Oxidative stress triggers Itch-mediated TXNIP degradation and NF-κB activation promoting chronic obstructive pulmonary disease

Abstract

Background: Chronic inflammatory lung diseases, including chronic obstructive pulmonary disease (COPD), are characterized by pulmonary structural changes, narrowing of the small airways, and destruction of the lung parenchyma caused by prolonged inflammation. Sustained inflammation mediated by macrophages is considered to play a critical role in COPD pathogenesis, while the inductive mechanisms of persistent inflammation remain unclear.

Methods: In vitro, RAW264.7 cells were treated with cigarette smoke extract (CSE), hydrogen peroxide, and 12-O-tetradecanoylphorbol-13-acetate. Loss-of-function assays were performed using MAPK inhibitors and Itch-specific knockdown. In vivo, lung tissues from mice exposed to whole-body cigarette smoke (CS) for 12 weeks, as well as clinical samples from healthy non-smokers, a healthy smoker, and COPD patients, were analyzed.

Results: Our findings demonstrated that thioredoxin-interacting protein (TXNIP) participates in CS-induced NF-κB activation in macrophages, which may contribute to pulmonary inflammation. CSE markedly inhibited TXNIP expression in RAW264.7 cells through MAPK-dependent regulation, accompanied by the induction of iNOS/NO and COX-2. The decrease in TXNIP was also detected in lung tissues and macrophages obtained from smoking mice, while higher NF-κB activation and lung inflammation occurred simultaneously. Additionally, CS-induced oxidative stress triggered MAPK-dependent proteasomal degradation of TXNIP, leading to subsequent NF-κB activation. The expression of E3 ligase Itch was elevated in smoking mouse lungs and in hydrogen peroxide-stimulated cells, whereas specific silencing Itch significantly attenuated TXNIP degradation as well as NF-κB activation. Moreover, Itch expression was increased in lung tissues, whereas TXNIP was markedly reduced in lung tissues, bronchoalveolar lavage fluid cells, and peripheral blood mononuclear cells from patients with COPD.

Conclusion: Accordingly, CS-induced oxidative stress promotes Itch-mediated TXNIP degradation, leading to NF-κB-driven inflammation in macrophages and potentially contributing to COPD pathogenesis.

Keywords: COPD; Itch; NF-κB; ROS; TXNIP.

Fig. 1

CSE initiates TXNIP downregulation. A RAW264.7 cells were treated with indicated dosages of CSE for 24 h, the expressions of TXNIP, iNOS, and COX-2 were detected. GAPDH was used as an internal control. B NO release was detected after 24 h CSE exposure. ***p < 0.001 vs. the untreated group. C The expression of phospho-JNK, JNK, phospho-ERK, ERK, phospho-p38, and p38 after CSE exposure were detected by western blot analysis. GAPDH was used as an internal control. D Cells were pretreated with or without SP600125 (SP), SB203580 (SB), and PD98059 (PD) for 1 h followed by CSE exposure for 24 h. The expression of TXNIP and GAPDH was detected. Protein molecular weights are indicated as kilodaltons (kDa)

Fig. 2

Smoking mice present lung inflammation, NF-κB activation, and TXNIP downregulation. A The hematoxylin and eosin (H&E) staining of lung sections from mice with or without a 12-week time course of smoking exposure were shown. Microphotographs are shown at 100× magnification. B Immunofluorescence staining was performed to detect the expression of phospho-NF-κB p65 (Ser536) in lung tissues of normal and smoking mice using specific antibodies followed by Alexa594-conjugated secondary antibodies staining (Red). DAPI staining was used to determine the nuclei. Scale bar is 200 μm. C TXNIP expression in total protein isolated from normal (n = 3) and smoking mouse lung tissues (n = 3) were measured by immunoblotting. β-actin was used as an internal control, and the ratios of TXNIP to β-actin were shown as the means ± SEM. D Lung tissues obtained from normal and smoking mice were stained with TXNIP antibodies followed by Alexa488-conjugated secondary and CD11b-Alexa594-conjugated antibodies. DAPI was used for nuclear staining. The immunofluorescence and differential interference contrast (DIC) images were determined by confocal microscope and shown. Scale bar is 25 μm

