Department of Ophthalmology, Peking University Third Hospital, Beijing, China.
Beijing Key Laboratory of Restoration of Damaged Ocular Nerve, Peking University Third Hospital, Beijing, China.
Invest Ophthalmol Vis Sci. 2024 Jan 2;65(1):4. doi: 10.1167/iovs.65.1.4.
To investigate the impact of transmembrane protein CMTM6 on the pathogenesis of dry eye disease (DED) and elucidate its potential mechanisms.
CMTM6 expression was confirmed by database analysis, real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. Tear secretion was measured using the phenol red thread test. Immune cell infiltration was assessed through flow cytometry. Barrier function was evaluated by fluorescein sodium staining, immunofluorescence staining of zonula occludens 1 (ZO-1), and electric cell-substrate impedance sensing (ECIS) assessment. For silencing CMTM6 expression, siRNA and shRNA were employed, along with lentiviral vector-mediated overexpression of CMTM6. Proinflammatory cytokine levels were analyzed by RT-PCR and cytometric bead array (CBA) analysis.
CMTM6 showed high expression in healthy human and mouse corneal and conjunctival epithelium but was notably reduced in DED. Notably, this downregulation was correlated with disease severity. Cmtm6-/- dry eye (DE) mice displayed reduced tear secretion, severe corneal epithelial defects, decreased conjunctival goblet cell density, and upregulated inflammatory response. Additionally, Cmtm6-/- DE mice and CMTM6 knockdown human corneal epithelial cell-transformed (HCE-T) cells showed more severe barrier disruption and reduced expression of ZO-1. Knockdown of CMTM6 in HCE-T cells increased inflammatory responses induced by hyperosmotic stress, which was significantly mitigated by CMTM6 overexpression. Moreover, the level of phospho-p65 in hyperosmolarity-stimulated HCE-T cells increased after silencing CMTM6. Nuclear factor kappa B (NF-κB) p65 inhibition (JSH-23) reversed the excessive inflammatory responses caused by hyperosmolarity in CMTM6 knockdown HCE-T cells.
The reduction in CMTM6 expression on the ocular surface contributes to the pathogenesis of DED. The CMTM6-NF-κB p65 signaling pathway may serve as a promising therapeutic target for DED.
研究跨膜蛋白 CMTM6 对干眼病（DED）发病机制的影响，并阐明其潜在机制。
通过数据库分析、实时聚合酶链反应（RT-PCR）、western blot 和免疫组织化学验证 CMTM6 的表达。使用酚红棉线试验测量泪液分泌。通过流式细胞术评估免疫细胞浸润。通过荧光素钠染色、紧密连接蛋白 1（ZO-1）免疫荧光染色和电细胞-基质阻抗感应（ECIS）评估屏障功能。使用 siRNA 和 shRNA 以及慢病毒载体介导的 CMTM6 过表达来沉默 CMTM6 表达。通过 RT-PCR 和流式细胞术分析细胞因子分析分析促炎细胞因子水平。
CMTM6 在健康人眼角膜和结膜上皮中表达较高，但在 DED 中明显降低。值得注意的是，这种下调与疾病严重程度相关。Cmtm6-/-干眼（DE）小鼠表现出泪液分泌减少、严重的角膜上皮缺损、结膜杯状细胞密度降低和炎症反应增强。此外，Cmtm6-/-DE 小鼠和 CMTM6 敲低的人角膜上皮细胞转化（HCE-T）细胞表现出更严重的屏障破坏和 ZO-1 表达降低。在 HCE-T 细胞中敲低 CMTM6 会增加高渗应激诱导的炎症反应，而过表达 CMTM6 可显著减轻这种反应。此外，在沉默 CMTM6 后，高渗刺激的 HCE-T 细胞中磷酸化 p65 的水平增加。核因子 kappa B（NF-κB）p65 抑制剂（JSH-23）逆转了 CMTM6 敲低的 HCE-T 细胞中高渗引起的过度炎症反应。
眼表面 CMTM6 表达的减少导致 DED 的发病机制。CMTM6-NF-κB p65 信号通路可能成为 DED 的有前途的治疗靶点。
