Splicing factor SRSF6 mediates pleural fibrosis
Li-Mei Liang, Liang Xiong, Pei-Pei Cheng, Shuai-Jun Chen, Xiao Feng, Ya-Ya Zhou, Qian Niu, Meng Wang, Qianlan Chen, Lin-Jie Song, Fan Yu, Xin-Liang He, Fei Xiang, Xiaorong Wang, Hong Ye, Wan-Li Ma
Li-Mei Liang, Liang Xiong, Pei-Pei Cheng, Shuai-Jun Chen, Xiao Feng, Ya-Ya Zhou, Qian Niu, Meng Wang, Qianlan Chen, Lin-Jie Song, Fan Yu, Xin-Liang He, Fei Xiang, Xiaorong Wang, Hong Ye, Wan-Li Ma
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Research Article Pulmonology

Splicing factor SRSF6 mediates pleural fibrosis

Abstract

Pleural fibrosis is defined as an excessive deposition of extracellular matrix that results in destruction of the normal pleural tissue architecture and compromised function. Tuberculous pleurisy, asbestos injury, and rheumatoid pleurisy are main causes of pleural fibrosis. Pleural mesothelial cells (PMCs) play a key role in pleural fibrosis. However, detailed mechanisms are poorly understood. Serine/arginine-rich protein SRSF6 belongs to a family of highly conserved RNA-binding splicing-factor proteins. Based on its known functions, SRSF6 should be expected to play a role in fibrotic diseases. However, the role of SRSF6 in pleural fibrosis remains unknown. In this study, SRSF6 protein was found to be increased in cells of tuberculous pleural effusions (TBPE) from patients, and decellularized TBPE, bleomycin, and TGF-β1 were confirmed to increase SRSF6 levels in PMCs. In vitro, SRSF6 mediated PMC proliferation and synthesis of the main fibrotic protein COL1A2. In vivo, SRSF6 inhibition prevented mouse experimental pleural fibrosis. Finally, activated SMAD2/3, increased SOX4, and depressed miRNA-506-3p were associated with SRSF6 upregulation in PMCs. These observations support a model in which SRSF6 induces pleural fibrosis through a cluster pathway, including SRSF6/WNT5A and SRSF6/SMAD1/5/9 signaling. In conclusion, we propose inhibition of the splicing factor SRSF6 as a strategy for treatment of pleural fibrosis.

Authors

Li-Mei Liang, Liang Xiong, Pei-Pei Cheng, Shuai-Jun Chen, Xiao Feng, Ya-Ya Zhou, Qian Niu, Meng Wang, Qianlan Chen, Lin-Jie Song, Fan Yu, Xin-Liang He, Fei Xiang, Xiaorong Wang, Hong Ye, Wan-Li Ma

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Figure 5

SMAD2/3 and SOX4 upregulated SRSF6 expression in PMCs.

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SMAD2/3 and SOX4 upregulated SRSF6 expression in PMCs. (A–F) Human PMCs ...
(A–F) Human PMCs were incubated with or without TBPE (5%), bleomycin (0.2 μg/mL), and TGF-β1 (5 ng/mL) for 24 hours, after which intracellular protein levels of p-SMAD2/3 (phosphorylated), t-SMAD2/3 (total), and SOX4 were measured by Western blotting (A, C, and E). Bar graphs revealed changes in the relative ratio of p-SMAD2/3 to t-SMAD2/3 and GAPDH, or SOX4 to GAPDH (B, D, and F). (B) n = 6; (D) n = 4; and (F) n = 5 (p-SMAD2/3), n = 6 (SOX4). (G and H) PMCs were treated with bleomycin (0.2 μg/mL) in the presence or absence of SB413542 (10 ng/mL) for 24 hours, after which intracellular protein levels were measured by Western blotting. n = 4. (I) A schematic diagram illustrating the 2 putative SOX4 binding sites in the SRSF6 promoter. (J and K) ChIP assay of PMCs. Binding of SOX4 to the 2 sites was confirmed by PCR (J) and qPCR (K) with primers specific for the 2 sites, n = 4. (L) ChIP assay of PMCs treated with or without TBPE (5%), bleomycin (0.2 μg/mL), and TGF-β1 (5 ng/mL) for 24 hours. SOX4 binding to site 1 was confirmed by qPCR with specific primers, n = 4. Data are shown as mean ± SEM of n individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P values were determined by Student’s t test (B, D, F, and K) or 1-way ANOVA (H and L).