Fig. 3

Oxidative stress mediates TXNIP proteasomal degradation. A Immunofluorescence staining was performed to detect the expression of 4-hydroxynonenal (4-HNE) and nitrotyrosine (NitroTyr) in lung tissues of normal and smoking mice respectively using specific antibodies followed by Alexa488-conjugated secondary antibodies staining (Green). DAPI staining was used to determine the nuclei. Scale bar is 200 μm. B The expression of 4-HNE and NitroTyr in total protein isolated from normal (n = 3) and smoking mouse lung tissues (n = 3) were measured, and β-actin was used as an internal control. C) RAW264.7 cells were treated with various dosages of H₂O₂ for indicated time points, and the protein expression of TXNIP and GAPDH were detected. D In the presence of MG132 (10 µM) and lactacystin (10 µM), cells were treated with H₂O₂ (1 mM) for 2 h followed by the determination of TXNIP and GAPDH expression. E RAW264.7 cells were treated with TPA (50 ng/ml) for indicated time points followed by the ROS detection. The relative percentages of ROS production were measured, and shown as the means ± SEM from triplicate cultures. *p < 0.05, **p < 0.01, ***p < 0.001. F TXNIP expression in TPA (50 ng/ml)-treated RAW264.7 cells was detected at indicated time points. G Cells were pretreated with or without NAC (5 mM) for 1 h followed by TPA (50 ng/ml) stimulation for 2 h. TXNIP expression was detected, and GAPDH was used as an internal control. H RAW264.7 cells were exposed to indicated dosages of CSE for 24 h followed by the measurements of LDH release. I In the presence or absence of MG132 (10 µM), lactacystin (10 µM), and NAC (5 mM), cells were exposed to indicated dosages of CSE for 24 h followed by detecting TXNIP expression. GAPDH was used as an internal control. Protein molecular weights are indicated as kilodaltons (kDa)

Fig. 4

MAP-Kinases regulate oxidative stress-mediated TXNIP degradation and NF-κB activation. A The nuclear translocation of NF-κB in H₂O₂-treated RAW264.7 cells were assayed at indicated time points using a specific antibody against NF-κB (Green) followed by fluorescence microscopic observation. DAPI was used as a nuclear staining, and scale bar was shown. Quantitative measurements of NF-κB nuclear translocation (percentages based on more than 150 cells in total) were performed and shown as the means ± SEM of triplicate cultures. **p < 0.01. B RAW264.7 cells were treated with H₂O₂ (1 mM) for 4 h and subsequently stained with TXNIP (Red) and NF-κB (Green) respectively. DAPI was used as a nuclear staining, and scale bar was shown. C RAW264.7 cells were treated with different dosages of H₂O₂ for indicated time points, the expressions of TXNIP, phospho-JNK (Thr183/Tyr185), JNK, phospho-ERK (Thr202/Tyr204), ERK, phospho-p38 (Thr180/Tyr182), and p38 were determined. GAPDH was used as an internal control. D RAW264.7 cells were preincubated with 25 µM of SP600125 (SP), PD98059 (PD), and SB 253580 (SB) for 1 h followed by 1 mM of H₂O₂ treatment for 2 h. The expression of TXNIP and GAPDH were measured. Protein molecular weights are indicated as kilodaltons (kDa). E The luciferase reporter assay of NF-κB was measured after 1 mM of H₂O₂ treatment for 4 h in the presence or absence of inhibitors. Triplicate cultures were performed and shown as the means ± SEM. **p < 0.01 as compared to control; ^## p < 0.01 and ^#p < 0.05 as compared to H₂O₂ groups

