Van Aken Elisabeth H, De Wever Olivier, Van Hoorde Leen, Bruyneel Erik, De Laey Jean-Jacques, Mareel Marc M
Department of Ophthalmology, Ghent University Hospital, Gent, Belgium.
Invest Ophthalmol Vis Sci. 2003 Feb;44(2):463-72. doi: 10.1167/iovs.01-1096.
To investigate the role of N-cadherin and hepatocyte growth factor (HGF) in the invasion of collagen type I by human retinal pigment epithelial (RPE) cells.
RPE sheets from eight human eyes were used for characterization through Western blot analysis of the expression of cadherin in total lysates or after immunoprecipitation with anti beta-catenin antibody. First-passage primary cultures of RPE sheets were successfully established from 28 of 56 human eyes. First-passage primary RPE cell cultures on glass substrate, consisting of patches of cells, were used for immunocytochemistry. Fifteen first-passage primary RPE cell cultures in culture vessels were grown to confluence. Four of the 15 first-passage primary RPE cell cultures were investigated for cadherin expression by immunocytochemistry, and the other 11 were further subcultured for two to six passages. These 11 cultures were used for functional assays and investigated for expression of cadherin at regular time intervals. Cells from passage-3 to -4 primary RPE cell cultures were tested for invasion into collagen type I gels, with or without neutralizing antibodies for HGF and N-cadherin, respectively. Activation of the c-Met receptor for HGF and of focal adhesion kinase (FAK) was investigated by immunoprecipitation with anti-phosphotyrosine antibody, gel electrophoresis, and immunostaining on Western blot. Levels of HGF in conditioned medium (CM) of RPE cells were determined by ELISA.
RPE cells in culture displayed two phenotypes: Both fibroblast-like and epithelioid cells were present in all 15 first-passage primary cultures and in further passaged cultures derived therefrom. When seeded on collagen, all RPE cells acquired a fibroblast-like phenotype and invaded the collagen type I gel. RPE cells also stimulated branching morphogenesis of MDCK/AZ epithelial canine kidney cell colonies inside collagen. HGF was found in RPE CM, suggesting an autocrine loop for invasion through its c-Met receptor. HGF-neutralizing antibody inhibited invasion of collagen. The major cadherin expressed by RPE cells in culture was N(euronal)-cadherin. Invasion of collagen by RPE cells was also inhibited by an N-cadherin-neutralizing antibody. N-cadherin and c-Met coimmunoprecipitated in RPE cells. FAK and c-Met were both phosphorylated in RPE cells in culture, and phosphorylation was inhibited by antibodies neutralizing either N-cadherin or HGF.
The present investigation provides evidence for an autocrine HGF/c-Met loop that stimulates RPE cell invasion into collagen through FAK. The invasion-stimulatory molecule N-cadherin also activates FAK in invasive RPE cells.
研究N-钙黏蛋白和肝细胞生长因子（HGF）在人视网膜色素上皮（RPE）细胞侵袭I型胶原中的作用。
通过对总裂解物中钙黏蛋白表达或用抗β-连环蛋白抗体免疫沉淀后的表达进行蛋白质印迹分析，对来自8只人眼的RPE片层进行特征分析。从56只人眼中的28只成功建立了RPE片层的第一代原代培养物。在玻璃基质上的第一代原代RPE细胞培养物，由细胞斑块组成，用于免疫细胞化学。将培养容器中的15个第一代原代RPE细胞培养物培养至汇合。对15个第一代原代RPE细胞培养物中的4个进行免疫细胞化学检测钙黏蛋白表达，另外11个进一步传代培养2至6代。这11个培养物用于功能测定，并定期检测钙黏蛋白表达。分别用或不用HGF和N-钙黏蛋白的中和抗体，检测第3至4代原代RPE细胞培养物中的细胞对I型胶原凝胶的侵袭。通过用抗磷酸酪氨酸抗体免疫沉淀、凝胶电泳和蛋白质印迹免疫染色，研究HGF的c-Met受体和黏着斑激酶（FAK）的激活情况。用酶联免疫吸附测定法测定RPE细胞条件培养基（CM）中的HGF水平。
培养的RPE细胞表现出两种表型：在所有15个第一代原代培养物及其衍生的传代培养物中均存在成纤维细胞样和上皮样细胞。当接种在胶原上时，所有RPE细胞均获得成纤维细胞样表型并侵袭I型胶原凝胶。RPE细胞还刺激了胶原内MDCK/AZ上皮犬肾细胞集落的分支形态发生。在RPE CM中发现了HGF，提示通过其c-Met受体存在自分泌侵袭环路。HGF中和抗体抑制了对胶原的侵袭。培养的RPE细胞表达的主要钙黏蛋白是N（神经元）-钙黏蛋白。N-钙黏蛋白中和抗体也抑制了RPE细胞对胶原的侵袭。N-钙黏蛋白和c-Met在RPE细胞中共免疫沉淀。培养的RPE细胞中FAK和c-Met均被磷酸化，并且磷酸化被中和N-钙黏蛋白或HGF的抗体抑制。
本研究为自分泌HGF/c-Met环路通过FAK刺激RPE细胞侵袭胶原提供了证据。侵袭刺激分子N-钙黏蛋白也激活侵袭性RPE细胞中的FAK。
