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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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Related in: MedlinePlus

Polymer formation and an (exaggerated) ER stress response are not causing the augmented NF-κB response. (A) Total α1-antitrypsin (AAT) and α1-antitrypsin polymer production of fully differentiated primary bronchial epithelial cells stimulated with OSM-mix for 48 h. Note lack of polymer signal with the 2C1 antibody (mean, n = 6). (B) Total α1-antitrypsin and α1-antitrypsin polymer production of the overexpressing Tet-On A549 cells after inducing for 48 h with doxycycline (dox; mean ± SEM, n = 3). (C) Total α1-antitrypsin levels produced by ZZ lung epithelial cells compared with ZZ liver homogenate (n = 3 from one individual). (D) Quantitative RT-PCR of fully differentiated primary bronchial epithelial cells treated with OSM-mix for 48 h as indicated. Four hours before harvesting, cells were stimulated with tunicamycin (Tm; 1 µg/ml). XBP1 splicing, CHOP and GADD34 mRNA levels are displayed normalized to the housekeeping genes RPL13A and ATP5B (mean, n = 6). (E) Western blot for GRP94 and GRP78 using anti-KDEL antibody. Cells were treated as in D but stimulated for 16 h with tunicamycin (mean, n = 6). N.D. not detectable. *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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DDT487F2: Polymer formation and an (exaggerated) ER stress response are not causing the augmented NF-κB response. (A) Total α1-antitrypsin (AAT) and α1-antitrypsin polymer production of fully differentiated primary bronchial epithelial cells stimulated with OSM-mix for 48 h. Note lack of polymer signal with the 2C1 antibody (mean, n = 6). (B) Total α1-antitrypsin and α1-antitrypsin polymer production of the overexpressing Tet-On A549 cells after inducing for 48 h with doxycycline (dox; mean ± SEM, n = 3). (C) Total α1-antitrypsin levels produced by ZZ lung epithelial cells compared with ZZ liver homogenate (n = 3 from one individual). (D) Quantitative RT-PCR of fully differentiated primary bronchial epithelial cells treated with OSM-mix for 48 h as indicated. Four hours before harvesting, cells were stimulated with tunicamycin (Tm; 1 µg/ml). XBP1 splicing, CHOP and GADD34 mRNA levels are displayed normalized to the housekeeping genes RPL13A and ATP5B (mean, n = 6). (E) Western blot for GRP94 and GRP78 using anti-KDEL antibody. Cells were treated as in D but stimulated for 16 h with tunicamycin (mean, n = 6). N.D. not detectable. *P < 0.05, **P < 0.01, ***P < 0.001 versus—or 0 with a two-way repeated-measurements ANOVA (Bonferroni post hoc).

