JAM-C in retinal pigment epithelium

视网膜色素上皮中的 JAM-C

• 批准号: 7734645
• 负责人: Sheldon Miller
• 金额: $ 46.05万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734645
• 关键词: Adherence; Adult; Apical; Biological Assay; CD18 Antigens; Calcium; Cells; Chemicals; Confocal Microscopy; E-Cadherin; Extracellular Space; Germ Cells; Hour; Human; Immigration; Immunofluorescence Immunologic; Integral Membrane Protein; Leukocytes; Ligands; Localized; Macrophage-1 Antigen; Measures; Migration Assay; Monitor; N-Cadherin; Purpose; Recovery; Recruitment Activity; Resistance; Role; Site; Staging; Staining method; Stains; Structure of retinal pigment epithelium; System; Tight Junctions; Time; Western Blotting; chemokine; citrate carrier; cytokine; desmoplakin; ezrin; fetal; inhibitor/antagonist; monocyte; monolayer; neutrophil; occludin; protein expression; reconstitution; research study

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.

应用免疫荧光共聚焦显微镜对人胎儿视网膜色素上皮（hfRPE）融合培养物和亚融合培养物以及成人视网膜色素上皮（RPE）全细胞培养物中JAM-C、ZO-1、N-钙粘蛋白和埃兹蛋白的定位进行了研究。Western blot检测JAM-C蛋白表达。用EVOM法测量跨上皮阻力。通过监测跨上皮电阻（TER）的变化，使用钙开关试验来确定JAM-C在连接形成中的重要性。hfRPE培养物的稳态电阻为935+/- 283 ohm.cm2（平均SD; n=9）。使用跨上皮迁移测定（基底至顶端）来研究JAM-C在白细胞迁移通过hfRPE单层中的作用。 在培养的hfRPE细胞和成人天然RPE的紧密连接中发现了JAM-C，其中它与ZO-1共定位。此外，JAM-C与E-cadherin或桥粒斑蛋白只有部分共定位，而促炎细胞因子刺激细胞并不改变JAM-C的定位或表达。抑制JAM-C导致钙耗竭诱导TER减少后hfRPE连接的重新组装显著延迟。在对照实验中，该回收率为初始TER的90.7 +/-3.9%，而在存在JAM-C抑制剂的情况下，在Ca重建后24小时的相同时间范围内，回收率仅为初始TER的67.9+/-9.8%（n = 3; p=0.01）。在连接重建期间，JAM-C被募集到最初的细胞-细胞接触，并且在JAM-C敲低后，细胞-细胞接触位点处的N-钙粘蛋白和ZO-1的募集减少。此外，JAM-C敲低导致hfRPE细胞极化延迟，如在选定时间点埃兹蛋白顶端染色减少所示。在其他系统中已经显示，JAM-C可以作为白细胞上β 2-整联蛋白Mac-1的配体。我们研究了人单核细胞和中性粒细胞通过hfRPE单层从基底到顶端的迁移。与对照相比，JAM-C抑制显著降低了趋化因子诱导的白细胞穿过hfRPE单层的迁移（p= 0.03; n =3）。 JAM-C特异性定位于hfRPE和成人天然RPE的紧密连接中。JAM-C在hfRPE紧密连接形成的初始阶段起重要作用，可能通过调节细胞间接触的N-cadherin和ZO-1的募集，并在hfRPE细胞的极化中起作用，最后，JAM-C促进白细胞通过hfRPE单层从基底向顶端的迁移。