| It is known that Rab5directs the transport and fusion of endocytic vesicles to and with early endosomes. Previous studies have demonstrasted that endocytosis was involved the down-regulation of receptors in many cells. However, the effect of Rab5a on endocytosis and down-regulation of beta-adrenoceptors (β-ARs) and thereafter regulating the endothelial barrier function in pulmonary microvascular endothelial cells is not known. We investigated the role of Rab5a, a sub-type of Rab5that coordinates protein transport from the membrane to the early endosome and regulates the function of endogenous β-ARs in RPMECs in the presence of lipopolysaccharide (LPS).We found that selective activation of β2-AR signaling had protective effects on the tight junction and adheren junction and therefore partly prevented the endothelial barrier function from LPS disruption. The localization analysis suggested that expression of Rab5a small interfering RNA (siRNA) significantly inhibits the agonist-stimulated transport of β2-AR-GFP from membrane to cytoplasm. In addition, the expression of wild-type of Rab5a (Rab5a WT) promoted the trafficking of β2-AR-GFP from membrane to cytoplasm when agonist exist without affecting the sublocalization of β2-AR when agonist did not exist. The quantified analysis demonstrated that the expression of dominant-negative mutant Rab5a (Rab5a S34N) and Rab5a small interfering RNA (siRNA) in RPMECs enhanced the expression of β-ARs especially β2-AR on the cell surface, whereas the wild-type of Rab5a (Rab5a WT) did not alter the β-ARs expression on the cell surface. Besides, LPS treatment significantly reduced β2-AR expression on the cell surface in RPMECs, which could be reversed by expression of dominant-negative mutant Rab5a (Rab5a S34N) and Rab5a small interfering RNA (siRNA). Meanwhile, the internalization and down-regulation of β-ARs could be inhibited by expression of Rab5a small interfering RNA (siRNA). In contrast, the expression of Rab5a WT did not change the process. Morever, the expression of Rab5a small interfering RNA (siRNA) strengthened the effect of β2-AR signaling in the presence of LPS on barrier function and permeability of RPMECs. Our data indicate that, Rab5a modulates the trafficking from membrane to cytoplasm and the cell surface targeting of β-ARs, thereby regulating the function of β-AR signaling on integrity of RPMECs. Methods:1. RPMECs were cultured using traditional way. Cells were quantified by immunocytochemical staining with anti-CD34antigen, anti-human factor VIII-related antigen and binding of Lectin BSI. The cell morphology and ultrastructure were observed with transmission electron microscope.2. RPMECs were seeded on E-plate L8. The endothelial cell barrier function was measured using iCELLigence system.3. Expression of VE-cadherin and occludin on membrane were measured by western blot.4. Cells were transfected with wild-type Rab5a, dominant-negative mutant Rab5a S34N plasmid construct and Rab5a siRNA. Thereafter, the localization of β2-AR-GFP were visualized under fluorescence microscopy.5. Cells were transfected with wild-type Rab5a, dominant-negative mutant Rab5a S34N plasmid construct and Rab5a siRNA. Then, the receptor binding site was measured using radiative way.6. RPMECs were transiently transfected with Rab5a siRNA or wild-type Rab5a plasmid construct, then internalization assay and receptor down-regulation assay were done.7. RPMECs were seeded on transwell dishes. The permeability of monolayer was determined by pervasion of biotinylated bovine serum albumin (biotin-BSA) and biotin-BSA concentrations were measured by enzyme-linked immunosorbent assay after RPMECs were treated with LPS and transfected with Rab5a siRNA.Results:1. Positive expression of factor VIII-related antigen, CD34antigen and BSI binding in RPMECs was displayed in the cytoplasm. Transmission electron microscope showed the cell membrane, cell nuclei, endoplasmic reticulum, mitochondria and lysosome.2. LPS induced decrease in normalized cell index of RPMECs in a dose-and time-dependent manner. The expression of occludin and VE-cadherin on membrane was decreased by LPS in a time-dependent manner, while cytosol proteins expression was unchangeable. The decreased normalized cell index and protein levels of occludin and VE-cadherin in membrane by LPS were reversed partly by activation of β2-AR signaling.3. β2-AR-GFP mainly distributed on cell surface in normal condition and localized along perinuclear regions when stimulated with agonist. When co-transfected with Rab5a siRNA, the β2-AR-GFP was mainly observed on cell surface