情况下，本研究旨在研究 SF 条件培养基（SFCM）和 P 物质（SP）对角膜上皮细胞功能的影响。采用抗体微阵列分析 SFCM 和 SP 存在时细胞表面标记物和细胞因子的差异表达。抗体微阵列数据分析显示，SP 刺激后角膜上皮细胞中 ITGB1 的表达增强，而 SFCM 诱导大量表达 IL-8、ITGB1、PD1L1、PECAA1、IL-15、BDNF、ICAM1、CD8A、CD44 和 NTF4。所有这些蛋白在组织再生过程中都直接或间接地参与上皮细胞的生长、运动和黏附相关信号级联反应。我们还观察到 MAPK 信号通路的激活以及粘着斑激酶（FAK）、桩蛋白、波形蛋白、β-连环蛋白和血管扩张刺激磷蛋白（VASP）磷酸化的增加。此外，在 SFCM 存在的情况下，上皮-间充质转化（EMT）调节转录因子 Slug 和 ZEB1 的表达增强。SP 增强了整合素亚基 α4、α5、αV、β1 和 β3 的表达，而 SFCM 增加了 α4、α5、αV、β1 和 β5 整合素亚基的表达。我们还观察到 SP 和 SFCM 处理后 Serpin E1 的表达增加。划痕实验表明，添加 SFCM 后上皮细胞的迁移能力增强。综上所述，我们得出结论，SFCM 介导的 ZEB1 和 Slug 的持续激活，与上调的迁移相关整合素和 ERK（细胞外信号调节激酶）-FAK-桩蛋白轴一起，可能导致角膜上皮伤口愈合过程中诱导 2 型 EMT 样变化。
