JAM-C in retinal pigment epithelium

用免疫荧光共聚焦显微镜研究了JAM-C、ZO-1、N-钙粘蛋白和Ezrin在融合和亚融合培养的人胎儿RPE(HfRPE)和成人天然RPE整细胞中的定位。Western印迹分析JAM-C蛋白表达。EVOM法测定跨上皮阻力。钙开关试验通过监测跨上皮阻力(TER)的变化来确定JAM-C在连接形成中的重要性。HfRPE培养的稳态电阻为935+/-283ohm.cm2(平均SD；n=9)。跨上皮游走实验(从基础到顶端)被用来研究JAM-C在白细胞通过hfRPE单层迁移中的作用。 JAM-C存在于培养的hfRPE细胞和成人天然RPE细胞的紧密连接中，并与ZO-1共存。此外，仅发现JAM-C与E-钙粘附素或桥粒蛋白部分共定位。促炎症细胞因子刺激细胞不改变JAM-C的定位或表达。抑制JAM-C导致钙耗竭诱导的TER减少后，hfRPE连接的重组明显延迟。在对照实验中，这一回收率是初始TER的90.7+/-3.9%，而在JAM-C抑制剂存在的情况下，在同一时间段，即钙重建后24小时，回收率仅为初始TER的67.9+/-9.8%(n=3；p=0.01)。在连接重建过程中，JAM-C被募集到最初的细胞-细胞接触处，在JAM-C被敲除后，N-钙粘素和ZO-1在细胞-细胞接触部位的募集减少。此外，JAM-C基因敲除导致hfRPE细胞极化延迟，在选定的时间点，Ezrin的顶端染色减少。在其他系统中已经表明，JAM-C可能作为白细胞上的β2-整合素Mac-1的配体。我们研究了人单核细胞和中性粒细胞通过hfRPE单层向心尖的迁移。与对照组相比，抑制JAM-C可显著减少趋化因子诱导的白细胞通过hfRPE单层的迁移(p=0.03；n=3)。 JAM-C专门定位于hfRPE和成人天然RPE的紧密连接。JAM-C在hfRPE紧密连接形成的初始阶段起重要作用，可能是通过调节细胞-细胞接触处N-钙粘蛋白和ZO-1的募集，并在hfRPE细胞的极化过程中发挥作用。最后，JAM-C通过hfRPE单层促进白细胞从底部向顶端的迁移。

The localization of JAM-C, ZO-1, N-cadherin and ezrin was studied by immunofluorescence confocal microscopy in confluent and subconfluent cultures of human fetal RPE (hfRPE) and in adult native RPE wholemounts. Western blot was used to analyze JAM-C protein expression. The transepithelial resistance was measured by EVOM. A calcium switch assay was used to determine the importance of JAM-C in junction formation by monitoring changes in transepithelial resistance (TER). The steady-state resistance of the hfRPE cultures was 935+/- 283 ohm.cm2 (mean SD; n=9). A transepithelial migration assay (basal to apical) was used to study the role of JAM-C in the migration of leukocytes through the hfRPE monolayer. JAM-C was found in the tight junctions of both cultured hfRPE cells and adult native RPE where it colocalized with ZO-1. In addition, only partial colocalization of JAM-C with E-cadherin or desmoplakin was found. JAM-C localization or expression was not altered by stimulation of the cells with proinflammatory cytokines. The inhibition of JAM-C resulted in a significant delay in the reassembly of the hfRPE junctions after calcium depletion-induced reduction in TER. In control experiments, this recovery was 90.7 +/- 3.9% of the initial TER while in the presence of the JAM-C inhibitor the recovery was only 67.9+/-9.8% of the initial TER in the same time-frame, 24 hours after Ca reconstitution (n = 3; p=0.01). During junction reformation JAM-C was recruited to the initial cell-cell contacts and after JAM-C knockdown, the recruitment of N-cadherin and ZO-1 at the site of cell-cell contact was reduced. Furthermore, JAM-C knockdown caused a delay in the hfRPE cell polarization, as shown by the reduced apical staining of ezrin at selected time points. It has been shown in other systems that JAM-C may act as a ligand for the beta2-integrin Mac-1 on leukocytes. We studied the basal to apical transmigration of human monocytes and neutrophils through the hfRPE monolayer. JAM-C inhibition significantly decreased the chemokine induced transmigration of leukocytes through the hfRPE monolayer compared to control (p= 0.03; n =3). JAM-C localizes specifically in the tight junctions of hfRPE and adult native RPE. JAM-C is important in the initial stages of tight junction formation in hfRPE possibly by regulating the recruitment of N-cadherin and ZO-1 at the cell-cell contacts and has a role in the polarization of hfRPE cells.Finally, JAM-C promotes the basal to apical transmigration of leukocytes through the hfRPE monolayer.