Department of Internal Medicine, SMG-SNU Boramae Medical Center, Seoul 07061, Korea.
Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Seoul National University Hospital, Seoul 03080, Korea.
Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Korea.
Department of Convergence Medicine, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul 05505, Korea.
Abstract
Cereblon (CRBN) has been shown to play an essential role in regulating inflammatory response and endoplasmic reticulum stress, thus mediating the development of various diseases. However, little is known about the roles of CRBN in chronic obstructive pulmonary disease (COPD) pathogenesis. We found that the protein levels of CRBN in lung homogenates from patients with COPD were lower than those from never smokers and smokers. The CRBN protein level was positively correlated with the forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC). To investigate the role of CRBN in modulating elastase-induced emphysema, we used Crbn knockout (KO) mice. Elastase-induced emphysematous changes were significantly aggravated in Crbn KO mice. Neutrophil infiltration, lung cell injury, and protein leakage into the bronchoalveolar space were more severe in Crbn KO mice than in wild-type (WT) mice. Furthermore, Crbn KO resulted in the elevated release of neutrophilic chemokines and inflammatory cytokines in lung epithelial cells and macrophages. The transcriptional activity of nuclear factor-κB (NF-κB) was significantly increased in Crbn knocked-down cells. In conclusion, Crbn deficiency might be involved in the development of emphysema by enhancing NF-κB activation, suggesting that targeting CRBN might be an effective therapeutic approach for the treatment of COPD.
Keywords
NF-κBcereblonemphysemalung inflammation
Figures
Figure 1The level of CRBN expression was decreased in lung tissues of patients with COPD and correlated with the FEV1/FVC ratio. (a) Lung homogenates from never smokers (n = 8), smokers (n = 8), and patients with COPD (n = 8) were subjected to Western blot analysis for CRBN and GAPDH. (b) Gel data were quantified using Scion image densitometry. Data represent the mean ± SE. * p < 0.05, ** p < 0.01. (c–k) Pearson correlation coefficient, r, was calculated for the expression level of CRBN (y-axis) and clinical parameters (x-axis) of 24 subjects.Figure 2Crbn KO exaggerated elastase-induced emphysema in mice. WT and Crbn KO mice were given 0.5 units of elastase via intratracheal injection. (Day 14: WT-saline n = 5, WT-elastase n = 5, KO-saline n = 4, KO-elastase n = 3) (a) RT-PCR for Crbn and Gapdh in the lung tissues from WT and Crbn KO BALB/c mice. (b) Effect of Crbn deficiency on elastase-induced emphysema formation. Representative images of H&E staining in lungs from WT and Crbn KO mice 14 days after intratracheal instillation of elastase (×40). (c) The MLI was measured. Data represent means ± SE. * p < 0.05, and *** p < 0.001.Figure 3Crbn KO increased elastase-induced inflammation and cellular injury. WT and Crbn KO mice were given 0.5 units of elastase via intratracheal injection. (Day 2: WT-saline n = 5, WT-elastase n = 5, KO-saline n = 5, KO-elastase n = 5, Day 14: WT-saline n = 5, WT-elastase n = 5, KO-saline n = 4, KO-elastase n = 3). (a) Representative images of H&E staining in lungs from WT and Crbn KO mice on day 2 (d2) and day 14 (d14) after intratracheal instillation of elastase (×100). (b,c) BALF was collected on days 2 and 14 after intratracheal instillation of elastase. Total cells (b) and differential cells (c) including macrophages and neutrophils in BALF were counted. Data represent the mean ± SE. The concentrations of LDH (d) and protein (e) in BALF were measured. (f–i) Real-time PCR analysis of KC, TNF-α, IL-6, IL-1β, and GAPDH expression in the lung tissues of WT and Crbn KO mice. (j-m) The concentrations of KC, TNF-α, IL-6, and IL-1β were measured. Data represent the mean ± SE. