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ORIGINAL ARTICLE
Year : 2018  |  Volume : 131  |  Issue : 16  |  Page : 1917-1925

Follistatin-Like 1 Promotes Bleomycin-Induced Pulmonary Fibrosis through the Transforming Growth Factor Beta 1/Mitogen-Activated Protein Kinase Signaling Pathway


1 Department of Respiratory and Critical Care Medicine, Beijing Chao-Yang Hospital, Capital Medical University; Department of Respiratory Disease, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Capital Medical University; Department of Respiratory Disease, Capital Medical University, Beijing 100020, China
2 Department of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China
3 Department of Respiratory Disease, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Capital Medical University; Department of Physiology and Pathophysiology, Capital Medical University, Beijing 100069, China
4 Department of Respiratory Disease, Capital Medical University, Beijing 100020, China
5 Department of Respiratory Disease, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Capital Medical University; Department of Respiratory Disease, Capital Medical University, Beijing 100020, China

Correspondence Address:
Prof. Chen Wang
Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Capital Medical University, Beijing 100020
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0366-6999.238151

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Background: Follistatin-like 1 (FSTL1) is a novel profibrogenic factor that induces pulmonary fibrosis (PF) through the transforming growth factor-beta 1 (TGF-β1)/Smad signaling. Little is known about its effects on PF through the non-Smad signaling, like the mitogen-activated protein kinase (MAPK) pathway. Therefore, this study aimed to investigate the role of FSTL1 in PF through the MAPK signaling pathway and its mechanisms in lung fibrogenesis. Methods: PF was induced in Fstl1+/−and wild-type (WT) C57BL/6 mice with bleomycin. After 14 days, the mice were sacrificed, and lung tissues were stained with hematoxylin and eosin; the hydroxyproline content was measured to confirm PF. The mRNA and protein level of FSTL1 and the change of MAPK phosphorylation were measured by quantitative polymerase chain reaction and Western blotting. The effect of Fstl1 deficiency on fibroblasts differentiation was measured by Western blotting and cell immunofluorescence. MAPK signaling activation was measured by Western blotting in Fstl1+/− and WT fibroblasts treated with recombinant human FSTL1 protein. We pretreated mouse lung fibroblast cells with inhibitors of the extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) signaling and analyzed their differentiation, proliferation, migration, and invasion by Western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, and transwell assays. The Student's t-test was used to compare the differences between two groups. Results: Fstl1 deficiency attenuated phosphorylation of the ERK, p38, and JNK signaling in bleomycin-induced fibrotic lung tissue 14 days after injury (0.67 ± 0.05 vs. 1.22 ± 0.03, t = 14.92, P = 0.0001; 0.41 ± 0.01 vs. 1.15 ± 0.07; t = 11.19; P = 0.0004; and 0.41 ± 0.01 vs. 1.07 ± 0.07, t = 8.92, P = 0.0009; respectively), compared with WT lungs at the same time and in primary lung fibroblasts (0.82 ± 0.01 vs. 1.01 ± 0.04, t = 4.06, P = 0.0150; 1.04 ± 0.03 vs. 1.24 ± 0.03, t = 4.44, P = 0.0100; and 0.76 ± 0.05 vs. 0.99 ± 0.05, t = 4.48, P = 0.0100; respectively), compared with TGF-β1-stimulated WT group. Recombinant human FSTL1 protein in lung fibroblasts enhanced TGF-β1-mediated phosphorylation of the ERK (1.19 ± 0.08 vs. 0.55 ± 0.04, t = 6.99, P = 0.0020), p38 (1.18 ± 0.04 vs. 0.66 ± 0.03, t = 11.20, P = 0.0020), and JNK (1.11 ± 0.01 vs. 0.84 ± 0.04, t = 6.53, P = 0.0030), compared with the TGF-β1-stimulated WT group. Fstl1-deficient fibroblasts showed reduced alpha-smooth muscle actin (α-SMA) expression (0.70 ± 0.06 vs. 1.28 ± 0.11, t = 4.65, P = 0.0035, compared with the untreated WT group; 1.40 ± 0.05 vs. 1.76 ± 0.02, t = 6.31, P = 0.0007; compared with the TGF-β1-treated WT group). Compared with the corresponding condition in the control group, the TGF-β1/FSTL1-mediated α-SMA expression was significantly suppressed by pretreatment with an inhibitor of p38 (0.73 ± 0.01 vs. 1.13 ± 0.10, t = 3.92, P = 0.0078) and JNK (0.78 ± 0.03 vs. 1.08 ± 0.06, t = 4.40, P = 0.0046) signaling. The proliferation of mouse lung fibroblast cells (MLgs) significantly decreased after treatment of an inhibitor of p38 (0.30 ± 0.01 vs. 0.46 ± 0.03, t = 4.64, P = 0.0009), JNK (0.30 ± 0.01 vs. 0.49 ± 0.01, t = 12.84, P = 0.0001), and Smad2/3 (0.18 ± 0.02 vs. 0.46 ± 0.02, t = 12.69, P = 0.0001) signaling compared with the dimethylsulfoxide group. The migration and invasion cells of MLgs significantly decreased in medium pretreated with an inhibitor of p38 (70.17 ± 3.28 vs. 116.30 ± 7.11, t = 5.89, P = 0.0042 for the migratory cells; 19.87 ± 0.84 vs. 32.70 ± 0.95, t = 10.14, P = 0.0005 for the invasive cells), JNK (72.30 ± 3.85 vs. 116.30 ± 7.11, t = 5.44, P = 0.0056 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ± 0.95, t = 11.00, P = 0.0004 for the invasive cells), and Smad2/3 (64.76 ± 1.41 vs. 116.30 ± 7.11, t = 7.11, P = 0.0021 for the migratory cells; 18.03 ± 0.94 vs. 32.70 ± 0.95, t = 13.29, P = 0.0002 for the invasive cells) signaling compared with the corresponding condition in the dimethylsulfoxide group. Conclusion: FSTL1 affects lung fibroblast differentiation, proliferation, migration, and invasion through p38 and JNK signaling, and in this way, it might influence the development of PF.

