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Regulation of β-adrenergic receptor trafficking and lung microvascular endothelial cell permeability by Rab5 GTPase.

Yang J, Sun H, Zhang J, Hu M, Wang J, Wu G, Wang G - Int. J. Biol. Sci. (2015)

Bottom Line: Our data demonstrate that lipopolysaccharide (LPS) treatment disrupts LMEC barrier function and reduces the cell surface expression of β-ARs.Importantly, knockdown of Rab5 not only inhibits the LPS-induced effects on β-ARs but also protects the LMEC monolayer permeability.All together, these data provide strong evidence indicating a crucial role of Rab5-mediated internalization of β-ARs in functional regulation of LMECs.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.

ABSTRACT
Rab5 GTPase modulates the trafficking of the cell surface receptors, including G protein-coupled β-adrenergic receptors (β-ARs). Here, we have determined the role of Rab5 in regulating the internalization of β-ARs in lung microvascular endothelial cells (LMECs) and in maintaining the integrity and permeability of endothelial cell barrier. Our data demonstrate that lipopolysaccharide (LPS) treatment disrupts LMEC barrier function and reduces the cell surface expression of β-ARs. Furthermore, the activation of β-ARs, particularly β2-AR, is able to protect the LMEC permeability from LPS injury. Moreover, siRNA-mediated knockdown of Rab5 inhibits both the basal and agonist-provoked internalization of β-ARs, therefore, enhancing the cell surface expression of the receptors and receptor-mediated ERK1/2 activation. Importantly, knockdown of Rab5 not only inhibits the LPS-induced effects on β-ARs but also protects the LMEC monolayer permeability. All together, these data provide strong evidence indicating a crucial role of Rab5-mediated internalization of β-ARs in functional regulation of LMECs.

No MeSH data available.


Related in: MedlinePlus

Rab5a-mediated β-AR trafficking protects the LMEC permeability from LPS injury. (A) The LMEC barrier function (CI) was enhanced by the activation of β-ARs. After transfection with Rab5a siRNA for 48 h, the LMECs were pretreated with vehicle, atenolol or ICI118,551 for 1 h; with ISO, atenolol plus ISO or ICI118,551 plus ISO for 0.5 h, and then stimulated with or without LPS (10 μg/ml) at time 1 h. The responses of the LMECs were monitored with the iCELLigence System. The data are expressed as the means ± S.E. from three individual experiments. (B) Quantitative analysis of the normalized CI values of LMECs after the LPS challenge for 6 h. The data are expressed as the means ± S.E., n = 3. *p< 0.05 versus the LMEC control group; ^p< 0.05 versus the LMECs treated with LPS alone group. (C) Rab5a siRNA inhibited LPS-mediated the monolayer hyperpermeability of LMECs. The LMECs were grown to confluence on 0.4-μm polyester membranes in the upper chambers of coculture wells and transfected with control or Rab5a siRNA for 48 h. The cultures were pretreated with ISO, ISO plus atenolol, ISO plus ICI118,551 or vehicle, and then subjected to LPS (10 μg/ml) for 6 h. Biotin-BSA (500 μg/ml) was added to the upper chamber wells. Aliquots of the lower chamber media were aspirated at 0.5 h after treatment. The biotin-BSA concentrations in the media were determined via an enzyme-linked immunosorbent assay. The data are expressed as the means ± S.E., n = 4. *p< 0.05 versus the LMEC control group; ^p< 0.05 versus the LMECs treated with LPS alone group.
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Figure 7: Rab5a-mediated β-AR trafficking protects the LMEC permeability from LPS injury. (A) The LMEC barrier function (CI) was enhanced by the activation of β-ARs. After transfection with Rab5a siRNA for 48 h, the LMECs were pretreated with vehicle, atenolol or ICI118,551 for 1 h; with ISO, atenolol plus ISO or ICI118,551 plus ISO for 0.5 h, and then stimulated with or without LPS (10 μg/ml) at time 1 h. The responses of the LMECs were monitored with the iCELLigence System. The data are expressed as the means ± S.E. from three individual experiments. (B) Quantitative analysis of the normalized CI values of LMECs after the LPS challenge for 6 h. The data are expressed as the means ± S.E., n = 3. *p< 0.05 versus the LMEC control group; ^p< 0.05 versus the LMECs treated with LPS alone group. (C) Rab5a siRNA inhibited LPS-mediated the monolayer hyperpermeability of LMECs. The LMECs were grown to confluence on 0.4-μm polyester membranes in the upper chambers of coculture wells and transfected with control or Rab5a siRNA for 48 h. The cultures were pretreated with ISO, ISO plus atenolol, ISO plus ICI118,551 or vehicle, and then subjected to LPS (10 μg/ml) for 6 h. Biotin-BSA (500 μg/ml) was added to the upper chamber wells. Aliquots of the lower chamber media were aspirated at 0.5 h after treatment. The biotin-BSA concentrations in the media were determined via an enzyme-linked immunosorbent assay. The data are expressed as the means ± S.E., n = 4. *p< 0.05 versus the LMEC control group; ^p< 0.05 versus the LMECs treated with LPS alone group.

