UCD Clinical Research Centre, Mater Misericordiae University Hospital, D07 R2WY Dublin, Ireland.
Department of Ophthalmology, Mater Misericordiae University Hospital, D07 R2WY Dublin, Ireland.
Int J Mol Sci. 2024 Nov 13;25(22):12173. doi: 10.3390/ijms252212173.
Lamina cribrosa (LC) cells play an integral role in extracellular matrix remodeling and fibrosis in human glaucoma. LC cells bear similarities to myofibroblasts that adopt an apoptotic-resistant, proliferative phenotype, a process linked to dysregulation of tumor suppressor-gene p53 pathways, including ubiquitin-proteasomal degradation via murine-double-minute-2 (MDM2). Here, we investigate p53 and MDM2 in glaucomatous LC cells. Primary human LC cells were isolated from glaucomatous donor eyes (GLC) and age-matched normal controls (NLC) (n = 3 donors/group). LC cells were cultured under standard conditions ± 48-h treatment with p53-MDM2-interaction inhibitor RG-7112. Markers of p53-MDM2, fibrosis, and apoptosis were analyzed by real-time polymerase chain reaction (qRT-PCR), western blotting, and immunofluorescence. Cellular proliferation and viability were assessed using colorimetric methyl-thiazolyl-tetrazolium salt assays (MTS/MTT). In GLC versus NLC cells, protein expression of p53 was significantly decreased ( < 0.05), MDM2 was significantly increased, and immunofluorescence showed reduced p53 and increased MDM2 expression in GLC nuclei. RG-7112 treatment significantly increased p53 and significantly decreased MDM2 gene and protein expression. GLC cells had significantly increased protein expression of αSMA, significantly decreased caspase-3 protein expression, and significantly increased proliferation after 96 h. RG-7112 treatment significantly decreased COL1A1 and αSMA, significantly increased BAX and caspase-3 gene expression, and significantly decreased proliferation in GLC cells. MTT-assay showed equivocal cellular viability in NLC/GLC cells with/without RG-7112 treatment. Our data suggests that proliferation and the ubiquitin-proteasomal pathway are dysregulated in GLC cells, with MDM2-led p53 protein degradation negatively impacting its protective role.
筛板（LC）细胞在人类青光眼的细胞外基质重塑和纤维化中起着重要作用。LC 细胞与肌成纤维细胞相似，具有抗凋亡、增殖的表型，这一过程与肿瘤抑制基因 p53 途径的失调有关，包括通过鼠双微体-2（MDM2）的泛素-蛋白酶体降解。在这里，我们研究了青光眼 LC 细胞中的 p53 和 MDM2。从青光眼供体眼（GLC）和年龄匹配的正常对照（NLC）中分离出原代人 LC 细胞（n = 3 个供体/组）。LC 细胞在标准条件下培养，± 48 小时用 p53-MDM2 相互作用抑制剂 RG-7112 处理。通过实时聚合酶链反应（qRT-PCR）、Western 印迹和免疫荧光分析 p53-MDM2、纤维化和细胞凋亡的标志物。使用比色甲噻唑基四唑盐测定法（MTS/MTT）评估细胞增殖和活力。在 GLC 与 NLC 细胞相比，p53 的蛋白表达明显降低（<0.05），MDM2 明显增加，免疫荧光显示 GLC 核中 p53 减少，MDM2 表达增加。RG-7112 处理显著增加了 p53 的表达，并显著降低了 MDM2 的基因和蛋白表达。GLC 细胞的 αSMA 蛋白表达显著增加，caspase-3 蛋白表达显著降低，96 小时后增殖显著增加。RG-7112 处理显著降低了 COL1A1 和 αSMA，显著增加了 BAX 和 caspase-3 的基因表达，并显著降低了 GLC 细胞的增殖。MTT 测定法显示，在有/没有 RG-7112 处理的 NLC/GLC 细胞中，细胞活力不确定。我们的数据表明，GLC 细胞中的增殖和泛素蛋白酶体途径失调，MDM2 介导的 p53 蛋白降解对其保护作用产生负面影响。