Mentions: NF-κB activation by mutant serpins has previously been associated with the accumulation of protein polymers within the ER (20–22,29). This accumulation has also been shown to exaggerate ER stress upon a second hit (20,22). To verify whether this mechanism was responsible for the enhanced NF-κB signalling in fully differentiated ZZ primary bronchial epithelial cells, we first measured total secreted and intracellular α1-antitrypsin. Resting primary bronchial epithelial cells produced unquantifiable amounts of α1-antitrypsin, but after stimulation with OSM-mix for 48 h, α1-antitrypsin was detectable in both the apical washes and basal supernatant (Fig. 2A). Similar results were obtained after exclusion of the current smokers, indicating that the differences in smoking status of the patients between MM and ZZ patients from which cells were obtained did not explain the differences in production of α1-antitrypsin (data not shown). Using the 2C1 monoclonal antibody that specifically detects naturally occurring polymers of Z α1-antitrypsin (32), we found no evidence of polymer formation (Fig. 2A). Accordingly, we could detect no differences in ER protein mobility, which we have previously shown occurs in cells containing ER luminal polymers of α1-antitrypsin [(22) Supplementary Material, Fig. S2]. To determine whether the absence of polymer formation was a feature of lung epithelial cells, we generated stable transfected A549 lung carcinoma cell lines that conditionally expressed either M or Z α1-antitrypsin under the control of a Tet-On responsive promoter. As expected, M α1-antitrypsin-expressing A549 cells secreted five-times more α1-antitrypsin than did Z α1-antitrypsin-expressing A549 cells (Fig. 2B). Again, we were unable to detect protein polymers in either the supernatant or cellular lysates (Fig. 2B). As polymer formation is dependent upon α1-antitrypsin concentration (8), we compared the relative levels of α1-antitrypsin in tissue from an explanted cirrhotic ZZ liver (Fig. 2C) with those in cultured airway epithelial cells. This revealed a 100-fold higher level of α1-antitrypsin in hepatic tissue and significant polymer accumulation (Fig. 2C). While polymerization of α1-antitrypsin in vitro is highly dependent upon protein concentration, the concentration dependence of polymerization within the crowded environment of the ER in vivo is not known. Therefore, to determine the critical concentration for the polymerization of Z α1-antitrypsin in living cells, we induced the expression of Z α1-antitrypsin in Tet-On stable CHO stable cells (22) and measured both total α1-antitrypsin and polymer levels (Supplementary Material, Fig. S3A). This revealed that levels of 300 ng α1-antitrypsin per 1 mg of total lysate protein are necessary before intracellular polymers can be detected in these cells (Supplementary Material, Fig. S3A). To test this finding in lung epithelial-derived cells, we induced expression of Z α1-antitrypsin in Tet-On A549 cells with doxycycline and augmented the protein level by inhibiting ERAD with lactacystin, a selective proteasome inhibitor. This increased the concentration of intracellular Z α1-antitrypsin of >300 ng α1-antitrypsin per 1 mg of total lysate, whereupon polymers were detected (Supplementary Material, Fig. S3B). It therefore seems likely that the low levels of α1-antitrypsin produced by airway epithelia are insufficient to generate detectable polymers.Figure 2.


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)

Polymer formation and an (exaggerated) ER stress response are not causing the augmented NF-κB response. (A) Total α1-antitrypsin (AAT) and α1-antitrypsin polymer production of fully differentiated primary bronchial epithelial cells stimulated with OSM-mix for 48 h. Note lack of polymer signal with the 2C1 antibody (mean, n = 6). (B) Total α1-antitrypsin and α1-antitrypsin polymer production of the overexpressing Tet-On A549 cells after inducing for 48 h with doxycycline (dox; mean ± SEM, n = 3). (C) Total α1-antitrypsin levels produced by ZZ lung epithelial cells compared with ZZ liver homogenate (n = 3 from one individual). (D) Quantitative RT-PCR of fully differentiated primary bronchial epithelial cells treated with OSM-mix for 48 h as indicated. Four hours before harvesting, cells were stimulated with tunicamycin (Tm; 1 µg/ml). XBP1 splicing, CHOP and GADD34 mRNA levels are displayed normalized to the housekeeping genes RPL13A and ATP5B (mean, n = 6). (E) Western blot for GRP94 and GRP78 using anti-KDEL antibody. Cells were treated as in D but stimulated for 16 h with tunicamycin (mean, n = 6). N.D. not detectable. *P < 0.05, **P < 0.01, ***P < 0.001 versus—or 0 with a two-way repeated-measurements ANOVA (Bonferroni post hoc).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4007119&req=5

