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P38/MAPK contributes to endothelial barrier dysfunction via MAP4 phosphorylation-dependent microtubule disassembly in inflammation-induced acute lung injury.

Li L, Hu J, He T, Zhang Q, Yang X, Lan X, Zhang D, Mei H, Chen B, Huang Y - Sci Rep (2015)

Bottom Line: Microtubule-associated protein 4 (MAP4), a tubulin assembly-promoting protein, is important for maintaining the microtubule (MT) cytoskeleton and cell-cell junctional structures.In contrast, MKK6 (Glu), which constitutively activated p38/MAPK, resulted in microtubule depolymerisation and, subsequently, hyperpermeability.Our findings reveal a novel role of MAP4 in endothelial barrier dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China.

ABSTRACT
Excessive activation of inflammation and the accompanying lung vascular endothelial barrier disruption are primary pathogenic features of acute lung injury (ALI). Microtubule-associated protein 4 (MAP4), a tubulin assembly-promoting protein, is important for maintaining the microtubule (MT) cytoskeleton and cell-cell junctional structures. However, both the involvement and exact mechanism of MAP4 in the development of endothelial barrier disruption in ALI remains unknown. In this study, lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α) were applied to human pulmonary microvascular endothelial cells (HPMECs) to mimic the endothelial damage during inflammation in vitro. We demonstrated that the MAP4 (Ser696 and Ser787) phosphorylation increased concomitantly with the p38/MAPK pathway activation by the LPS and TNF-α stimulation of HPMECs, which induced MT disassembly followed by hyperpermeability. Moreover, the application of taxol, the overexpression of a MAP4 (Ala) mutant, or the application of the p38/MAPK inhibitor SB203580 inhibited the MT disruption and the intracellular junction dysfunction. In contrast, MKK6 (Glu), which constitutively activated p38/MAPK, resulted in microtubule depolymerisation and, subsequently, hyperpermeability. Our findings reveal a novel role of MAP4 in endothelial barrier dysfunction.

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LPS- and TNF-α-induced MAP4 phosphorylation and its effect on endothelial barrier function and MTs.(a) HPMECs were treated with LPS or TNF-α (500 ng/ml) for 1, 3, 6, and 12 hr and examined using Western blotting. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. the control. (b) Confirmation of MAP4 (Ala) transfection at comparable levels in HPMECs. (c) The permeability of endothelial cells was assessed by measuring the influx of FITC-conjugated dextran and the TER. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (d) For the immunofluorescence confocal micrographs, all the cells were transfected with CMV- or MAP4 (Ala) and then treated or untreated with LPS or TNF-α for 6 hr. Bar, 10 μm. The inserts show high-magnification images of the peripheral MT network. (e) Cells were treated with LPS or TNF-α after being transfected with CMV- or MAP4 (Ala). The Western blot shows polymerised and free tubulin; VDAC and GAPDH were used as the marker proteins. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the MAP4 (Ala), LPS, or TNF-α group. (f) Determination of MAP4 binding to tubulin in cells with or without CMV- or MAP4 (Ala) overexpression under LPS or TNF-α treatment by IP; Isotype control is as the negative control (n = 3).
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f3: LPS- and TNF-α-induced MAP4 phosphorylation and its effect on endothelial barrier function and MTs.(a) HPMECs were treated with LPS or TNF-α (500 ng/ml) for 1, 3, 6, and 12 hr and examined using Western blotting. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. the control. (b) Confirmation of MAP4 (Ala) transfection at comparable levels in HPMECs. (c) The permeability of endothelial cells was assessed by measuring the influx of FITC-conjugated dextran and the TER. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (d) For the immunofluorescence confocal micrographs, all the cells were transfected with CMV- or MAP4 (Ala) and then treated or untreated with LPS or TNF-α for 6 hr. Bar, 10 μm. The inserts show high-magnification images of the peripheral MT network. (e) Cells were treated with LPS or TNF-α after being transfected with CMV- or MAP4 (Ala). The Western blot shows polymerised and free tubulin; VDAC and GAPDH were used as the marker proteins. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the MAP4 (Ala), LPS, or TNF-α group. (f) Determination of MAP4 binding to tubulin in cells with or without CMV- or MAP4 (Ala) overexpression under LPS or TNF-α treatment by IP; Isotype control is as the negative control (n = 3).

Mentions: We and others have determined that the amino acid residues S696, S768, and S787 in MAP4 are the critical sites responsible for the binding of MAP4 to tubulin; the phosphorylation of these sites leads to the dissociation of MAP4 from tubulin12131920. To determine whether the above observed MT disassembly (Fig. 1) was caused by MAP4 phosphorylation, the MAP4 phosphorylation was investigated using Western blots of HPMECs with or without LPS or TNF-α treatment (Fig. 3a). MAP4 was found to show weak basal levels of phosphorylation in cultured HPMECs and that LPS (500 ng/ml) or TNF-α (500 ng/ml) stimulation induced robust phosphorylation at S696 and S787 (Fig. 3a) in a time-dependent manner but not at S768 (Supplementary Fig. S1a); the total level of MAP4 remained unchanged before and after LPS or TNF-α treatment (Fig. 3a).


