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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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Related in: MedlinePlus

Role of p38/MAPK activation in LPS- and TNF-α-induced endothelial barrier dysfunction and MT disassembly.(a) Cells were pretreated with SB203580 (5 μM) for 1 hr, and CMV- or MKK6 (Glu) was transfected into HPMECs for 72 hr before the LPS or TNF-α (500 ng/ml) treatment. The permeability of endothelial cells was assessed by measuring the influx of FITC-conjugated dextran and the TER across the cells. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (b) For the immunofluorescence confocal micrographs, HPMECs were transfected with CMV- or MKK6 (Glu) and pretreated with or without SB203580 during an LPS or TNF-α challenge. (c) The Western blot shows poly/free tubulin with and without SB203580 pretreatment before an LPS or TNF-α challenge; MKK6 (Glu) was transfected into HPMECs for 72 hr before treatment. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (d) The HPMEC monolayer permeability was assessed by measuring the influx of FITC-conjugated dextran and the TER after the cells were transfected or cotransfected with CMV-, MAP4 (Ala), or MKK6 (Glu). The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. the CMV- and MAP4 (Ala) groups; #P < 0.05 vs. the CMV- + MKK6 (Glu) group. (e) Cells were studied using immunofluorescence staining with the anti-α-tubulin antibody. The boxed areas are shown at higher magnification in the inserts to provide a detailed illustration of the MTs. Bar, 10 μm. (f) Cells were transfected or cotransfected with CMV-, MAP4 (Ala), or MKK6 (Glu). The poly/free tubulin fractions were determined by Western blot analysis; VDAC and GAPDH were used as the internal controls. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. the CMV- and MAP4 (Ala) groups; #P < 0.05 vs. the CMV- + MKK6 (Glu) group.
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f5: Role of p38/MAPK activation in LPS- and TNF-α-induced endothelial barrier dysfunction and MT disassembly.(a) Cells were pretreated with SB203580 (5 μM) for 1 hr, and CMV- or MKK6 (Glu) was transfected into HPMECs for 72 hr before the LPS or TNF-α (500 ng/ml) treatment. The permeability of endothelial cells was assessed by measuring the influx of FITC-conjugated dextran and the TER across the cells. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (b) For the immunofluorescence confocal micrographs, HPMECs were transfected with CMV- or MKK6 (Glu) and pretreated with or without SB203580 during an LPS or TNF-α challenge. (c) The Western blot shows poly/free tubulin with and without SB203580 pretreatment before an LPS or TNF-α challenge; MKK6 (Glu) was transfected into HPMECs for 72 hr before treatment. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (d) The HPMEC monolayer permeability was assessed by measuring the influx of FITC-conjugated dextran and the TER after the cells were transfected or cotransfected with CMV-, MAP4 (Ala), or MKK6 (Glu). The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. the CMV- and MAP4 (Ala) groups; #P < 0.05 vs. the CMV- + MKK6 (Glu) group. (e) Cells were studied using immunofluorescence staining with the anti-α-tubulin antibody. The boxed areas are shown at higher magnification in the inserts to provide a detailed illustration of the MTs. Bar, 10 μm. (f) Cells were transfected or cotransfected with CMV-, MAP4 (Ala), or MKK6 (Glu). The poly/free tubulin fractions were determined by Western blot analysis; VDAC and GAPDH were used as the internal controls. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. the CMV- and MAP4 (Ala) groups; #P < 0.05 vs. the CMV- + MKK6 (Glu) group.

Mentions: We then observed the effect of p38/MAPK activation and inhibition on endothelial barrier function and MT dynamics in HPMECs. SB203580 (5 uM) was found largely to abolish the LPS- or TNF-α-induced endothelial hyperpermeability, which resulted in 1.16- and 1.20-fold reduction in dextran leakage, and 1.31- and 1.46-fold increase in TER compared with those for LPS and TNF-α, respectively, whereas the transduction with MKK6 (Glu) under normal conditions resulted in hyperpermeability (3.59-fold increase in dextran leakage and 2.07-fold reduction in TER compared with CMV- values) (Fig. 5a), suggesting the involvement of p38/MAPK in LPS- or TNF-α-induced endothelial barrier dysfunction. Concomitant with the prevention of hyperpermeability, SB203580 preserved the MT network in the LPS- or TNF-α-challenged cells (Fig. 5b), whereas the MKK6 (Glu) transfection resulted in complete destruction of the MT structure from the cell membrane to the nearby nuclei (Fig. 5b). Additionally, the quantification of the tubulin fractions (Fig. 5c) further confirmed that the transduction with MKK6 (Glu) led to significant MT depolymerisation, as represented by decreased polymerised tubulin and increased free tubulin. In contrast, the control cells expressing CMV- showed no changes in their MT dynamics (Fig. 5c). Similarly, the SB203580 treatment significantly increased the amount of polymeric tubulin and decreased the amount of free tubulin under inflammatory conditions (Fig. 5c).


