α1-Antitrypsin activates protein phosphatase 2A to counter lung inflammatory responses

Abstract

Rationale: α1-Antitrypsin (A1AT) was identified as a plasma protease inhibitor; however, it is now recognized as a multifunctional protein that modulates immunity, inflammation, proteostasis, apoptosis, and cellular senescence. Like A1AT, protein phosphatase 2A (PP2A), a major serine-threonine phosphatase, regulates similar biologic processes and plays a key role in chronic obstructive pulmonary disease.

Objectives: Given their common effects, this study investigated whether A1AT acts via PP2A to alter tumor necrosis factor (TNF) signaling, inflammation, and proteolytic responses in this disease.

Methods: PP2A activity was measured in peripheral blood neutrophils from A1AT-deficient (PiZZ) and healthy (PiMM) individuals and in alveolar macrophages from normal (60 mg/kg) and high-dose (120 mg/kg) A1AT-treated PiZZ subjects. PP2A activation was assessed in human neutrophils, airway epithelial cells, and peripheral blood monocytes treated with plasma purified A1AT protein. Similarly, lung PP2A activity was measured in mice administered intranasal A1AT. PP2A was silenced in lung epithelial cells treated with A1AT and matrix metalloproteinase and cytokine production was then measured following TNF-α stimulation.

Measurements and main results: PP2A was significantly lower in neutrophils isolated from PiZZ compared with PiMM subjects. A1AT protein activated PP2A in human alveolar macrophages, monocytes, neutrophils, airway epithelial cells, and in mouse lungs. This activation required functionally active A1AT protein and protein tyrosine phosphatase 1B expression. A1AT treatment acted via PP2A to prevent p38 and IκBα phosphorylation and matrix metalloproteinase and cytokine induction in TNF-α-stimulated epithelial cells.

Conclusions: Together, these data indicate that A1AT modulates PP2A to counter inflammatory and proteolytic responses induced by TNF signaling in the lung.

Keywords: PP2A; cell signaling; inflammation; phosphorylation; α1-antitrypsin.

Figure 1.

Neutrophils from PiZZ subjects have reduced protein phosphatase 2A (PP2A) activity. Neutrophils were isolated from PiMM and PiZZ α₁-antitrypsin (A1AT) homozygote individuals. Serine-threonine phosphatase activity for PP2A and mitogen-activated protein kinase phosphatase-1 (MKP-1) was determined from each individual and represented as picomoles of phosphate liberated per minute on the y axis. Graphs represent mean ± SEM of three measurements from each subject (n = 9 per group). P values shown, comparing both treatments connected by a line. Representative immunoblots are shown demonstrating equal cellular protein content.

Figure 2.

α₁-Antitrypsin (A1AT) stimulation promotes cellular protein phosphatase 2A (PP2A) activity. (A) Treatment of human peripheral blood neutrophils and monocytes, human small airway epithelial (SAE) cells, and airway epithelial cell line A549 cells with 2 μM A1AT for 24 hours enhances PP2A activity. (B) Oxidized A1AT cannot form complexes with neutrophil elastase (NE) demonstrated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and could not (C) induce a PP2A cellular response in A549 cells. Recombinant/inactive A1AT (rA1AT) also had no effect on PP2A activity. (D) An A1AT dose response stimulation of A549 cells demonstrated that an A1AT concentration of greater than 1 μM significantly increases PP2A activity. (E) A1AT-induced PP2A activity was observed from 1 hour after stimuli with dephosphorylation of PP2A_C greatest 24 hours after stimuli. (F) A1AT dephosphorylated PP2A_C at Tyr site 307 in A549 cells. Densitometry analysis was performed for p-PP2A_C(Tyr307) phosphorylation on immunoblots from 3 separate days and was represented as densitometry units (DU). Graphs represent mean ± SEM of three measurements from each subject (n = 6 per group). P values shown, comparing both treatments connected by a line. *P < 0.05 compared with baseline levels of activity. Representative PP2A_C immunoblots are shown demonstrating equal cellular protein content.

Figure 3.

Protein tyrosine phosphatase 1B (PTP1B) is required for α₁-antitrypsin (A1AT) to activate protein phosphatase 2A (PP2A). Neutrophils isolated from PiZZ A1AT homozygote individuals have (A) less PTP1B activity and (B) greater PP2A_C phosphorylation than PiMM individuals (n = 10 per group). Loss of PTP1B expression in A549 cells prevents A1AT-induced (C) activation of PP2A and (D) dephosphorylation of p-PP2A_C(Tyr307). Densitometry analysis was performed on immunoblots from 3 separate days. Graphs represent mean ± SEM of three measurements from each subject (n = 10 per group). P values shown, comparing both treatments connected by a line. Representative immunoblots are shown demonstrating equal cellular protein content. DU = densitometry units.

Figure 4.

Airway administration of α₁-antitrypsin (A1AT) protein activates protein tyrosine phosphatase 1B (PTP1B) and protein phosphatase 2A (PP2A) in mice. FVB/NJ and Ptp1b^−/− mice were administered either 2 μM of albumin or A1AT intranasally. (A) PP2A and PTP1B activity levels and (B) PP2A_C phosphorylation at Tyr site 307 was examined in mouse lungs. Densitometry analysis was performed for p-PP2A_C(Tyr307) phosphorylation. (C) Neutrophils from Ptp1b^−/− mice were undergoing cell death at a faster rate than wild-type (WT) neutrophils. Graphs represent mean ± SEM of three measurements from each animal (n = 10 per group). P values shown, comparing both treatments connected by a line. DU = densitometry units.

Figure 5.

