7-酮胆固醇(7KC)的积累发生在年龄相关性黄斑变性(AMD),以前发现促进纤维化,无法治愈的视力丧失原因,部分通过诱导内皮-间质转化。为了解决7KC引起视网膜色素上皮细胞(RPE)间质转化的假设,我们将人类原发性RPE(hRPE)暴露于7KC或对照。7KC处理的hRPE没有表现出增加的间充质标志物,而是维持RPE特异性蛋白,并表现出衰老迹象,组蛋白H3的丝氨酸磷酸化增加,哺乳动物雷帕霉素靶蛋白(p-mTOR)的丝氨酸/苏氨酸磷酸化增加,p16和p21,β-半乳糖苷酶标记,和减少层B1,表明衰老。细胞还发展了衰老相关的分泌表型(SASP),确定增加的IL-1β,IL-6和VEGF通过mTOR介导的NF-κB信号传导,并降低了通过mTOR抑制剂恢复的屏障完整性,雷帕霉素.7KC诱导的p21,VEGF,和IL-1β被蛋白激酶C抑制剂抑制。激酶调节IQGAP1丝氨酸磷酸化。此外,在7KC注射和激光诱导损伤后,与同窝对照小鼠相比,IQGAP1丝氨酸1441点突变的小鼠纤维化显著减少.我们的结果提供了证据,玻璃疣中7KC的年龄相关积累介导了RPE中的衰老和SASP,而IQGAP1丝氨酸磷酸化在惹起AMD纤维化中具有重要意义。
Accumulation of 7-ketocholesterol (7KC) occurs in age-related macular degeneration (AMD) and was found previously to promote fibrosis, an untreatable cause of vision loss, partly through induction of endothelial-mesenchymal transition. To address the hypothesis that 7KC causes mesenchymal transition of retinal pigment epithelial cells (RPE), we exposed human primary RPE (hRPE) to 7KC or a control. 7KC-treated hRPE did not manifest increased mesenchymal markers, but instead maintained RPE-specific proteins and exhibited signs of senescence with increased serine phosphorylation of histone H3, serine/threonine phosphorylation of mammalian target of rapamycin (p-mTOR), p16 and p21, β-galactosidase labeling, and reduced LaminB1, suggesting senescence. The cells also developed senescence-associated secretory phenotype (SASP) determined by increased IL-1β, IL-6, and VEGF through mTOR-mediated NF-κB signaling, and reduced barrier integrity that was restored by the mTOR inhibitor, rapamycin. 7KC-induced p21, VEGF, and IL-1β were inhibited by an inhibitor of protein kinase C. The kinase regulates IQGAP1 serine phosphorylation. Furthermore, after 7KC injection and laser-induced injury, mice with an IQGAP1 serine 1441-point mutation had significantly reduced fibrosis compared to littermate control mice. Our results provide evidence that age-related accumulation of 7KC in drusen mediates senescence and SASP in RPE, and IQGAP1 serine phosphorylation is important in causing fibrosis in AMD.