Mentions: Our previous data demonstrate that Rab5a knockdown inhibits agonist-induced β-AR internalization and the activation of β-AR protects the endothelial barrier from LPS injury. Based on these findings, we determined the effect of Rab5a on LPS-induced endothelial barrier dysfunction by monitoring the normalized CI using the iCELLigence System. The LPS-induced change of the normalized CI in the LPS alone group was similar to the LPS combined treatment with ISO plus ICI 118,551 pretreatment group after transfection with Rab5a siRNA and the control (NC) siRNA at 7 h. Under normal condition, the whole CI in the cells after transfection with Rab5a siRNA was similar to that after transfection with NC siRNA (Fig. 7A and Supplementary Fig. 3A). After 6 h of LPS treatment, the normalized CI was not significantly different in the Rab5a siRNA group compared to NC siRNA group. However, there was a significant difference in the normalized CI in the Rab5a siRNA cells pretreated with ISO or ISO plus atenolol compared to the corresponding control cells (P<0.05, Fig. 7B). ISO and ICI 118,551 pretreatment/LPS treatment produced no significant effect. Rab5a knockdown did not affect LMEC proliferation compared to NC siRNA (Supplementary Fig. 3B). Thus, Rab5a knockdown did enhance the protective effect of β2-AR activation.


Regulation of β-adrenergic receptor trafficking and lung microvascular endothelial cell permeability by Rab5 GTPase.

Yang J, Sun H, Zhang J, Hu M, Wang J, Wu G, Wang G - Int. J. Biol. Sci. (2015)