whether stimulated with agonist or not. Receptors accumulated as granular vesicles in cytoplasm with agonist stimulated while remain at cell surface without agonist stimulated in cells transfected with Rab5a WT.4. Expression of total β-ARs was increased by23.9%and28.3%, respectively, in RPMECs transfected with Rab5a S34N and Rab5a siRNA compare with normal cells. Morever, expression of β2-AR was enhanced more significantly by32.1%and35.6%. Rab5a WT did not alter β-ARs or β2-AR expression at the cell surface. The expression of β2-AR was significantly decreased by42%at6h after LPS treatment in RPMECs, which could be reversed by39%and37%, respectively, by Rab5a S34N and Rab5a siRNA, wheras Rab5a WT have no distinct effects.5. The radioactive ligand [3H]-CGP12177induced β-AR internalization. The percentage of cell surface β-AR internalized as a consequence of [3H]-CGP12177incubation for2min and30min were10.40±3.8%and32.5±3.3%, respectively. The percentage of internalized receptor at30min were respectively,17.1±2.8%and33.8±2.1%when transfected with Rab5a siRNA and Rab5a WT. As for β2-AR, the percentage of P2-AR remaining on cell surface after ISO incubation for2h,6h and10h were83.9±5.1%,72.5±5.8%and64.7±5.3%, respectively. And the maximal extent of β2-AR down-regulation appeared when ISO incubation for16h (61.4±4.4%), which was close to that of24h. There was no obvious differernce between the control group and Rab5a WT group at each timepoint. When transfected with Rab5a siRNA, however, the percentage of β2-AR remaining on cell surface at every timepoint besides2h were higher than that of both control group and Rab5a WT group. The maximal extent of β2-AR down-regulation also appeared at16h, namely84.5±4.7%.6. The descending rate of normalized CI in Isoproterenol/LPS group and Isoproterenol/Atenolol/LPS group was lower than that of LPS alone group and Isoproterenol/ICI118,551/LPS group. The tendency was more evident when transfected with Rab5a siRNA. The normalized CI at6h after LPS treatment shows, the normalized CI decreased to0.712as a consequence of knockdown of Rab5a, compared with NC siRNA group, in which the normalized CI was0.718, there was no significant difference. In contrast, the normalized CI in Isoproterenol/LPS group and Isoproterenol/Atenolol/LPS group transfected with Rab5a siRNA decreased to0.897and0.889, respectively, obviously higher than that of cells transfected with NC siRNA, in which the normalized CI were respectively0.79and0.792. Besides, the normalized CI in Isoproterenol/ICI118,551/LPS group were alike when transfected with NC siRNA or Rab5a siRNA.The pervasion of BSA in LPS alone group was323.7±28.5ng/ml or313.0±36.2ng/ml, respectively, when transfected with NC siRNA or Rab5a siRNA, significantly exceed that of control group, namely76.1±6.6ng/ml and68.9±8.7ng/ml. The pervasion of BSA was207.2±24.2ng/ml and208.6±20.2ng/ml, respectively by pretrement with ISO or ISO plus atenolol after transfecting with NC siRNA. In contrast, after transfected with Rab5a siRNA, the pervasion of BSA was124.8±26.4ng/ml and125.9±11.3ng/ml, respectively, in cells pretreated with ISO or ISO plus atenolol. However, there was no difference between NC siRNA and Rab5a siRNA when pretreated with ISO plus ICI118,551.Conclusion:1. These data suggest that the response to LPS develops gradually over the course of several hours and achieve its maximum between6h and8h post-LPS addition. Consistent with EC barrier function disruption, expression of both occludin and VE-cadherin on membrane decreased significantly after LPS challenge for6h and8h. Otherwise, cytosol proteins were at a low level and kept unchangeable when treated with LPS.2. These data indicate that in parrel with normalized CI change, the decreased expression of both occludin and VE-cadherin on membrane could also be inhibited by activation of β2-AR instead of β1-AR.3. These data suggest that Rab5a could regulate the localization of β2-AR.4. These data suggest that Rab5a could modify expression of β2-AR on the cell surface of RPMECs.5. These data indicate that agonist-induced β-AR internalization and down-regulation could be suppressed by expression of Rab5a siRNA, whereas Rab5a WT did not have evident effects.6. These data suggest that although Rab5a did not immediately influence the endothelial barrier function, the protective effects of activation of β2-AR were augmented by konckdown of Rab5a. Rab5a regulates the barrier function and monolayer permeability by adjusting the trafficking and cell surface expression of β2-AR in RPMECs. |
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