All-trans retinoic acid attenuates pulmonary fibrosis through inhibition of EphA2-EphrinA1 signal in bleomycin-induced pulmonary fibrosis mouse model

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible, and usually lethal lung disease of unknown pathophysiology. All-trans retinoic acid (ATRA) is a biologically active metabolite of vitamin A, and is known to affect cell differentiation, proliferation, and development. Eph-Ephrin signaling mediates various cellular processes including vasculogenesis, angiogenesis, cell migration, axon guidance, fluid homeostasis and repair after injury. Only a few studies about the effect of ATRA of pulmonary fibrosis have been reported. And the relationship between pulmonary fibrosis and ephrin type-A receptor 2 (EphA2) signaling remains unclear. Purpose: The aim of this study is to evaluation the role and related signal pathways of ATRA in the bleomycin-induced pulmonary fibrosis mouse model and whether the mechanism involves the regulation of pre-existing pathways such as and Eph-Ephrin, PI3k-Akt or Wnt/ß-catenin signaling pathway. Methods and Materials: A549 epithelial cells and CCD-11 Lu fibroblast were incubated and stimulated with or without ATRA, TGF-β1, and PI3K inhibitor, respectively. The levels of Smad2/3 and p-Smad2/3 were analyzed by western blotting. For animal models, we studied four experimental mice groups. There were PBS control group (PBS exposure), bleomycin + PBS group (PBS instillation after bleomycin exposure), bleomycin + ATRA group (peritoneal ATRA injection after bleomycin exposure), bleomycin + EphA2 monoclonal antibody group (EphA2 mAb instillation posttreatment after bleomycin exposure). On day 21, eight mice of each group were sacrificed. The cell counts and protein concentration in the bronchoalveolar lavage fluid (BALF), changes in histopathology, hydroxyproline assay, and the expression of several signal pathway proteins including EphA2-EphrinA1, PI3K-Akt, Wnt/ß-catenin, and cytokine levels were compared among the groups. Results: In this study, ATRA attenuates PI3K/Akt signaling in fibroblast. We report that bleomycin exposure significantly upregulated EphA2 and EphrinA1 expression at day 21 after bleomycin exposure. ATRA posttreatment attenuated lung injury score and reduced protein concentration of BALF. The expression of EphA2, Ephrin, and PI3K 110γ protein was significantly increased after bleomycin instillation, and decreased after ATRA posttreatment. ATRA led to a decrease of IL-6 and TNF-α production in the bleomycin + ATRA group compared to the bleomycin + PBS group (all, P < 0.05). Inhibiting EphA2 receptor by intranasal EphA2 mAb instillation attenuated pulmonary fibrosis, reduced cell counts and protein concentration of BALF (all, P < 0.05). Furthermore, bleomycin exposure upregulated the expression of Wnt5a and PI3K, and inhibiting EphA2 receptor downregulated both of them. Conclusion: The present data suggest that ATRA attenuated bleomycin-induced pulmonary fibrosis, and that it may regulate the EphA2-EphrinA1 signaling and PI3K dependent pathway. Furthermore, EphA2 antagonism attenuates the PI3K dependent pathway and Wnt/ß-catenin-independent pathway, and decreased pulmonary fibrosis. Therefore, ATRA and inhibitor for EphA2 may have a protective effect in bleomycin-induced pulmonary fibrosis.

배경: 특발성 폐섬유화증은 만성적, 비가역적으로 진행하며, 종종 치명적인 결과를 초래하는 폐 질환으로, 그 병태생리는 명확하게 알려져 있지 않다. All-trans retinoic acid (ATRA)는 비타민 A의 활성화된 대사물로, 세포의 분화와 증식, 발달과 관계되는 것으로 알려져 있다. 한편, Eph-Ephrin 신호 전달 체계는 혈관의 형성, 세포의 이동, 세액의 항상성 및 손상 후의 복구를 포함한 다양한 세포 과정을 매개한다. 폐섬유화에 대한 ATRA의 효과에 대하여 소수의 연구가 보고된 바 있으며, 폐섬유화외 EphA2 신호 전달 체계의 상관 관계에 대하여는 아직 명확하게 밝혀져 있지 않다. 목적: 본 연구에서는 bleomycin으로 유도된 폐섬유화 마우스 모델에서 ATRA 의 역할과, 그 메커니즘이 Eph-Ephrin, PI3K-Akt 혹은 Wnt/ß-catenin과 같은 기존에 존재하는 신호 전달 체계와 연관되는지를 밝히고자 하였다. 방법: 세포 실험에서 epithelial cell과 fibroblast를 ATRA, TGF-β1, 그리고 PI3K 억제제의 유무에 따라 배양하였다. 동물 실험은 대조군, bleomycin 그룹, ATRA 그룹, EphA2 monoclonal antibody 그룹의 4 그룹으로 나누어 진행하였다. 21일째에 각 그룹의 mice로부터 기관지폐포세척액을 획득하여 세포 수와 단백질 농도를 그룹별로 비교, 분석하였다. 또한 폐 조직을 얻어 각 그룹의 조직학적 변화를 비교하고, hydroxyproline assay, Eph...