Transcriptome network analysis identifies protective role of the LXR/SREBP-1c axis in murine pulmonary fibrosis
Shigeyuki Shichino, Satoshi Ueha, Shinichi Hashimoto, Mikiya Otsuji, Jun Abe, Tatsuya Tsukui, Shungo Deshimaru, Takuya Nakajima, Mizuha Kosugi-Kanaya, Francis H.W. Shand, Yutaka Inagaki, Hitoshi Shimano, Kouji Matsushima
Shigeyuki Shichino, Satoshi Ueha, Shinichi Hashimoto, Mikiya Otsuji, Jun Abe, Tatsuya Tsukui, Shungo Deshimaru, Takuya Nakajima, Mizuha Kosugi-Kanaya, Francis H.W. Shand, Yutaka Inagaki, Hitoshi Shimano, Kouji Matsushima
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Research Article Inflammation Pulmonology

Transcriptome network analysis identifies protective role of the LXR/SREBP-1c axis in murine pulmonary fibrosis

Abstract

Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Although lung fibroblasts play a central role in PF, the key regulatory molecules involved in this process remain unknown. To address this issue, we performed a time-course transcriptome analysis on lung fibroblasts of bleomycin- and silica-treated murine lungs. We found gene modules whose expression kinetics were associated with the progression of PF and human idiopathic PF (IPF). Upstream analysis of a transcriptome network helped in identifying 55 hub transcription factors that were highly connected with PF-associated gene modules. Of these hubs, the expression of Srebf1 decreased in line with progression of PF and human IPF, suggesting its suppressive role in fibroblast activation. Consistently, adoptive transfer and genetic modification studies revealed that the hub transcription factor SREBP-1c suppressed PF-associated gene expression changes in lung fibroblasts and PF pathology in vivo. Moreover, therapeutic pharmacological activation of LXR, an SREBP-1c activator, suppressed the Srebf1-dependent activation of fibroblasts and progression of PF. Thus, SREBP-1c acts as a protective hub of lung fibroblast activation in PF. Collectively, the findings of the current study may prove to be valuable in the development of effective therapeutic strategies for PF.

Authors

Shigeyuki Shichino, Satoshi Ueha, Shinichi Hashimoto, Mikiya Otsuji, Jun Abe, Tatsuya Tsukui, Shungo Deshimaru, Takuya Nakajima, Mizuha Kosugi-Kanaya, Francis H.W. Shand, Yutaka Inagaki, Hitoshi Shimano, Kouji Matsushima

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Figure 4

Effect of sterol regulatory element–binding protein 1 on lung fibroblast proliferation and pulmonary fibrosis pathology.

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Effect of sterol regulatory element–binding protein 1 on lung fibroblast...
(A) Experimental scheme of the intratracheal transfer of genetically modified lung fibroblasts. (B) Role of active form of Srebf1c (trSrebf1c) in activated fibroblasts in bleomycin-injured lung. Donor fibroblasts were identified by flow cytometry. Graphs show the mean ± SEM (n = 6). A representative result of 2 independent experiments is shown. ***P < 0.001 (2-tailed unpaired Student’s t test). ΔhLNGFRhi gate was defined by induction rates of BFP2 ≥ 80% in all of the control donor fibroblasts. (C) Effect of trSrebf1c on lung fibroblast proliferation in vitro. Cell cycle of genetically modified lung fibroblasts was analyzed by the fluorescence ubiquitination cell cycle indicator. Graphs show the mean ± SEM (n = 5). A representative result of 2 independent experiments is shown. ***P < 0.001 (2-tailed unpaired Student’s t test). (D–F) Lung fibroblast activation and pulmonary fibrosis pathology in Srebf1–/–Col-GFP mice. (D) Number of lung fibroblasts and myofibroblasts in bleomycin- or silica-treated right lungs on day 14. Graphs show the mean ± SEM of n = 5 (BLM WT Col-GFP, SiO2 Srebf1–/–Col-GFP), n = 6 (SiO2 WT Col-GFP), n = 7 (BLM Srebf1–/–Col-GFP). A representative result of 3 independent experiments is shown. *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed unpaired Student’s t test). (E) Quantification of hydroxyproline content in the whole left lung of bleomycin- or silica-treated Srebf1–/–Col-GFP and WT Col-GFP mice. Graphs show the mean ± SEM of n = 5 (BLM WT Col-GFP, BLM Srebf1–/–Col-GFP, SiO2 Srebf1–/–Col-GFP), n = 6 (SiO2 WT Col-GFP). A representative result of 3 independent experiments is shown. *P < 0.05 (2-tailed unpaired Student’s t test). (F) Masson’s trichrome staining of bleomycin- or silica-treated left lung sections from Srebf1–/–Col-GFP and WT Col-GFP mice at day 14. Scale bars: 100 μm. Representative images of n = 8 (BLM WT Col-GFP, SiO2 WT Col-GFP, SiO2 Srebf1–/–Col-GFP), n = 9 (BLM Srebf1–/–Col-GFP) from 2 independent experiments are shown. (B, C, and D) Effect size (d) is shown on the bottom of the graph. Col-GFP, Col1a2-GFP reporter; ΔhLNGFR, truncated form of human low-affinity nerve growth factor receptor; hPGK, human phosphoglycerate kinase 1; IRES2, internal ribosomal entry site 2; BFP2, mTagBFP2; AG, azami green, KO2, kusabira orange 2; BLM, bleomycin model; SiO2, silica model.