Department of Nephrology, Ankara University School of Medicine, Ankara, Turkey.
PLoS One. 2013 Jul 23;8(7):e70276. doi: 10.1371/journal.pone.0070276. Print 2013.
Free light chains (LCs) are among the many ligands that bind to cubilin/megalin for endocytosis via the clathrin-dependent endosomal/lysosomal pathway. Receptor associated protein (RAP), is a 39 kDA high-affinity, chaperone-like ligand for megalin that assists in the proper folding and functioning of megalin/cubilin. Although RAP is known to inhibit ligand binding to megalin/cubilin, its effect on LC endocytosis has not been shown directly.
We investigated whether RAP can block the endocytosis of LC in cultured human proximal tubule cells and whether this can prevent LC cytotoxicity. Immunofluorescence microscopy and flow cytometry showed that fluorescently labeled LC endocytosis was markedly inhibited in HK-2 cells pretreated with human RAP. The effect of RAP was dose-dependent, and was predominantly on endocytosis as it had no effect on the small acid-washable fraction of LC bound to cell membrane. RAP significantly inhibited LC induced cytokine production and phosphorylation of ERK1/2 and p38 MAPK. Prolonged exposure to LC for 48 h resulted in epithelial-to-mesenchymal transformation in HK-2 cells as evidenced by marked reduction in the expression of the epithelial cell marker E-cadherin, and increased the expression of the mesenchymal marker α-SMA, which was also prevented by RAP in the endocytosis medium.
RAP inhibited LC endocytosis by ∼88% and ameliorated LC-induced cytokine responses and EMT in human PTCs. The results not only provide additional evidence that LCs endocytosis occurs via the megalin/cubilin endocytic receptor system, but also show that blocking LC endocytosis by RAP can protect proximal tubule cells from LC cytotoxicity.
游离轻链 (LC) 是许多与巨球蛋白/内收蛋白结合通过网格蛋白依赖的内体/溶酶体途径进行内吞作用的配体之一。受体相关蛋白 (RAP) 是一种 39kDa 的高亲和力、伴侣样配体，可协助巨球蛋白/内收蛋白的正确折叠和功能。虽然已知 RAP 可抑制配体与巨球蛋白/内收蛋白结合，但尚未直接显示其对 LC 内吞作用的影响。
我们研究了 RAP 是否可以阻断培养的人近端肾小管细胞中 LC 的内吞作用，以及这是否可以防止 LC 的细胞毒性。免疫荧光显微镜和流式细胞术显示，在用人 RAP 预处理的 HK-2 细胞中，荧光标记的 LC 内吞作用明显受到抑制。RAP 的作用呈剂量依赖性，主要作用于内吞作用，因为它对与细胞膜结合的 LC 的小酸可洗分数没有影响。RAP 显著抑制 LC 诱导的细胞因子产生和 ERK1/2 和 p38 MAPK 的磷酸化。LC 延长暴露 48 小时导致 HK-2 细胞上皮-间充质转化，表现为上皮细胞标志物 E-钙粘蛋白表达明显减少，间充质标志物 α-SMA 表达增加，而 RAP 在摄取培养基中也可预防这种情况。
RAP 通过约 88%抑制 LC 的内吞作用，并改善 LC 诱导的人 PTC 细胞因子反应和 EMT。结果不仅提供了额外的证据表明 LC 的内吞作用是通过巨球蛋白/内收蛋白内吞受体系统发生的，而且表明通过 RAP 阻断 LC 的内吞作用可以保护近端肾小管细胞免受 LC 的细胞毒性。
