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Increased ERK signalling promotes inflammatory signalling in primary airway epithelial cells expressing Z α1-antitrypsin.

van 't Wout EF, Dickens JA, van Schadewijk A, Haq I, Kwok HF, Ordóñez A, Murphy G, Stolk J, Lomas DA, Hiemstra PS, Marciniak SJ - Hum. Mol. Genet. (2013)

Bottom Line: Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling.Moreover, the mechanism of NF-κB activation has not yet been elucidated.Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge CB2 0XY, United Kingdom.

ABSTRACT
Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here, we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared with wild-type (MM) controls, and this appears to be mediated by mitogen-activated protein/extracellular signal-regulated kinase, EGF receptor and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung.

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Increased NF-κB response in ZZ primary bronchial epithelial cells is dependent on the ERK/MEK/EGFR pathway. (A) Representative western blot of the activation of the MAP kinases ERK1/2, JNK and p38 MAPK of whole cell lysates from undifferentiated primary bronchial epithelial cells knocked-out for AAT with siRNA. Cells were cultured overnight and transfected for 24 h and left 48 h before harvesting. Neuroserpin (NS) siRNA served as a control. Densitometry of four independent experiments in duplicate (mean, n = 4). (B) ZZ primary bronchial epithelial cells treated for 24 h with 1 mg/ml purified plasma M α1-antitrypsin normalized ERK1/2 levels. Densitometry of four independent experiments in duplicate (mean, n = 4). (C) ZZ primary bronchial epithelial cells treated with 10 µM U0126 (a specific MEK inhibitor) for 8 h or 2 µg/ml anti-EGFR blocking antibody for 24 h. Densitometry of three independent experiments in duplicate (mean, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus—or 0 with a two-way repeated-measurements ANOVA (Bonferroni post hoc).
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DDT487F3: Increased NF-κB response in ZZ primary bronchial epithelial cells is dependent on the ERK/MEK/EGFR pathway. (A) Representative western blot of the activation of the MAP kinases ERK1/2, JNK and p38 MAPK of whole cell lysates from undifferentiated primary bronchial epithelial cells knocked-out for AAT with siRNA. Cells were cultured overnight and transfected for 24 h and left 48 h before harvesting. Neuroserpin (NS) siRNA served as a control. Densitometry of four independent experiments in duplicate (mean, n = 4). (B) ZZ primary bronchial epithelial cells treated for 24 h with 1 mg/ml purified plasma M α1-antitrypsin normalized ERK1/2 levels. Densitometry of four independent experiments in duplicate (mean, n = 4). (C) ZZ primary bronchial epithelial cells treated with 10 µM U0126 (a specific MEK inhibitor) for 8 h or 2 µg/ml anti-EGFR blocking antibody for 24 h. Densitometry of three independent experiments in duplicate (mean, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus—or 0 with a two-way repeated-measurements ANOVA (Bonferroni post hoc).

Mentions: In order to understand the mechanism of inflammatory signalling in ZZ epithelial cells, we next evaluated activation of the NF-κB pathway components, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), IκBα and p65. To evaluate MAPK signalling, we also measured JNK, p38 MAPK and ERK1/2. This revealed a significant difference only in the activation of ERK (P < 0.05; Fig. 3A and Supplementary Material, Fig. S4). Interestingly, depletion of α1-antitrypsin by siRNA caused phosphorylation of ERK in MM primary bronchial epithelial cells, but did not alter the phosphorylation of ERK in ZZ cells (Fig. 3A). This effect was specific for ablation of α1-antitrypsin, because silencing a non-specific serpin, neuroserpin (NS), did not increase phosphorylation of ERK in MM cells. This suggested that it was the lack of (M) α1-antitrypsin, rather than the presence of Z α1-antitrypsin, that might be responsible for the phosphorylation of ERK in ZZ primary bronchial epithelial cells. To test this, we treated ZZ primary bronchial epithelial cells with plasma-purified M α1-antitrypsin and observed a suppression of ERK phosphorylation (P < 0.05; Fig. 3B). We wished to determine whether this loss of function phenotype reflected a lack of anti-inflammatory activity in Z α1-antitrypsin or if cells secreted insufficient Z α1-antitrypsin. As concentration of plasma-purified Z α1-antitrypsin to a degree required for this experiment would result in its polymerization, we instead transiently transfected HeLa cells with either M or Z α1-antitrypsin or empty vector as control. After transfection, the cells produced high levels of α1-antitrypsin, with ZZ cells producing ∼20% of the amount that MM cells produced (444 ng/mg α1-antitrypsin in the total lysate versus 1995 ng/mg α1-antitrypsin in the total lysate respectively; Supplementary Material, Fig. S5A). Although 14% (33 ng/mg total lysate) of the extracellular Z α1-antitrypsin formed polymers (data not shown), the protein was able to inhibit phosphorylation of ERK1/2 to a similar degree as M α1-antitrypsin (Supplementary Material, Fig. S5B). This result is consistent with a model in which ZZ primary bronchial epithelial cells secrete insufficient α1-antitrypsin to inhibit ERK1/2 phosphorylation, rather than Z α1-antitrypsin lacking the anti-inflammatory activity per se.Figure 3.


