Youn Yong-Ha, Hong Jeehee, Burke Janice M
Department of Ophthalmology, Medical College of Wisconsin, Milwaukee 53226-4812, USA.
Invest Ophthalmol Vis Sci. 2006 Jun;47(6):2675-85. doi: 10.1167/iovs.05-1335.
Unlike most monolayer epithelial cells, cultured RPE are competent to form a zonular adhesion of N- rather than E-cadherin. To determine whether other normal epithelial cells do likewise, cells with high endogenous N-cadherin were cloned from the typically E-cadherin dominant epithelial line Madin-Darby canine kidney cells (MDCK) to analyze cell and junction phenotype in the presence of N-cadherin.
A MDCK subclonal line, clone-YH, was selected for high endogenous N-cadherin and was compared with the RPE line hTERT-RPE1 with regard to cell phenotype, cadherin gene expression and cadherin protein distribution, glycosylation state, and catenin complex composition.
In early cultures, hTERT-RPE1 cells are moderately epithelioid with junctional N-cadherin, but clone-YH cells are initially highly fusiform with N-cadherin in multiple sites. With time, N-cadherin in clone-YH becomes deglycosylated, resistant to detergent extraction, and zonular, and cells become epithelioid. Treatment with the N-glycosylation inhibitor tunicamycin induces an epithelioid phenotype in clone-YH, like time in culture but disrupts the hTERT-RPE1 phenotype. N-cadherin traffics to surface membranes and complexes with catenins regardless of cell type or glycosylation state, although catenin complex composition varied, showing enriched alpha-catenin under the cell-type-specific conditions in which N-cadherin was junctional. Clone-YH continued to express E-cadherin as a very minor cadherin, which trafficked to membranes but did not accumulate at junctions.
RPE cells are not unique in localizing N-cadherin to a zonular adhesion typical of a monolayer epithelium, because even epithelial cells derived from a typically E-cadherin dominant line (clone-YH) form a zonular N-cadherin junction if the protein is abundant. However, there are cell and cadherin differences in mechanisms of cadherin accumulation in a zonular pattern, and a previously unrecognized cell-type-specific role for protein glycosylation in epithelial phenotype development.
与大多数单层上皮细胞不同，培养的视网膜色素上皮（RPE）细胞能够形成以N-钙黏着蛋白而非E-钙黏着蛋白为主的小带黏附。为了确定其他正常上皮细胞是否也如此，从典型的以E-钙黏着蛋白为主导的上皮细胞系——马-达二氏犬肾细胞（MDCK）中克隆出具有高内源性N-钙黏着蛋白的细胞，以分析在N-钙黏着蛋白存在情况下的细胞和连接表型。
选择一个具有高内源性N-钙黏着蛋白的MDCK亚克隆系克隆-YH，并在细胞表型、钙黏着蛋白基因表达、钙黏着蛋白蛋白分布、糖基化状态和连环蛋白复合体组成方面，将其与RPE细胞系hTERT-RPE1进行比较。
在早期培养中，hTERT-RPE1细胞呈中度上皮样，具有连接性N-钙黏着蛋白，但克隆-YH细胞最初呈高度梭形，N-钙黏着蛋白分布在多个部位。随着时间推移，克隆-YH中的N-钙黏着蛋白去糖基化，对去污剂提取具有抗性，并形成小带，细胞变为上皮样。用N-糖基化抑制剂衣霉素处理可诱导克隆-YH出现上皮样表型，类似于培养过程中的情况，但会破坏hTERT-RPE1的表型。无论细胞类型或糖基化状态如何，N-钙黏着蛋白都会转运到表面膜并与连环蛋白形成复合体，尽管连环蛋白复合体组成有所不同，在N-钙黏着蛋白形成连接的细胞类型特异性条件下显示α-连环蛋白富集。克隆-YH继续表达E-钙黏着蛋白，但其作为一种非常少量的钙黏着蛋白，虽能转运到膜上但不会在连接处积累。
RPE细胞并非唯一能将N-钙黏着蛋白定位到单层上皮典型的小带黏附处的细胞，因为即使是源自典型的以E-钙黏着蛋白为主导的细胞系（克隆-YH）的上皮细胞，如果该蛋白丰富，也会形成小带N-钙黏着蛋白连接。然而，在钙黏着蛋白以小带模式积累的机制方面存在细胞和钙黏着蛋白的差异，并且在蛋白质糖基化在上皮表型发育中存在以前未被认识的细胞类型特异性作用。
