The effect of core fucosylation-mediated regulation of multiple signaling pathways on lung pericyte activation and fibrosis

Abstract

The main event in the progression of pulmonary fibrosis is the appearance of myofibroblasts. Recent evidence supports pericytes as a major source of myofibroblasts. TGFβ/Smad2/3 and PDGF/Erk signaling pathways are important for regulating pericyte activation. Previous studies have demonstrated that PDGFβR and TGFβR are modified by core fucosylation (CF) catalyzed by α-1,6-fucosyltransferase (FUT8). The aim of this study was to compare the effect of inhibiting CF versus the PDGFβR and TGFβR signaling pathways on pericyte activation and lung fibrosis. FUT8shRNA was used to knock down FUT8-mediated CF both in vivo and in isolated lung pericytes. The small molecule receptor antagonists, ST1571 (imatinib) and LY2109761, were used to block the PDGFβ/pErk and TGFβ/pSmad2/3 signaling pathways, respectively. Pericyte detachment and myofibroblastic transformation were assessed by immunofluorescence and Western blot. Histochemical and immunohistochemical staining were used to evaluate the effect of the intervention on pulmonary fibrosis. Our findings demonstrate that FUT8shRNA significantly blocked pericyte activation and the progression of pulmonary fibrosis, achieving intervention effects superior to the small molecule inhibitors. The PDGFβ and TGFβ pathways were simultaneously affected by the CF blockade. FUT8 expression was upregulated with the transformation of pericytes into myofibroblasts, and silencing FUT8 expression inhibited this transformation. In addition, there is a causal relationship between CF modification catalyzed by FUT8 and pulmonary fibrosis. Our findings suggest that FUT8 may be a novel therapeutic target for pulmonary fibrosis.

Introduction

The number of patients with pulmonary fibrosis continues to steadily increase, and the absence of an effective therapy has led to heavy economical and public health burdens throughout the world (Sköld et al., 2019; Lunardi et al., 2018; Feng et al., 2019). Activation of myofibroblasts and the production of an abundance of extracellular matrix (ECM) is the pathophysiological mechanism of pulmonary fibrosis (Urushiyama et al., 2019；Shao et al., 2018). Moreover, ECM has been found to increase lung ischemia and hypoxia, which further promotes pulmonary fibrosis (Kathiriya et al., 2017；Braun et al., 2018；Terashima et al., 2019).

Recent studies have demonstrated that pericytes, which regulate microvascular circulation and physiological function may be an important source of myofibroblasts (Wang et al., 2019a, 2019b；Sava et al., 2017；Kumar et al., 2017；Wang et al., 2017). The mechanism of pulmonary fibrosis involves pericyte transdifferentiation into myofibroblasts through the TGF-β/Smad2/3 and PDGFβ/Erk signaling pathways (Underly et al., 2017; Hirooka et al., 2017；Pozdzik et al., 2016; Castellano et al., 2019；Minutti et al., 2019). Thus, the blockade of a single signaling pathway may not significantly inhibit pericyte activation. Previous studies have reported that the key receptors (TGFβR and PDGFβR) are modified by α-1,6-core fucosylation (CF) catalyzed by fucosyltransferase8 (FUT8) (Sun et al., 2017). CF modification catalyzed by FUT8 is an important method of modified glycosylation (Chen et al., 2016; Shen et al., 2013; Hirano et al., 2017). However, CF modification in pulmonary fibrosis remains unknown. Therefore, we hypothesize that inhibition of CF modification of TGFβR and PDGFβR can block pericyte activation and thus alleviate pulmonary fibrosis.

To test our hypothesis, we established a bleomycin-induced murine model of pulmonary fibrosis and an adenovirus-encapsulated FUT8shRNA was used to knockdown C57BL/6 mice (CF modification of many key proteins in the mouse lungs are inhibited). The small-molecule receptor antagonists, imatinib (PDGFβR) and LY2109761 (TGFβR), were given to block the PDGFβ/pErk and TGF-β/pSmad2/3 signaling pathways, respectively as the single signal pathway inhibition groups. The migration of pericytes and the effect on the transdifferentiation of pericytes into myofibroblasts were compared. Finally, the effect of each intervention on pulmonary pathology and ECM between the multi-pathway group and single pathway groups were compared to further clarify the impact of CF modification on pericyte activation in the progression of pulmonary fibrosis.