br Conclusion br Ethics statements file br Conflict

Conclusion
Ethics statements file
Conflict of interest
Acknowledgements This study was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (Nos. 2011-0030074 and 2018R1D1A3B07050361) and Sookmyung Women’s University Specialization Program Funding.
Introduction Pulmonary fibrosis, such as idiopathic pulmonary fibrosis (IPF), is a disease in which persistent injury and tissue remodeling generate massive scars with excessive deposition of extracellular matrix (ECM) [1]. This fibrotic scar formation is usually progressive and intractable, eventually leading to respiratory failure. Because there are currently very few therapeutic options for pulmonary fibrosis, elucidating the mechanism of scar formation and progression is a pressing topic for the development of novel therapies. The lung has unique tissue structure, characterized by a reticular network of thin alveolar walls optimized for gas exchange [2]. Pulmonary fibrosis involves destruction of aspartame products sale and replaces normal tissue with thick scars, which reduce the surface area for gas exchange [3]. The precise mechanism of alveolar scarring is unknown. It is thought that repetitive microinjury to epithelium triggers subsequent tissue remodeling [3]. Subepithelial resident fibroblasts migrate into injured area and differentiate into myofibroblasts, followed by scar formation in the setting of wound healing [4]. Similar activation and migration of fibroblasts are implicated in pulmonary fibrosis [5]. In a previous study, we have shown that fibroblasts transferred into injured alveolar airspaces through an intratracheal route get activated to produce ECM and are eventually incorporated into scars [6]. This result suggests that activated fibroblasts in scars can originate from injured alveolar airspaces and scars can be formed by coalescence of injured alveolar walls. It is possible that activated alveolar fibroblasts play a leading role in the formation of such scars as described in wound healing of other organs [4,7]. Therefore, exploring activation signatures of fibroblasts in injured alveolar airspaces may reveal the mechanism of scar formation in pulmonary fibrosis. Glioma-associated oncogene (Gli) proteins are transcription factors which mediate hedgehog signalings and play important roles in fibroblast activation [8,9]. Growing evidence suggests that Gli proteins are also activated through non-canonical pathways such as transforming growth factor-β pathway [10]. Furthermore, a recent study has shown that pirfenidone, an approved drug for idiopathic pulmonary fibrosis, exerts its antifibrotic effects by suppressing the Gli signaling [11]. In this study, we utilized intratracheal transfer of fibroblasts in bleomycin (BLM)-induced lung fibrosis to investigate gene expression signatures of activated fibroblasts after exposure to alveolar airspaces. We identified Gli signaling as a possible upstream regulator for fibroblast activation after exposure to alveolar airspaces, and pharmacological inhibition of Gli signaling resulted in altered scar formation. These findings suggest an important role of Gli proteins in activated fibroblasts located at alveolar airspaces for scarring in pulmonary fibrosis.
Materials and methods
Results To investigate serial gene expression changes of lung fibroblasts which are exposed to injured alveolar airspaces, we performed intratracheal transfer of Col-GFP+ lung fibroblasts in BLM-induced lung fibrosis and acquired whole transcriptome of purified donor cells on day 2, 4, and 7 after the transfer (Fig. 1). Lineage markers (CD31, CD45, CD146, EpCAM, and Ter119)-negative Col-GFP+ cells from untreated mice were used as day 0. We then examined upregulated (>3 folds) genes between each time point and sought upstream regulators by analyzing enrichment of transcription factor binding motifs in the promoter regions of those upregulated genes (Fig. 1). The top 6 upstream transcription factors are shown in Table 1. A notable upstream regulator between day 0 and 2 was hypoxia-inducible factors 1a (Hif1a). Upstream regulators of later time points (day 4/day 2, day 7/day 4) were similarly represented by kruppel-like factor 4 (Klf4) and Gli signaling molecules. Of note, Klf4, Gli1, and Gli2 are identified in our recent report as hub transcription factors in lung fibroblast activation in BLM- and silica-induced pulmonary fibrosis [16]. We also performed GO enrichment analysis of the upregulated genes (Supplementary Table 1). Between day 0 and 2, GO terms related to translation or metabolic process were enriched. Consistent to the upstream regulators, GO terms for day 4/day 2 and day 7/day 4 were similar and marked by terms related to ECM organizations (Supplementary Tables 2 and 3). These data suggest that fibroblasts exposed to injured alveolar airspaces first underwent metabolic changes driven by Hif1a followed by activation of ECM production driven by Klf4 and Gli signaling molecules.