Follistatin-Like 1 Promotes Bleomycin-Induced Pulmonary Fibrosis through the Transforming Growth Factor Beta 1/Mitogen-Activated Protein Kinase Signaling Pathway
Authors: Chen Wang
Wei Zhang
Xiao-He Li
Jie Liu
Yan-Kun Jin
Hua-Ping Dai
Zhi-Fei Zhang
Wang Wang
Yin-Shan Fang
Wen Ning
Journal: Chinese medical journal (2018)
Discipline: CLINICAL MEDICINE
Keywords: Follistatin-Like 1; Mitogen-Activated Protein Kinase; Pulmonary Fibrosis; Transforming Growth Factor Beta 1.
Affiliations: 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., Department of Life Sciences, State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China., 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., Department of Respiratory Disease, Capital Medical University, Beijing 100020, China., 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.

Abstract:  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. 

Retracted: No

PubPeer comments:
 There are identical images for two different experiments in Figure 4. Could the authors please explain? ![file] , 
 We would like to thank Dr Rubioides for noting that the upper panel of Fig4C contains the same images. We have carefully checked our original images. We found that the image of FSTL1 DMSO is one of the 10 pictures collected from the FSTL1 DMSO treated cells (please see attached original images, the 5th image), indicating that the image of FSTL1 DMSO is a correct one. However, the image of TGF-b1 U0126 was not among these 10 pictures, showing that we reused an incorrect image by mistake. Ten images of TGF-b1 U0126 are attached. The authors will contact the editors of Chinese Medical Journal for corrigendum and will provide an updated version (the 8th image from the 10 original images). We thank you again for pointing out this mistake. ![file] ![file] , 
 Fig. 2b: There are some strange background signal with white edges around the bands of JNK panel. Could the author check or show the original data, pleaseuff1f ![file] , 
 I am the first author of the paper. We would like to thank Dr.u00a0Lacunalis for pointing outu00a0the white edges around the bands of JNK panelu00a0of Fig2B. We have checked our original imagesu00a0of JNK panels. We found that there are no white edges around the bands. We think that these white edges appear because the images have been sharpened when we made the illustration. , 
 #4 It would be appreciated if you would post the original images here. , 
 Original images of JNK panels are as below: ![file] ![file] , 
 Thanks for providing original blotting images. However, the p-JNK bands in the image your provided are obviously different from the bands in the published article. ![file] The p-JNK blot looks more identical as your original JNK blot, only with longer exposure time. So you probably need keep digging your raw data to find the correct one. Either way, more explanation are needed here. ,