P38/MAPK contributes to endothelial barrier dysfunction via MAP4 phosphorylation-dependent microtubule disassembly in inflammation-induced acute lung injury.

Li L, Hu J, He T, Zhang Q, Yang X, Lan X, Zhang D, Mei H, Chen B, Huang Y - Sci Rep (2015)

LPS- and TNF-α-induced MAP4 phosphorylation and its effect on endothelial barrier function and MTs.(a) HPMECs were treated with LPS or TNF-α (500 ng/ml) for 1, 3, 6, and 12 hr and examined using Western blotting. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. the control. (b) Confirmation of MAP4 (Ala) transfection at comparable levels in HPMECs. (c) The permeability of endothelial cells was assessed by measuring the influx of FITC-conjugated dextran and the TER. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (d) For the immunofluorescence confocal micrographs, all the cells were transfected with CMV- or MAP4 (Ala) and then treated or untreated with LPS or TNF-α for 6 hr. Bar, 10 μm. The inserts show high-magnification images of the peripheral MT network. (e) Cells were treated with LPS or TNF-α after being transfected with CMV- or MAP4 (Ala). The Western blot shows polymerised and free tubulin; VDAC and GAPDH were used as the marker proteins. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the MAP4 (Ala), LPS, or TNF-α group. (f) Determination of MAP4 binding to tubulin in cells with or without CMV- or MAP4 (Ala) overexpression under LPS or TNF-α treatment by IP; Isotype control is as the negative control (n = 3).
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f3: LPS- and TNF-α-induced MAP4 phosphorylation and its effect on endothelial barrier function and MTs.(a) HPMECs were treated with LPS or TNF-α (500 ng/ml) for 1, 3, 6, and 12 hr and examined using Western blotting. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. the control. (b) Confirmation of MAP4 (Ala) transfection at comparable levels in HPMECs. (c) The permeability of endothelial cells was assessed by measuring the influx of FITC-conjugated dextran and the TER. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (d) For the immunofluorescence confocal micrographs, all the cells were transfected with CMV- or MAP4 (Ala) and then treated or untreated with LPS or TNF-α for 6 hr. Bar, 10 μm. The inserts show high-magnification images of the peripheral MT network. (e) Cells were treated with LPS or TNF-α after being transfected with CMV- or MAP4 (Ala). The Western blot shows polymerised and free tubulin; VDAC and GAPDH were used as the marker proteins. The graph shows the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the MAP4 (Ala), LPS, or TNF-α group. (f) Determination of MAP4 binding to tubulin in cells with or without CMV- or MAP4 (Ala) overexpression under LPS or TNF-α treatment by IP; Isotype control is as the negative control (n = 3).
Mentions: We and others have determined that the amino acid residues S696, S768, and S787 in MAP4 are the critical sites responsible for the binding of MAP4 to tubulin; the phosphorylation of these sites leads to the dissociation of MAP4 from tubulin12131920. To determine whether the above observed MT disassembly (Fig. 1) was caused by MAP4 phosphorylation, the MAP4 phosphorylation was investigated using Western blots of HPMECs with or without LPS or TNF-α treatment (Fig. 3a). MAP4 was found to show weak basal levels of phosphorylation in cultured HPMECs and that LPS (500 ng/ml) or TNF-α (500 ng/ml) stimulation induced robust phosphorylation at S696 and S787 (Fig. 3a) in a time-dependent manner but not at S768 (Supplementary Fig. S1a); the total level of MAP4 remained unchanged before and after LPS or TNF-α treatment (Fig. 3a).

Bottom Line: Microtubule-associated protein 4 (MAP4), a tubulin assembly-promoting protein, is important for maintaining the microtubule (MT) cytoskeleton and cell-cell junctional structures.In contrast, MKK6 (Glu), which constitutively activated p38/MAPK, resulted in microtubule depolymerisation and, subsequently, hyperpermeability.Our findings reveal a novel role of MAP4 in endothelial barrier dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, Third Military Medical University, Chongqing, China.

ABSTRACT
Excessive activation of inflammation and the accompanying lung vascular endothelial barrier disruption are primary pathogenic features of acute lung injury (ALI). Microtubule-associated protein 4 (MAP4), a tubulin assembly-promoting protein, is important for maintaining the microtubule (MT) cytoskeleton and cell-cell junctional structures. However, both the involvement and exact mechanism of MAP4 in the development of endothelial barrier disruption in ALI remains unknown. In this study, lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α) were applied to human pulmonary microvascular endothelial cells (HPMECs) to mimic the endothelial damage during inflammation in vitro. We demonstrated that the MAP4 (Ser696 and Ser787) phosphorylation increased concomitantly with the p38/MAPK pathway activation by the LPS and TNF-α stimulation of HPMECs, which induced MT disassembly followed by hyperpermeability. Moreover, the application of taxol, the overexpression of a MAP4 (Ala) mutant, or the application of the p38/MAPK inhibitor SB203580 inhibited the MT disruption and the intracellular junction dysfunction. In contrast, MKK6 (Glu), which constitutively activated p38/MAPK, resulted in microtubule depolymerisation and, subsequently, hyperpermeability. Our findings reveal a novel role of MAP4 in endothelial barrier dysfunction.

Show MeSH
Related in: MedlinePlus