MLN4924 protects against bleomycin-induced pulmonary fibrosis by inhibiting the early inflammatory process

Abstract

Pulmonary fibrosis is a complex pathological process characterized by massive destruction of the structure of lung tissues and aggravated pulmonary function impairment. The underlying mechanisms of pulmonary fibrosis are incompletely understood and therefore limited treatment options are available currently. Here, we report that MLN4924, an NEDD8 activation enzyme (NAE) activity-inhibiting molecule, blocks the maintenance and progression of established pulmonary fibrosis. We found that MLN4924 acts against bleomycin-induced pulmonary fibrosis mainly at the early inflammatory stage. Pharmacologically targeting the neddylation of Cullin-Ring E3 ligase (CRL) by MLN4924, significantly abrogated NF-κB responses, suppressed MAPK activity, and reduced secretion of TNF-α-elicited pro-inflammatory cytokines and MCP1-induced chemokines. MLN4924 inhibited pro-inflammatory responses while maintaining or increasing the production of the anti-inflammatory mediators such as anti-inflammatory interleukins (ILs) following bleomycin administration, which is closely correlated to its blocking NF-κB-mediated signaling. Consistently, our studies identified MLN4924 as a promising therapeutic drug for pulmonary fibrosis and suggested a potential role of MLN4924 that fine tunes the MAPK signaling pathway controlling the inflammatory reactions at the early stages of pulmonary fibrosis. In addition, our findings may broaden the potential practical application of MLN4924 as an effective therapeutic strategy against other inflammation-associated diseases.

Keywords: MLN4924; bleomycin; inflammation; pulmonary fibrosis.

Figure 1

MLN4924 ameliorates bleomycin-induced pulmonary fibrosis. (A) Representative histology of HE and Masson’s trichrome staining of lungs from mice treated with one intratracheal dose of PBS or bleomycin (15 mg/kg), or with a intraperitoneal injection of MLN4924 (10 mg/kg) after bleomycin treatment, respectively (n=8). (B) Fibrosis scores based on histopathological assessment as in (A). (C, D) Hydroxyproline content and relative mRNA expression of mice collagen 1 and collagen 3 from lungs treated as in (A), respectively. Error bars represent the mean ± SD, representative of three experiments, *P < 0.05. (E) Western blotting of A549 cell lines treated with TGF (5 ng/ml) and MLN4924 (0.5 μM or 1 μM) for the indicated times, respectively.

Figure 2

MLN4924 ameliorates LPS-induced acute lung injury. (A) Representative histology of HE staining of lungs from mice treated with a single intratracheal dose of PBS or LPS (5 mg/kg), or with intraperitoneal injection of MLN4924 (10 mg/kg) after LPS treatment, respectively (n=8). (B) Determination of lung wet-to-dry ratio for mice treated as in (A). (C) Protein concentrations of broncho alveolar lavage fluid in lungs of mice treated as in (A). (D-F) Relative mRNA levels of IL-6, IL-1β and TNFα in lung lysates of mice treated as in (A). (G-I) Total cell counts and neutrophil and macrophage numbers in the broncho alveolar lavage fluid of lungs in mice treated with PBS or MLN4924 after LPS intratracheal injection. Error bars represent the mean ± SD, representative of three experiments, *P < 0.05.

Figure 3

MLN4924 inhibits the expression of pro-inflammatory cytokines and chemokines in vitro. A, B. Determination of iNOS mRNA expression and NO production in LPS (1 μg/ml)-treated RAW264.7 cells, together with MLN4924 (0.5 μM) or PBS treatment for 24 hrs. C-E. Relative mRNA levels of IL-1β, TNFα and MCP-1 in LPS (1 μg/ml)-treated RAW264.7 cells, together with MLN4924 (0.5 μM) or PBS treatment for 12 hrs. F-H. Relative mRNA levels of IL-8, CXCL5 and KC in an A549 cell line treated with IL-1β (10 ng/ml) and MLN4924 at the indicated doses for 24 hrs. Error bars represent the mean ± SD, representative of three experiments, *P < 0.05.

Figure 4

MLN4924 functions through the NF-κB signaling pathway. A, B. Western blotting analysis of NF-κB and MAPK signaling in RAW264.7 cells under LPS (1 μg/ml) and MLN4924 treatment at the indicated doses. C, D. Western blotting analysis of NF-κB and MAPK signaling in an A549 cell line under IL-1β (10 ng/ml) and MLN4924 treatment at the indicated doses.

Figure 5

Graphic abstract of MLN4924 protects against bleomycin-induced pulmonary fibrosis by inhibiting the early inflammatory process. When lung injury occurs, the airway epithelia firstly detect the injury and recruit inflammatory cells by secreting chemokines, such as KC, IL-8 and CXCL5. Recruited inflammatory cells, including macrophages and neutrophils, trigger inflammation by producing NO and several cytokines, including IL-6, IL-1β and TNFα, and thus contributing to acute lung injury. When the injury proceeds, extracellular matrix (ECM) begins to form and deposit, which at last leads to pulmonary fibrosis. MLN4924 can effectively block the production of chemokines from the airway epithelia as well as NO and cytokines from the inflammatory cells. By this mechanism MLN4924 suppresses the recruitment of inflammatory cells and the initiation of inflammation responses.