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.Figure 4Crbn KO enhanced the production of inflammatory cytokines/chemokines in lung epithelial cells and macrophages. (a,b) BEAS-2B cells were transiently transfected with control siRNA or Crbn siRNA. Forty-eight hours after transfection, the cells were treated with VC or NE (1 U/mL) (a) and CSE (1%, v/v) (b) for 24 h. The concentration of IL-8 in cell supernatants was measured with an ELISA. Data represent the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 (c–f) WT and Crbn KO BMDMs were treated with VC or NE (1 U/mL) for 24 h. (g-j) WT and Crbn KO BMDMs were treated with CSE (1%) for 24 h. (k–n) WT and Crbn KO BMDMs were treated with LPS (100 ng/mL) for 6 or 24 h. The levels of inflammatory cytokines/chemokines in cell culture media were measured with an ELISA. Data represent the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.Figure 5Crbn KO enhanced inflammatory cytokine/chemokine production by increasing NF-κB activation. (a,b) BEAS-2B cells were transiently transfected with control siRNA or Crbn siRNA. Forty-eight hours after transfection, the cells were treated with VC or NE (1 U/mL) (a) and CSE (1%) (b) for the indicated times. Total cell lysates were subjected to Western blot analysis for IκBα, p-SAPK/JNK, total SAPK/JNK, p-p38, total p38, p-ERK, total ERK, CRBN, and GAPDH. (c) WT and Crbn KO BMDMs were stimulated with LPS (100 ng/mL) for the indicated times. Total cell lysates were subjected to Western blot analysis for IκBα and GAPDH. (d) BEAS-2B cells were co-transfected with the NF-κB-luciferase reporter construct, control plasmid, control siRNA, and Crbn siRNA using the Neon electroporation kit. Forty-eight hours after transfection, cells were treated with CSE (1%) for 24 h. Luciferase activity was detected and normalized with Renilla activity. Data represent the mean ± SD. ** p < 0.01, and *** p < 0.001.Figure 6Crbn KO increased elastase-induced oxidative damage. WT and Crbn KO mice were given 0.5 units elastase via intratracheal injection. (Day 2: WT-saline n = 5, WT-elastase n = 5, KO-saline n = 5, KO-elastase n = 5). Representative images of 8-OHdG (a) and 4HNE (c) immunohistochemical staining in lungs from WT and Crbn KO mice on day 2 (d2) after intratracheal instillation of elastase. (b,d) Quantity of 8-OHdG positive cells and 4HNE positive cells in ×100 field of the whole group. Data represent the mean ± SE. * p < 0.05, ** p < 0.01, and *** p < 0.001, n.s., not significant.Figure 7Crbn KO increased elastase-induced MMP9 expression. WT and Crbn KO mice were given 0.5 units of elastase via intratracheal injection. (Day 2: WT-saline n = 5, WT-elastase n = 5, KO-saline n = 5, KO-elastase n = 5). (a) Representative images of MMP9 immunohistochemical staining in lungs from WT and Crbn KO mice on day 2 (d2) after intratracheal instillation of elastase (×40). (b) Quantity of MMP9 positive cells in ×100 field of the whole group. Data represent the mean ± SE. * p < 0.05, and *** p < 0.001. (c) BEAS-2B cells were transiently transfected with control siRNA or Crbn siRNA. Forty-eight hours after transfection, the cells were treated with VC or NE (1 U/mL) for 24 h. (d) WT and Crbn KO BMDMs were treated with LPS (100 ng/mL) for 24 h. Total cell lysates were subjected to Western blot analysis for MMP9, CRBN, and GAPDH.Figure 8Proposed mechanism for the role of CRBN on the development of emphysema. Crbn deficiency enhances elastase-, CSE-, or LPS-induced inflammation, oxidative damage, and MMP9 expression in lung epithelial cells and macrophages. However, cellular aging and apoptosis in lung epithelial cells and the surface expression levels of scavenger receptors such as MARCO and SR-A in macrophages were not affected by CRBN expression. Increased inflammation, oxidative damage, and MMP9 expression induced by Crbn deficiency might augment the development of emphysema.
그림 출처: PubMed Central 공개 논문 (PMC9598989)
Conflict of interest statement
The authors declare that there is no conflict of interest regarding the publication of this paper.