Rab5a-mediated β-AR trafficking protects the LMEC permeability from LPS injury. (A) The LMEC barrier function (CI) was enhanced by the activation of β-ARs. After transfection with Rab5a siRNA for 48 h, the LMECs were pretreated with vehicle, atenolol or ICI118,551 for 1 h; with ISO, atenolol plus ISO or ICI118,551 plus ISO for 0.5 h, and then stimulated with or without LPS (10 μg/ml) at time 1 h. The responses of the LMECs were monitored with the iCELLigence System. The data are expressed as the means ± S.E. from three individual experiments. (B) Quantitative analysis of the normalized CI values of LMECs after the LPS challenge for 6 h. The data are expressed as the means ± S.E., n = 3. *p< 0.05 versus the LMEC control group; ^p< 0.05 versus the LMECs treated with LPS alone group. (C) Rab5a siRNA inhibited LPS-mediated the monolayer hyperpermeability of LMECs. The LMECs were grown to confluence on 0.4-μm polyester membranes in the upper chambers of coculture wells and transfected with control or Rab5a siRNA for 48 h. The cultures were pretreated with ISO, ISO plus atenolol, ISO plus ICI118,551 or vehicle, and then subjected to LPS (10 μg/ml) for 6 h. Biotin-BSA (500 μg/ml) was added to the upper chamber wells. Aliquots of the lower chamber media were aspirated at 0.5 h after treatment. The biotin-BSA concentrations in the media were determined via an enzyme-linked immunosorbent assay. The data are expressed as the means ± S.E., n = 4. *p< 0.05 versus the LMEC control group; ^p< 0.05 versus the LMECs treated with LPS alone group.
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Figure 7: Rab5a-mediated β-AR trafficking protects the LMEC permeability from LPS injury. (A) The LMEC barrier function (CI) was enhanced by the activation of β-ARs. After transfection with Rab5a siRNA for 48 h, the LMECs were pretreated with vehicle, atenolol or ICI118,551 for 1 h; with ISO, atenolol plus ISO or ICI118,551 plus ISO for 0.5 h, and then stimulated with or without LPS (10 μg/ml) at time 1 h. The responses of the LMECs were monitored with the iCELLigence System. The data are expressed as the means ± S.E. from three individual experiments. (B) Quantitative analysis of the normalized CI values of LMECs after the LPS challenge for 6 h. The data are expressed as the means ± S.E., n = 3. *p< 0.05 versus the LMEC control group; ^p< 0.05 versus the LMECs treated with LPS alone group. (C) Rab5a siRNA inhibited LPS-mediated the monolayer hyperpermeability of LMECs. The LMECs were grown to confluence on 0.4-μm polyester membranes in the upper chambers of coculture wells and transfected with control or Rab5a siRNA for 48 h. The cultures were pretreated with ISO, ISO plus atenolol, ISO plus ICI118,551 or vehicle, and then subjected to LPS (10 μg/ml) for 6 h. Biotin-BSA (500 μg/ml) was added to the upper chamber wells. Aliquots of the lower chamber media were aspirated at 0.5 h after treatment. The biotin-BSA concentrations in the media were determined via an enzyme-linked immunosorbent assay. The data are expressed as the means ± S.E., n = 4. *p< 0.05 versus the LMEC control group; ^p< 0.05 versus the LMECs treated with LPS alone group.
Mentions: Our previous data demonstrate that Rab5a knockdown inhibits agonist-induced β-AR internalization and the activation of β-AR protects the endothelial barrier from LPS injury. Based on these findings, we determined the effect of Rab5a on LPS-induced endothelial barrier dysfunction by monitoring the normalized CI using the iCELLigence System. The LPS-induced change of the normalized CI in the LPS alone group was similar to the LPS combined treatment with ISO plus ICI 118,551 pretreatment group after transfection with Rab5a siRNA and the control (NC) siRNA at 7 h. Under normal condition, the whole CI in the cells after transfection with Rab5a siRNA was similar to that after transfection with NC siRNA (Fig. 7A and Supplementary Fig. 3A). After 6 h of LPS treatment, the normalized CI was not significantly different in the Rab5a siRNA group compared to NC siRNA group. However, there was a significant difference in the normalized CI in the Rab5a siRNA cells pretreated with ISO or ISO plus atenolol compared to the corresponding control cells (P<0.05, Fig. 7B). ISO and ICI 118,551 pretreatment/LPS treatment produced no significant effect. Rab5a knockdown did not affect LMEC proliferation compared to NC siRNA (Supplementary Fig. 3B). Thus, Rab5a knockdown did enhance the protective effect of β2-AR activation.

Bottom Line: Our data demonstrate that lipopolysaccharide (LPS) treatment disrupts LMEC barrier function and reduces the cell surface expression of β-ARs.Importantly, knockdown of Rab5 not only inhibits the LPS-induced effects on β-ARs but also protects the LMEC monolayer permeability.All together, these data provide strong evidence indicating a crucial role of Rab5-mediated internalization of β-ARs in functional regulation of LMECs.

View Article: PubMed Central - PubMed

Affiliation: 1. Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.

ABSTRACT
Rab5 GTPase modulates the trafficking of the cell surface receptors, including G protein-coupled β-adrenergic receptors (β-ARs). Here, we have determined the role of Rab5 in regulating the internalization of β-ARs in lung microvascular endothelial cells (LMECs) and in maintaining the integrity and permeability of endothelial cell barrier. Our data demonstrate that lipopolysaccharide (LPS) treatment disrupts LMEC barrier function and reduces the cell surface expression of β-ARs. Furthermore, the activation of β-ARs, particularly β2-AR, is able to protect the LMEC permeability from LPS injury. Moreover, siRNA-mediated knockdown of Rab5 inhibits both the basal and agonist-provoked internalization of β-ARs, therefore, enhancing the cell surface expression of the receptors and receptor-mediated ERK1/2 activation. Importantly, knockdown of Rab5 not only inhibits the LPS-induced effects on β-ARs but also protects the LMEC monolayer permeability. All together, these data provide strong evidence indicating a crucial role of Rab5-mediated internalization of β-ARs in functional regulation of LMECs.

No MeSH data available.


Related in: MedlinePlus