 

 Abstract in Chinese

FSTL1通过TGF-β1/MAPK信号通路影响肺纤维化发生发展

摘要

背景:卵泡抑素样蛋白1(Follistatin-like 1, FSTL1)具有促纤维化作用,能够通过转化生长因子β1(TGF-β1)/Smad信号通路诱发肺纤维化。但FSTL1是否也能通过非Smad信号通路,如丝裂原活化蛋白激酶(MAPK)信号通路,影响肺纤维化发生发展,目前鲜有报道。基于此,本研究的目的是探讨FSTL1是否通过TGF-β1/MAPK信号通路影响肺纤维化发生、发展的影响及其可能的作用机制。

方法:Fstl1+/-和野生型C57BL/6小鼠中,气管内注入博来霉素建立小鼠肺纤维化模型;对照组气管内注入生理盐水。14天后,取肺组织,用HE染色及羟脯氨酸含量测定方法验证纤维化模型是否构建成功。用qPCR和Western blotting的方法分别检测小鼠肺组织内FSTL1的mRNA和蛋白水平变化,以及MAPK信号通路磷酸化水平变化。从Fstl1+/-和野生型小鼠肺组织中分离原代肺成纤维细胞,对比两组MAPK信号通路磷酸化水平变化。用Western blotting和细胞荧光免疫的方法观察肺成纤维细胞向肌成纤维细胞转分化情况及FSTL1缺失对其分化的影响。通过Western blotting方法观察Fstl1+/-小鼠成纤维细胞和在野生型同窝小鼠成纤维细胞中,外源加入FSTL1蛋白后,MAPK信号通路磷酸化水平变化。在小鼠肺成纤维细胞系(MLgs)中,分别加入ERK、p38及JNK的抑制剂U0126、SB202190及SP600125后,运用Western blotting、MTT法和Transwell小室分别观察MLgs的分化、增殖、迁移和侵袭情况。两组数据间差异用t检验进行分析。

结果:博来霉素处理14天后,与对照组相比,FSTL1的缺失能减弱肺组织中ERK (0.67 ± 0.05 vs 1.22 ± 0.03; t = 14.92; P = 0.0001),p38 (0.41 ± 0.01 vs 1.15 ± 0.07; t = 11.19; P = 0.0004),JNK (0.41 ± 0.01 vs 1.07 ± 0.07; t = 8.92; P = 0.0009)信号通路的磷酸化水平。在细胞学实验中,与TGF-β1刺激的原代小鼠成纤维细胞相比,FSTL1的缺失减弱ERK,p38,JNK信号通路的磷酸化水平(分别减弱0.82 ± 0.01 vs 1.01 ± 0.04; t = 4.06; P = 0.0150; 1.04 ± 0.03 vs 1.24 ± 0.03; t = 4.44; P = 0.0100; 0.76 ± 0.05 vs 0.99 ± 0.05; t = 4.48; P = 0.0100);与单纯使用TGF-β1刺激MLgs相比,外源加入FSTL1蛋白后能增强MLgs中ERK (1.19 ± 0.08 vs 0.55 ± 0.04; t = 6.99; P = 0.0020), p38 (1.18 ± 0.04 vs 0.66 ± 0.03; t = 11.2; P = 0.0020), JNK (1.11 ± 0.01 vs 0.84 ± 0.04; t = 6.53; P = 0.0030)信号通路的磷酸化水平。FSTL1的缺失可减少α-SMA的表达 (0.70 ± 0.06 vs 1.28 ± 0.11; t = 4.65; P = 0.0030,与对照组相比;1.40 ± 0.05 vs 1.76 ± 0.02; t = 6.31; P = 0.0007,与TGF-β1组相比)。与DMSO组中相对应组别相比,p38及JNK信号通路抑制剂可抑制肌成纤维细胞的分化(分别为0.73 ± 0.01 vs 1.13 ± 0.10; t = 3.92; P = 0.0078和0.78 ± 0.03 vs 1.08 ± 0.06; t = 4.40; P = 0.0046) ;还能抑制MLgs细胞系的增殖p38 (0.30 ± 0.01 vs 0.46 ± 0.03; t = 4.64; P = 0.0009), JNK (0.30 ± 0.01 vs 0.49 ± 0.01; t = 12.84; P = 0.0001), Smad2/3 (0.18 ± 0.02 vs 0.46 ± 0.02; t = 12.69; P = 0.0001)、迁移p38 (70.17 ± 3.28 vs 116.30 ± 7.11; t = 5.89; P = 0.0042), JNK (72.30 ± 3.85 vs 116.30 ± 7.11; t = 5.44; P = 0.0056), Smad2/3 (64.76 ± 1.41 vs 116.30 ± 7.11; t = 7.11; P = 0.0021)及侵袭p38 (19.87 ± 0.84 vs 32.70 ± 0.95; t = 10.14; P = 0.0005), JNK (18.03 ± 0.94 vs 32.70 ± 0.95; t = 11.00; P = 0.0004), Smad2/3 (18.03 ± 0.94 vs 32.70 ± 0.95; t = 13.29; P = 0.0002)能力。

结论:FSTL1可能通过TGF-β1/p38/JNK信号通路影响肺成纤维细胞的分化、增殖、迁移和侵袭能力ʌ



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