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.

通过免疫荧光共聚焦显微镜在人类胎儿RPE（HFRPE）和成年天然RPE Wholemounts中进行了免疫荧光共聚焦显微镜研究JAM-C，ZO-1，N-钙粘蛋白和Ezrin的定位。蛋白质印迹用于分析JAM-C蛋白表达。通过EVOM测量旋转的抗性。使用钙开关测定法来通过监测跨越电阻（TER）的变化来确定JAM-C在结中的重要性。 HFRPE培养物的稳态抗性为935 +/- 283 OHM.CM2（平均SD； n = 9）。使用了跨越迁移测定法（基础至顶端）来研究JAM-C在白细胞通过HFRPE单层迁移中的作用。 在培养的HFRPE细胞和成年天然RPE的紧密连接中发现了JAM-C，并与ZO-1共定位。另外，仅发现JAM-C与E-钙粘蛋白或去氨木蛋白进行部分共定位。 JAM-C定位或表达不会因促炎细胞因子的细胞刺激而改变。 JAM-C的抑制作用导致钙耗尽诱导的TER降低后HFRPE连接的重新组装显着延迟。在对照实验中，这种恢复是初始TER的90.7 +/- 3.9％，而在JAM-C抑制剂的存在下，恢复仅为CA重组后24小时在同一时间段内的初始TER的67.9 +/- 9.8％（n = 3; p = 0.01）。在连接改革期间，JAM-C被招募到初始细胞 - 细胞接触，JAM-C敲低后，在细胞 - 细胞接触部位募集N-钙粘着蛋白和ZO-1。此外，JAM-C敲低导致HFRPE细胞极化延迟，如所选时间点Ezrin的根尖染色所示。在其他系统中已经显示，JAM-C可以充当白细胞上β2-整合素Mac-1的配体。我们通过HFRPE单层研究了人单核细胞和中性粒细胞的基础迁移。与对照相比，JAM-C抑制显着降低了通过HFRPE单层的白细胞诱导的白细胞迁移（p = 0.03; n = 3）。 JAM-C专门定位在HFRPE和成人本地RPE的紧密连接中。 JAM-C在HFRPE紧密连接形成的最初阶段很重要，这可能是通过调节细胞细胞接触中N-辅助蛋白和ZO-1的募集，并且在HFRPE细胞的极化中起作用。