Increased ERK signalling promotes inflammatory signalling in primary airway epithelial cells expressing Z α1-antitrypsin.

van 't Wout EF, Dickens JA, van Schadewijk A, Haq I, Kwok HF, Ordóñez A, Murphy G, Stolk J, Lomas DA, Hiemstra PS, Marciniak SJ - Hum. Mol. Genet. (2013)

Increased NF-κB response in ZZ primary bronchial epithelial cells is dependent on the ERK/MEK/EGFR pathway. (A) Representative western blot of the activation of the MAP kinases ERK1/2, JNK and p38 MAPK of whole cell lysates from undifferentiated primary bronchial epithelial cells knocked-out for AAT with siRNA. Cells were cultured overnight and transfected for 24 h and left 48 h before harvesting. Neuroserpin (NS) siRNA served as a control. Densitometry of four independent experiments in duplicate (mean, n = 4). (B) ZZ primary bronchial epithelial cells treated for 24 h with 1 mg/ml purified plasma M α1-antitrypsin normalized ERK1/2 levels. Densitometry of four independent experiments in duplicate (mean, n = 4). (C) ZZ primary bronchial epithelial cells treated with 10 µM U0126 (a specific MEK inhibitor) for 8 h or 2 µg/ml anti-EGFR blocking antibody for 24 h. Densitometry of three independent experiments in duplicate (mean, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus—or 0 with a two-way repeated-measurements ANOVA (Bonferroni post hoc).
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DDT487F3: Increased NF-κB response in ZZ primary bronchial epithelial cells is dependent on the ERK/MEK/EGFR pathway. (A) Representative western blot of the activation of the MAP kinases ERK1/2, JNK and p38 MAPK of whole cell lysates from undifferentiated primary bronchial epithelial cells knocked-out for AAT with siRNA. Cells were cultured overnight and transfected for 24 h and left 48 h before harvesting. Neuroserpin (NS) siRNA served as a control. Densitometry of four independent experiments in duplicate (mean, n = 4). (B) ZZ primary bronchial epithelial cells treated for 24 h with 1 mg/ml purified plasma M α1-antitrypsin normalized ERK1/2 levels. Densitometry of four independent experiments in duplicate (mean, n = 4). (C) ZZ primary bronchial epithelial cells treated with 10 µM U0126 (a specific MEK inhibitor) for 8 h or 2 µg/ml anti-EGFR blocking antibody for 24 h. Densitometry of three independent experiments in duplicate (mean, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 versus—or 0 with a two-way repeated-measurements ANOVA (Bonferroni post hoc).
Mentions: In order to understand the mechanism of inflammatory signalling in ZZ epithelial cells, we next evaluated activation of the NF-κB pathway components, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), IκBα and p65. To evaluate MAPK signalling, we also measured JNK, p38 MAPK and ERK1/2. This revealed a significant difference only in the activation of ERK (P < 0.05; Fig. 3A and Supplementary Material, Fig. S4). Interestingly, depletion of α1-antitrypsin by siRNA caused phosphorylation of ERK in MM primary bronchial epithelial cells, but did not alter the phosphorylation of ERK in ZZ cells (Fig. 3A). This effect was specific for ablation of α1-antitrypsin, because silencing a non-specific serpin, neuroserpin (NS), did not increase phosphorylation of ERK in MM cells. This suggested that it was the lack of (M) α1-antitrypsin, rather than the presence of Z α1-antitrypsin, that might be responsible for the phosphorylation of ERK in ZZ primary bronchial epithelial cells. To test this, we treated ZZ primary bronchial epithelial cells with plasma-purified M α1-antitrypsin and observed a suppression of ERK phosphorylation (P < 0.05; Fig. 3B). We wished to determine whether this loss of function phenotype reflected a lack of anti-inflammatory activity in Z α1-antitrypsin or if cells secreted insufficient Z α1-antitrypsin. As concentration of plasma-purified Z α1-antitrypsin to a degree required for this experiment would result in its polymerization, we instead transiently transfected HeLa cells with either M or Z α1-antitrypsin or empty vector as control. After transfection, the cells produced high levels of α1-antitrypsin, with ZZ cells producing ∼20% of the amount that MM cells produced (444 ng/mg α1-antitrypsin in the total lysate versus 1995 ng/mg α1-antitrypsin in the total lysate respectively; Supplementary Material, Fig. S5A). Although 14% (33 ng/mg total lysate) of the extracellular Z α1-antitrypsin formed polymers (data not shown), the protein was able to inhibit phosphorylation of ERK1/2 to a similar degree as M α1-antitrypsin (Supplementary Material, Fig. S5B). This result is consistent with a model in which ZZ primary bronchial epithelial cells secrete insufficient α1-antitrypsin to inhibit ERK1/2 phosphorylation, rather than Z α1-antitrypsin lacking the anti-inflammatory activity per se.Figure 3.

Bottom Line: Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling.Moreover, the mechanism of NF-κB activation has not yet been elucidated.Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge CB2 0XY, United Kingdom.

ABSTRACT
Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here, we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared with wild-type (MM) controls, and this appears to be mediated by mitogen-activated protein/extracellular signal-regulated kinase, EGF receptor and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung.

Show MeSH
Related in: MedlinePlus