Protein kinase A (PKA) inhibition prevents α₁-antitrypsin (A1AT) activation of protein tyrosine phosphatase 1B (PTP1B)/protein phosphatase 2A (PP2A). A1AT did not alter PP2A B subunit (A) protein or (B) gene expression in A549 cells. (C) A1AT enhances PP2A activity primarily in the cytoplasm without altering translocation of the protein. (D) A1AT stimuli did not alter binding of the PP2A inhibitor, SET, to PP2A, demonstrated by IP of PP2A_C and probing for SET and PP2A_C. (E) Inhibition of PKA/cAMP activity alters A1AT’s ability to activate PP2A. Graphs represent mean ± SEM of three measurements. Representative immunoblots are shown demonstrating equal cellular protein content. *P < 0.05 for A1AT-treated versus albumin-treated A549 cells. **P < 0.05 for A1AT-treated versus A1AT+PKA inhibitor–treated A549 cells.

Figure 6.

α₁-Antitrypsin (A1AT) from the serum of PiZZ A1AT subjects has decreased antielastase activity and less ability to activate protein phosphatase 2A (PP2A) in cells. (A) A549 cells have increased PP2A activity following treatment with purified A1AT or serum from PiMM individuals but not with serum from PiZZ individuals. Equal A1AT levels were administered in each group. Affinity chromatography was performed to isolate A1AT from PiMM and PiZZ subjects. (B) A1AT levels were examined in both PiMM and PiZZ A1AT isolations by Coomassie blue staining and immunoblots. (C) Antineutrophil elastase activity assays demonstrated that PiZZ A1AT has less antielastase activity and binding potential to neutrophil elastase (NE) than PiMM A1AT. (D) Treatment of A549 cells for 24 hours with 2 μM albumin, commercially available A1AT (com), or A1AT isolated from PiMM subjects enhances protein tyrosine phosphatase 1B (PTP1B) and PP2A activities, unlike PiZZ A1AT protein. The ability of A1AT to increase PP2A activity in cells correlated with its antielastase activity (right). Graphs represent mean ± SEM of three measurements from each subject (n = 10 per group). P values shown, comparing both treatments connected by a line. Representative immunoblots are shown demonstrating equal cellular protein content.

Figure 7.

α₁-Antitrypsin (A1AT) acts via protein phosphatase 2A (PP2A) to inhibit tumor necrosis factor (TNF)-α induced p38 and IκBα phosphorylation and subsequent nuclear factor (NF)-κB activation. A549 cells, which were stably transfected with lentivirus expressing negative control, PP2A_A or protein tyrosine phosphatase 1B (PTP1B) shRNA, were exposed to 2 μM A1AT for 1 hour before stimulation with 10 ng/ml TNF-α (1 h). (A) Immunoblots and densitometry analysis was performed for p38 and IκBα phosphorylation. (B) Transcription factor, NF-κB, and relative activation were examined in each treatment group, compared with negative control shRNA-treated cells. Graphs represent mean ± SEM, where each measurement was performed three times (n = 6 per group). P values shown, comparing both treatments connected by a line. DU = densitometry units. *P < 0.05 compared with A549 cells treated only with TNF-α. **P < 0.05 compared with A1AT+TNF-α–treated control shRNA A549 cells.

Figure 8.

Protein phosphatase 2A (PP2A) activity is required for α₁-antitrypsin (A1AT) to inhibit tumor necrosis factor (TNF)-α induced proteases and cytokines. A549 cells, which were stably transfected with lentivirus expressing control PP2A_A or protein tyrosine phosphatase 1B (PTP1B) shRNA, were exposed to 2 μM A1AT for 1 hour before 10 ng/ml TNF-α stimulation (24 h). Quantitative polymerase chain reaction and multiplex analysis were performed for (A) matrix metalloproteinase (MMP)-1 and (B) MMP-9. (C) Multiplex analysis was performed for IL-8 and monocyte chemotactic protein (MCP) 1 and quantitative polymerase chain reaction was performed for IL-1β. Graphs represent mean ± SEM, where each measurement was performed three times (n = 6 per group). *P < 0.05 compared with A549 cells treated only with TNF-α. **P < 0.05 compared with A1AT+TNF-α–treated control shRNA A549 cells.

Figure 9.

α₁-Antitrypsin (A1AT) augmentation therapy enhances protein phosphatase 2A (PP2A) activity and subdues protease and cytokine production in humans. (A) A1AT-deficient (PiZZ) subjects were administered weekly A1AT intravenously (60 mg/kg for 1 mo, followed by 1 mo of 120 mg/kg weekly, and then returning to 60 mg/kg weekly for 1 mo). Plasma, bronchoalveolar lavage fluid (BALF), BALF cells, and airway epithelial cells were collected on the final day of each month of A1AT therapy (n = 8 subjects). (B) Plasma A1AT levels were observed to be enhanced following 120 mg/kg A1AT treatment, demonstrated by Coomassie blue staining and A1AT immunoblots. (C) BALF matrix metalloproteinase (MMP)-1, -9, and -12 levels were altered following A1AT augmentation therapy. Graphs represent individual subject MMP BALF concentrations. (D) A1AT did not alter protein tyrosine phosphatase 1B (PTP1B), PP2A_A, or PP2A_C expression in airway epithelial cells collected from the patients but subdued IL-8, IL-1β, monocyte chemotactic protein (MCP)-1, and tumor necrosis factor (TNF)-α expression. (E) The 120 mg/kg A1AT treatment induced dephosphorylation of p-PP2A_C(Tyr307) in epithelial cells from patients. (F) Likewise, it enhanced PTP1B and PP2A activity in alveolar macrophages. Graphs represent mean ± SEM, where each measurement was performed three times (n = 8 per group). P values shown, comparing both treatments connected by a line. *P < 0.05.