Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5.
Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5.
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DOI：
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发表时间：
1998-12
影响因子：
4.4
通讯作者：
Kerrin C. Dunn;A. Marmorstein;V. Bonilha;E. Rodriguez-Boulan;F. Giordano;L. Hjelmeland
中科院分区：
文献类型：
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作者：
Kerrin C. Dunn;A. Marmorstein;V. Bonilha;E. Rodriguez-Boulan;F. Giordano;L. Hjelmeland
目的测定视网膜色素上皮细胞（retinal pigment epithelium，RPE）分泌的成纤维细胞生长因子5（fibroblast growth factor 5，FGF 5）的极性，并检测ARPE-19细胞系作为研究RPE极性模型的可行性和实用性。方法采用流感病毒感染和腺病毒介导的基因转移技术，将流感病毒血凝素（HA）、p75-NTR（一种神经营养因子受体）、低密度脂蛋白（LDL）受体（LDLR）和FGF 5基因转染ARPE-19细胞，并在融合单层细胞中表达。通过共聚焦显微镜确定HA、p75-NTR和LDLR的定位。使用结构域选择性生物素化测定来定量确定p75-NTR和LDLR的极性。通过条件培养基的免疫印迹分析来检查FGF 5分泌到ARPE-19培养物的顶端和基底培养基中。结果HA和p75-NTR均定位于感染和转导的ARPE-19细胞的顶端表面。与此相反，LDLR优先与ARPE-19细胞的基底外侧膜。生物素化研究表明，84%的p75-NTR存在于顶端表面，79%的LDLR是基底外侧极化的。在6小时的过程中，超过90%的总分泌的FGF 5蛋白在基底外侧培养基中积累。结论ARPE-19细胞在共聚焦显微镜或表面标记法检测时表现出细胞表面标记物的极化分布。这表明ARPE-19细胞系是研究RPE细胞极性的有效模型。FGF 5是一种通常由RPE细胞产生的分泌蛋白，仅在6小时后就优先在基础培养基中积累，表明它是从ARPE-19细胞的基底外侧表面以载体方式分泌的。
PURPOSE To determine the polarity of fibroblast growth factor 5 (FGF5) secretions from retinal pigment epithelium (RPE) cells and to examine the viability and utility of the ARPE-19 cell line as a model for the study of RPE polarity. METHODS Influenza infection and adenovirus-mediated gene transfer were used to deliver and express genes encoding influenza hemagglutinin (HA), p75-NTR (a neurotrophin receptor), low-density lipoprotein (LDL) receptor (LDLR), and FGF5 in confluent monolayers of ARPE-19 cells. The localization of HA, p75-NTR, and LDLR was determined by confocal microscopy. Domain selective biotinylation assays were used to quantitatively determine the polarities of p75-NTR and LDLR. The secretion of FGF5 into the apical and basal media of ARPE-19 cultures was examined by immunoblot analysis of conditioned media. RESULTS Hemagglutinin and p75-NTR were found to be localized on the apical surface of infected and transduced ARPE-19 cells. In contrast, LDLR was associated preferentially with the basolateral membrane of ARPE-19 cells. Biotinylation studies indicated that 84% of p75-NTR was present on the apical surface, and 79% of LDLR was basolaterally polarized. Over the course of 6 hours, more than 90% of the total secreted FGF5 protein accumulated in the basolateral media. CONCLUSIONS ARPE-19 cells exhibit a polarized distribution of cell surface markers when examined by either confocal microscopy or surface-labeling assays. This indicates that the ARPE-19 cell line is a valid model for studies of RPE cell polarity. FGF5, a secreted protein normally produced by RPE cells, is accumulated preferentially in the basal media after only 6 hours, suggesting that it is vectorially secreted from the basolateral surface of ARPE-19 cells.