Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, Bucharest 060031, Romania.
Biochem Cell Biol. 2014 Apr;92(2):127-36. doi: 10.1139/bcb-2013-0064. Epub 2014 Mar 31.
The receptor for advanced glycation end products (RAGE) is involved in multiple stages of tumor development and malignization. To gain further knowledge on the RAGE role in tumor progression, we investigated the receptor expression profile and its subcellular localization in melanoma cells at different stages of malignancy. We found that RAGE clustered at membrane ruffles and leading edges, and at sites of cell-to-cell contact in primary melanoma cells (e.g., MelJuSo), in contrast with a more dispersed localization in metastatic cells (e.g., SK-Mel28). RAGE silencing by RNAi selectively inhibited migration of MelJuSo cells, whilst having no influence on SK-Mel28 cell migration, in a "wound healing" assay. Western blot detection of RAGE showed a more complex RAGE oligomerization in MelJuSo cells compared to melanocytes and SK-Mel28 cells. By competing the binding of antibodies with recombinant soluble RAGE, an oligomeric form running at approximately 200 kDa was detected, as it was the monomeric RAGE of 55-60 kDa. SDS-PAGE electrophoresis under reducing versus nonreducing conditions indicated that the oligomer of about 200 kDa is formed by disulfide bonds, but other interactions are likely to be important for RAGE multimerization in melanoma cells. Immunofluorescence microscopy revealed that treatment with two cholesterol-chelating drugs, nystatin and filipin, significantly affected RAGE localization in MelJuSo cells. SK-Mel28 cells showed a reduced RAGE glycosylation and association with cholesterol-rich membranes and also a considerable downregulation of the soluble forms. Our results indicate that RAGE isoform expression and subcellular localization could be important determinants for the regulation of its function in tumor progression.
晚期糖基化终产物受体（RAGE）参与肿瘤发生和恶性转化的多个阶段。为了进一步了解 RAGE 在肿瘤进展中的作用，我们研究了不同恶性程度的黑色素瘤细胞中受体的表达谱及其亚细胞定位。我们发现 RAGE 在原代黑色素瘤细胞（如 MelJuSo）中聚集在细胞膜皱襞和前缘以及细胞间接触部位，而在转移性细胞（如 SK-Mel28）中则分布更为弥散。RNAi 沉默 RAGE 选择性地抑制了 MelJuSo 细胞的迁移，而对 SK-Mel28 细胞的迁移没有影响，在“伤口愈合”测定中。Western blot 检测 RAGE 显示，与黑素细胞和 SK-Mel28 细胞相比，MelJuSo 细胞中 RAGE 的寡聚化更为复杂。通过用重组可溶性 RAGE 竞争抗体结合，检测到大约 200 kDa 的寡聚形式，因为它是 55-60 kDa 的单体 RAGE。在还原与非还原条件下进行的 SDS-PAGE 电泳表明，大约 200 kDa 的寡聚体是通过二硫键形成的，但其他相互作用可能对黑色素瘤细胞中 RAGE 的多聚化很重要。免疫荧光显微镜显示，用两种胆固醇螯合剂，制霉菌素和 filipin 处理，显著影响了 MelJuSo 细胞中 RAGE 的定位。SK-Mel28 细胞显示 RAGE 糖基化减少，与富含胆固醇的膜结合减少，可溶性形式也显著下调。我们的结果表明，RAGE 同工型的表达和亚细胞定位可能是调节其在肿瘤进展中功能的重要决定因素。
