PPARγ controls ESCRT-dependent synoviocyte exosome biogenesis and Alleviates chondrocyte Osteoarthritis by Exosomal ANXA1

Osteoarthritis (OA) is characterized by synovitis, cartilage degeneration and exercise therapy has been recognized as first line therapy. The exercise related exosome involved in the interaction between fibroblast-like synoviocytes (FLSs) and chondrocytes could be a novel promising strategy for treating OA. In this study, PPARγ was upregulated in FLSs under exercise by single cell transcriptome sequencing. Then, we investigated the underlying mechanisms of PPARγ-treated FLSs derived exosome on OA in vivo and vitro. Our data revealed that overexpression PPARγ FLSs derived exosome could ameliorate the OA severity in vivo and vitro. But knockdown PPARγ FLSs derived exosome aggravate OA. Moreover, we found PPARγ controls the endosomal sorting complex required for the transport (ESCRT)-dependent exosome biogenesis pathway. Annexin A1 (ANXA1) was enriched in OE-PPARγ exosome by quantitative proteomics. By Chip-qPCR and Co-IP methods, PPARγ and its coactivator -1α (PGC-1α) acts with ESCRT subunits including HRS, STAM1, TSG101, CHMP7 and promotes their association to cargo ANXA1. As a therapeutic cargo, exosomal ANXA1 was confirmed be internalization by chondrocyte via exosome labeled experiment and ANXA1 could inhibit the phospharylation of ERK to activate the autophagy and decrease chondrocyte apoptosis. While the ANXA1 receptor blocker BOC-2 could reverse the therapic effect. In conclusion, PPARγ/ESCRT – FLSs exosomal ANXA1 – ERK axis provides a deeper theoretical basis for exercise therapy of OA and a new idea for the clinical transformation of exosomes into OA therapy.

骨关节炎（Osteoarthritis, OA）以滑膜炎、软骨退变为主要病理特征，运动疗法已被公认为其一线治疗方案。参与成纤维样滑膜细胞（fibroblast-like synoviocytes, FLSs）与软骨细胞相互作用的运动相关外泌体，有望成为治疗骨关节炎的新型潜在策略。本研究通过单细胞转录组测序发现，运动状态下成纤维样滑膜细胞中过氧化物酶体增殖物激活受体γ（PPARγ）表达上调。随后，我们在体内与体外模型中探究了经PPARγ处理的成纤维样滑膜细胞来源外泌体对骨关节炎的潜在作用机制。研究数据显示，过表达PPARγ的成纤维样滑膜细胞来源外泌体可在体内及体外减轻骨关节炎的严重程度；而敲低PPARγ的成纤维样滑膜细胞来源外泌体则会加重骨关节炎病变。此外，我们发现PPARγ可调控转运必需内体分选复合物（endosomal sorting complex required for transport, ESCRT）依赖的外泌体生物发生通路。通过定量蛋白质组学分析，我们发现膜联蛋白A1（Annexin A1, ANXA1）在过表达PPARγ的外泌体中显著富集。借助染色质免疫沉淀-定量PCR（ChIP-qPCR）与免疫共沉淀（Co-IP）实验，我们证实PPARγ与其辅助激活因子PGC-1α可与ESCRT亚基（包括HRS、STAM1、TSG101及CHMP7）结合，并促进其与货物蛋白ANXA1的相互作用。作为治疗性货物，外泌体携带的ANXA1可通过外泌体标记实验证实被软骨细胞内化；ANXA1可抑制细胞外调节蛋白激酶（Extracellular regulated protein kinases, ERK）的磷酸化水平，从而激活细胞自噬并减少软骨细胞凋亡。而ANXA1受体拮抗剂BOC-2可逆转这一治疗效应。综上，PPARγ/ESCRT-成纤维样滑膜细胞外泌体ANXA1-ERK轴为骨关节炎的运动疗法提供了更深层次的理论基础，也为外泌体用于骨关节炎治疗的临床转化提供了新思路。