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)

Role of p38/MAPK activation in LPS- and TNF-α-induced endothelial barrier dysfunction and MT disassembly.(a) Cells were pretreated with SB203580 (5 μM) for 1 hr, and CMV- or MKK6 (Glu) was transfected into HPMECs for 72 hr before the LPS or TNF-α (500 ng/ml) treatment. The permeability of endothelial cells was assessed by measuring the influx of FITC-conjugated dextran and the TER across the cells. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (b) For the immunofluorescence confocal micrographs, HPMECs were transfected with CMV- or MKK6 (Glu) and pretreated with or without SB203580 during an LPS or TNF-α challenge. (c) The Western blot shows poly/free tubulin with and without SB203580 pretreatment before an LPS or TNF-α challenge; MKK6 (Glu) was transfected into HPMECs for 72 hr before treatment. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (d) The HPMEC monolayer permeability was assessed by measuring the influx of FITC-conjugated dextran and the TER after the cells were transfected or cotransfected with CMV-, MAP4 (Ala), or MKK6 (Glu). The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. the CMV- and MAP4 (Ala) groups; #P < 0.05 vs. the CMV- + MKK6 (Glu) group. (e) Cells were studied using immunofluorescence staining with the anti-α-tubulin antibody. The boxed areas are shown at higher magnification in the inserts to provide a detailed illustration of the MTs. Bar, 10 μm. (f) Cells were transfected or cotransfected with CMV-, MAP4 (Ala), or MKK6 (Glu). The poly/free tubulin fractions were determined by Western blot analysis; VDAC and GAPDH were used as the internal controls. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. the CMV- and MAP4 (Ala) groups; #P < 0.05 vs. the CMV- + MKK6 (Glu) group.
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f5: Role of p38/MAPK activation in LPS- and TNF-α-induced endothelial barrier dysfunction and MT disassembly.(a) Cells were pretreated with SB203580 (5 μM) for 1 hr, and CMV- or MKK6 (Glu) was transfected into HPMECs for 72 hr before the LPS or TNF-α (500 ng/ml) treatment. The permeability of endothelial cells was assessed by measuring the influx of FITC-conjugated dextran and the TER across the cells. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (b) For the immunofluorescence confocal micrographs, HPMECs were transfected with CMV- or MKK6 (Glu) and pretreated with or without SB203580 during an LPS or TNF-α challenge. (c) The Western blot shows poly/free tubulin with and without SB203580 pretreatment before an LPS or TNF-α challenge; MKK6 (Glu) was transfected into HPMECs for 72 hr before treatment. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. CMV-; #P < 0.05 vs. the LPS or TNF-α group. (d) The HPMEC monolayer permeability was assessed by measuring the influx of FITC-conjugated dextran and the TER after the cells were transfected or cotransfected with CMV-, MAP4 (Ala), or MKK6 (Glu). The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. the CMV- and MAP4 (Ala) groups; #P < 0.05 vs. the CMV- + MKK6 (Glu) group. (e) Cells were studied using immunofluorescence staining with the anti-α-tubulin antibody. The boxed areas are shown at higher magnification in the inserts to provide a detailed illustration of the MTs. Bar, 10 μm. (f) Cells were transfected or cotransfected with CMV-, MAP4 (Ala), or MKK6 (Glu). The poly/free tubulin fractions were determined by Western blot analysis; VDAC and GAPDH were used as the internal controls. The data are represented as the mean ± SEM (n = 3). *P < 0.05 vs. the CMV- and MAP4 (Ala) groups; #P < 0.05 vs. the CMV- + MKK6 (Glu) group.
Mentions: We then observed the effect of p38/MAPK activation and inhibition on endothelial barrier function and MT dynamics in HPMECs. SB203580 (5 uM) was found largely to abolish the LPS- or TNF-α-induced endothelial hyperpermeability, which resulted in 1.16- and 1.20-fold reduction in dextran leakage, and 1.31- and 1.46-fold increase in TER compared with those for LPS and TNF-α, respectively, whereas the transduction with MKK6 (Glu) under normal conditions resulted in hyperpermeability (3.59-fold increase in dextran leakage and 2.07-fold reduction in TER compared with CMV- values) (Fig. 5a), suggesting the involvement of p38/MAPK in LPS- or TNF-α-induced endothelial barrier dysfunction. Concomitant with the prevention of hyperpermeability, SB203580 preserved the MT network in the LPS- or TNF-α-challenged cells (Fig. 5b), whereas the MKK6 (Glu) transfection resulted in complete destruction of the MT structure from the cell membrane to the nearby nuclei (Fig. 5b). Additionally, the quantification of the tubulin fractions (Fig. 5c) further confirmed that the transduction with MKK6 (Glu) led to significant MT depolymerisation, as represented by decreased polymerised tubulin and increased free tubulin. In contrast, the control cells expressing CMV- showed no changes in their MT dynamics (Fig. 5c). Similarly, the SB203580 treatment significantly increased the amount of polymeric tubulin and decreased the amount of free tubulin under inflammatory conditions (Fig. 5c).

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