DDT487F2: Polymer formation and an (exaggerated) ER stress response are not causing the augmented NF-κB response. (A) Total α1-antitrypsin (AAT) and α1-antitrypsin polymer production of fully differentiated primary bronchial epithelial cells stimulated with OSM-mix for 48 h. Note lack of polymer signal with the 2C1 antibody (mean, n = 6). (B) Total α1-antitrypsin and α1-antitrypsin polymer production of the overexpressing Tet-On A549 cells after inducing for 48 h with doxycycline (dox; mean ± SEM, n = 3). (C) Total α1-antitrypsin levels produced by ZZ lung epithelial cells compared with ZZ liver homogenate (n = 3 from one individual). (D) Quantitative RT-PCR of fully differentiated primary bronchial epithelial cells treated with OSM-mix for 48 h as indicated. Four hours before harvesting, cells were stimulated with tunicamycin (Tm; 1 µg/ml). XBP1 splicing, CHOP and GADD34 mRNA levels are displayed normalized to the housekeeping genes RPL13A and ATP5B (mean, n = 6). (E) Western blot for GRP94 and GRP78 using anti-KDEL antibody. Cells were treated as in D but stimulated for 16 h with tunicamycin (mean, n = 6). N.D. not detectable. *P < 0.05, **P < 0.01, ***P < 0.001 versus—or 0 with a two-way repeated-measurements ANOVA (Bonferroni post hoc).
Mentions: NF-κB activation by mutant serpins has previously been associated with the accumulation of protein polymers within the ER (20–22,29). This accumulation has also been shown to exaggerate ER stress upon a second hit (20,22). To verify whether this mechanism was responsible for the enhanced NF-κB signalling in fully differentiated ZZ primary bronchial epithelial cells, we first measured total secreted and intracellular α1-antitrypsin. Resting primary bronchial epithelial cells produced unquantifiable amounts of α1-antitrypsin, but after stimulation with OSM-mix for 48 h, α1-antitrypsin was detectable in both the apical washes and basal supernatant (Fig. 2A). Similar results were obtained after exclusion of the current smokers, indicating that the differences in smoking status of the patients between MM and ZZ patients from which cells were obtained did not explain the differences in production of α1-antitrypsin (data not shown). Using the 2C1 monoclonal antibody that specifically detects naturally occurring polymers of Z α1-antitrypsin (32), we found no evidence of polymer formation (Fig. 2A). Accordingly, we could detect no differences in ER protein mobility, which we have previously shown occurs in cells containing ER luminal polymers of α1-antitrypsin [(22) Supplementary Material, Fig. S2]. To determine whether the absence of polymer formation was a feature of lung epithelial cells, we generated stable transfected A549 lung carcinoma cell lines that conditionally expressed either M or Z α1-antitrypsin under the control of a Tet-On responsive promoter. As expected, M α1-antitrypsin-expressing A549 cells secreted five-times more α1-antitrypsin than did Z α1-antitrypsin-expressing A549 cells (Fig. 2B). Again, we were unable to detect protein polymers in either the supernatant or cellular lysates (Fig. 2B). As polymer formation is dependent upon α1-antitrypsin concentration (8), we compared the relative levels of α1-antitrypsin in tissue from an explanted cirrhotic ZZ liver (Fig. 2C) with those in cultured airway epithelial cells. This revealed a 100-fold higher level of α1-antitrypsin in hepatic tissue and significant polymer accumulation (Fig. 2C). While polymerization of α1-antitrypsin in vitro is highly dependent upon protein concentration, the concentration dependence of polymerization within the crowded environment of the ER in vivo is not known. Therefore, to determine the critical concentration for the polymerization of Z α1-antitrypsin in living cells, we induced the expression of Z α1-antitrypsin in Tet-On stable CHO stable cells (22) and measured both total α1-antitrypsin and polymer levels (Supplementary Material, Fig. S3A). This revealed that levels of 300 ng α1-antitrypsin per 1 mg of total lysate protein are necessary before intracellular polymers can be detected in these cells (Supplementary Material, Fig. S3A). To test this finding in lung epithelial-derived cells, we induced expression of Z α1-antitrypsin in Tet-On A549 cells with doxycycline and augmented the protein level by inhibiting ERAD with lactacystin, a selective proteasome inhibitor. This increased the concentration of intracellular Z α1-antitrypsin of >300 ng α1-antitrypsin per 1 mg of total lysate, whereupon polymers were detected (Supplementary Material, Fig. S3B). It therefore seems likely that the low levels of α1-antitrypsin produced by airway epithelia are insufficient to generate detectable polymers.Figure 2.

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