Fig. 5

Itch expression modulates TXNIP-mediated NF-κB activation. A Itch expression in total protein isolated from normal (n = 3) and smoking mouse lung tissues (n = 3) were measured by immunoblotting. β-actin was used as an internal control, and the ratios of Itch to β-actin were shown as the means ± SEM. B RAW264.7 cells were stimulated with CSE for 2 h, and Itch expression was subsequently measured. GAPDH was used as an internal control. C The expression of Itch and GAPDH were measured in RAW 264.7 cells treated with H₂O₂ (1 mM) and TPA (50 ng/ml) for 2 h. D In the presence of 5 mM NAC, the expression of Itch and GAPDH were detected in cells treated with TPA (50 ng/ml) for 2 h. E Cells were pretreated with or without SP600125 (SP), SB203580 (SB), and PD98059 (PD) for 1 h followed by H₂O₂ treatment for 2 h. The expression of Itch and GAPDH was detected. F In the presence or absence of MG132 (10 µM), and the expression of phospho-NF-κB (Ser536), NF-κB, and GAPDH were detected in cells treated with H₂O₂ (1 mM) for 4 h. G RAW264.7 cells were expressed with shRNA targeting luciferase (shLuc) and shRNA targeting Itch (shItch#1, shItch#2, and shItch#3), and the expression of Itch and GAPDH were subsequently confirmed by immunoblotting. H Cells expressed shLuc and shItch#1 were treated with or without H₂O₂ for 2 h. The expression of Itch, TXNIP, and GAPDH were detected. Protein molecular weights are indicated as kilodaltons (kDa). I The luciferase reporter assay of NF-κB was measured in shLuc- and shItch-cells stimulated with H₂O₂ (1 mM) for 4 h. The quantitative data are presented as the means ± SEM from triplicate cultures. ***p < 0.001 as compared to untreated. ^###p < 0.001 as compared to the shLuc-group

Fig. 6

Decreased expression of TXNIP in COPD patients. A Gene expression of TXNIP (GDS3496/201010_s_at) and ITCH (GDS3496/217094_s_at) in alveolar macrophages of cigarette smokers obtained from the public GEO profiles was assayed. Gene expression of TXNIP (GDS737/201010_s_at) and ITCH (GDS737/209743_s_at) in smoker lung tissues with no/mild and severe emphysema obtained from the public GEO profiles was assayed. Gene expression of TXNIP (GDS2468/201010_s_at) and ITCH (GDS2468/217094_s_at) in small airway epithelium of cigarette smokers obtained from the public GEO profiles was assayed. The relative fold changes were shown as means ± SEM. *p < 0.05, **p < 0.01. Immunohistochemical staining was performed using specific antibodies to detect the expression of TXNIP (B) and Itch (C) in the fixed lung tissue sections obtained from COPD patients. Normal controls were obtained from the noninvolved lung sections of the tumor lesion. Microphotographs are shown at 200× and enlarged magnification. The scale bar is shown as 30 μm. D Bronchoalveolar lavage fluid (BALF) cells were collected from donors with or without COPD, and subsequently performed the immunoblotting to measure TXNIP expression. GAPDH was used as an internal control. E Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors (n = 2), smokers (n = 1), mild COPD patients (n = 1), and severe COPD patients (n = 2). TXNIP expression was detected and β-actin was used as an internal control. Protein molecular weights are indicated as kilodaltons (kDa)

Fig. 7

A schematic model of TXNIP downregulation promoting NF-κB-mediated inflammation in response to CS stimulation. CS contains thousands of chemicals, including abundant free radicals. Oxidative stress initiates the activation of ERK, JNK, and p38, thereby leading to HECT-type E3 ubiquitin ligase Itch-regulated TXNIP degradation. The downregulation of TXNIP may effectively enable NF-κB activation and the subsequent induction of proinflammatory iNOS/NO and COX-2. Therefore, sustained exposure to CS and oxidative stress may potentially cause TXNIP downregulation and chronic inflammatory responses, potentiating the progression of COPD