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Mitogen activated protein kinase activated protein kinase 2 regulates actin polymerization and vascular leak in ventilator associated lung injury.

Damarla M, Hasan E, Boueiz A, Le A, Pae HH, Montouchet C, Kolb T, Simms T, Myers A, Kayyali US, Gaestel M, Peng X, Reddy SP, Damico R, Hassoun PM - PLoS ONE (2009)

Bottom Line: However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability.Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV.Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2(-/-) mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

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p38 MAP kinase and MK2 inhibition prevents phosphorylation of HSP25.Wild type or MK2−/− mice were exposed to MV at HVT for 0 or 60 minutes (peak of HSP25 phosphorylation), after which lungs were harvested for immunoblotting for phosphorylated HSP25. A subset of wild type mice were pretreated with the p38 MAP kinase inhibitor SB203580 or the MK2 inhibitor KKKALNRQLGVAA. A representative Western blot indicates that chemical inhibition of p38 MAP kinase or MK2 abrogates the phosphorylation of HSP25 and that MK2−/− mice fail to phosphorylate HSP25 after exposure to HVT MV. Densitometric analysis confirms that the ratio of phospho-HSP25/total-HSP25 peaks is significantly inhibited with inhibition of p38 MAP kinase and MK2 after 60 min of HVT MV. * P<0.05 (vehicle vs wild type counter parts). N = 3–4 mice per condition.
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pone-0004600-g003: p38 MAP kinase and MK2 inhibition prevents phosphorylation of HSP25.Wild type or MK2−/− mice were exposed to MV at HVT for 0 or 60 minutes (peak of HSP25 phosphorylation), after which lungs were harvested for immunoblotting for phosphorylated HSP25. A subset of wild type mice were pretreated with the p38 MAP kinase inhibitor SB203580 or the MK2 inhibitor KKKALNRQLGVAA. A representative Western blot indicates that chemical inhibition of p38 MAP kinase or MK2 abrogates the phosphorylation of HSP25 and that MK2−/− mice fail to phosphorylate HSP25 after exposure to HVT MV. Densitometric analysis confirms that the ratio of phospho-HSP25/total-HSP25 peaks is significantly inhibited with inhibition of p38 MAP kinase and MK2 after 60 min of HVT MV. * P<0.05 (vehicle vs wild type counter parts). N = 3–4 mice per condition.

Mentions: Since our prior work had shown that HSP27 phosphorylation was necessary for stress fiber formation in vitro [9], we next sought to determine the role of p38 MAP kinase and MK2 on HSP25 (murine homologue of HSP27) phosphorylation on actin polymerization and stress fiber formation in response to mechanical stress in vivo. Mice were pretreated with the p38 MAP kinase inhibitor SB203580 (2 mg/kg, i.p.) and MK2 inhibitory peptide KKKALNRQLGVAA (2 mg/kg, i.p.) given one hour prior to MV. Immunoblot analysis for phospho- and total HSP25 was performed on lung homogenates of mice exposed to one hour of HVT of MV, a time point when HSP25 phosphorylation is at its peak (Figure 2C). As shown in Figure 3, both chemical inhibitors effectively decreased HSP25 phosphorylation indicating that p38 MAP kinase and MK2 are upstream of, and required for, HSP25 phosphorylation. In parallel experiments, lung homogenates obtained from MK2−/− mice exposed to MV with HVT displayed normal amounts of total HSP25, however, without evidence of activation (i.e., phospho-HSP25) in response to HVT MV (Figure 3), further suggesting that MK2 is necessary for HSP25 phosphorylation in response to mechanical stress.


Mitogen activated protein kinase activated protein kinase 2 regulates actin polymerization and vascular leak in ventilator associated lung injury.

Damarla M, Hasan E, Boueiz A, Le A, Pae HH, Montouchet C, Kolb T, Simms T, Myers A, Kayyali US, Gaestel M, Peng X, Reddy SP, Damico R, Hassoun PM - PLoS ONE (2009)

p38 MAP kinase and MK2 inhibition prevents phosphorylation of HSP25.Wild type or MK2−/− mice were exposed to MV at HVT for 0 or 60 minutes (peak of HSP25 phosphorylation), after which lungs were harvested for immunoblotting for phosphorylated HSP25. A subset of wild type mice were pretreated with the p38 MAP kinase inhibitor SB203580 or the MK2 inhibitor KKKALNRQLGVAA. A representative Western blot indicates that chemical inhibition of p38 MAP kinase or MK2 abrogates the phosphorylation of HSP25 and that MK2−/− mice fail to phosphorylate HSP25 after exposure to HVT MV. Densitometric analysis confirms that the ratio of phospho-HSP25/total-HSP25 peaks is significantly inhibited with inhibition of p38 MAP kinase and MK2 after 60 min of HVT MV. * P<0.05 (vehicle vs wild type counter parts). N = 3–4 mice per condition.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2643011&req=5

pone-0004600-g003: p38 MAP kinase and MK2 inhibition prevents phosphorylation of HSP25.Wild type or MK2−/− mice were exposed to MV at HVT for 0 or 60 minutes (peak of HSP25 phosphorylation), after which lungs were harvested for immunoblotting for phosphorylated HSP25. A subset of wild type mice were pretreated with the p38 MAP kinase inhibitor SB203580 or the MK2 inhibitor KKKALNRQLGVAA. A representative Western blot indicates that chemical inhibition of p38 MAP kinase or MK2 abrogates the phosphorylation of HSP25 and that MK2−/− mice fail to phosphorylate HSP25 after exposure to HVT MV. Densitometric analysis confirms that the ratio of phospho-HSP25/total-HSP25 peaks is significantly inhibited with inhibition of p38 MAP kinase and MK2 after 60 min of HVT MV. * P<0.05 (vehicle vs wild type counter parts). N = 3–4 mice per condition.
Mentions: Since our prior work had shown that HSP27 phosphorylation was necessary for stress fiber formation in vitro [9], we next sought to determine the role of p38 MAP kinase and MK2 on HSP25 (murine homologue of HSP27) phosphorylation on actin polymerization and stress fiber formation in response to mechanical stress in vivo. Mice were pretreated with the p38 MAP kinase inhibitor SB203580 (2 mg/kg, i.p.) and MK2 inhibitory peptide KKKALNRQLGVAA (2 mg/kg, i.p.) given one hour prior to MV. Immunoblot analysis for phospho- and total HSP25 was performed on lung homogenates of mice exposed to one hour of HVT of MV, a time point when HSP25 phosphorylation is at its peak (Figure 2C). As shown in Figure 3, both chemical inhibitors effectively decreased HSP25 phosphorylation indicating that p38 MAP kinase and MK2 are upstream of, and required for, HSP25 phosphorylation. In parallel experiments, lung homogenates obtained from MK2−/− mice exposed to MV with HVT displayed normal amounts of total HSP25, however, without evidence of activation (i.e., phospho-HSP25) in response to HVT MV (Figure 3), further suggesting that MK2 is necessary for HSP25 phosphorylation in response to mechanical stress.

Bottom Line: However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability.Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV.Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2(-/-) mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

Show MeSH
Related in: MedlinePlus