PMID- 32828826 OWN - NLM STAT- MEDLINE DCOM- 20210303 LR - 20210303 IS - 1090-2422 (Electronic) IS - 0014-4827 (Linking) VI - 395 IP - 2 DP - 2020 Oct 15 TI - Autophagy regulates exosome secretion in rat nucleus pulposus cells via the RhoC/ROCK2 pathway. PG - 112239 LID - S0014-4827(20)30488-2 [pii] LID - 10.1016/j.yexcr.2020.112239 [doi] AB - Our present study investigated whether exosome secretion of nucleus pulposus cells (NPCs) is regulated by autophagy. Different autophagic states of NPCs were induced by rapamycin (Rap), bafilomycin A1 (Baf) and other agents, and it was found that exosomes were secreted in an autophagy-dependent manner. Activation or inhibition of autophagy increased or decreased, respectively, the amount of exosomes that were released into the extracellular space. In addition, in order to confirm that Rap-promoted release of exosomes was mediated by autophagy rather than other pathways, we used autophagy associated gene 5 (ATG5) small-interfering RNA (siRNA) to silence the expression of ATG5 gene, which is indispensable for autophagy. The results showed that siRNA against ATG5 (siATG5) induced an accumulation of intraluminal vesicles (ILVs) in NPCs and a concomitant decrease in the amount of exosomes isolated from supernatant. Ras homolog gene (Rho) and Rho-associated coiled-coil forming protein kinase (ROCK) family molecules are capable of cytoskeletal remodeling and affecting vesicle transport. Therefore, we carried out targeted interventions and evaluated the effects of the RhoC/ROCK2 pathway on the secretion of exosomes within autophagic environment. Knockdown of RhoC and ROCK2 with corresponding siRNA significantly inhibited the secretion of exosomes originating from ILVs in NPCs, even when NPCs were subsequently treated with Rap. Taken together, our findings suggest that autophagy positively regulates expression levels of RhoC and ROCK2, and that the RhoC/ROCK2 pathway exerts a key function on NPCs-derived exosome secretion. CI - Copyright (c) 2020 Elsevier Inc. All rights reserved. FAU - Hu, Shun-Qi AU - Hu SQ AD - Department of Orthopedics, Zhongshan Hospital, Fudan University, Shanghai, 200032, China. FAU - Zhang, Qi-Chen AU - Zhang QC AD - Department of Orthopedics, Zhongshan Hospital, Fudan University, Shanghai, 200032, China. FAU - Meng, Qing-Bing AU - Meng QB AD - Department of Orthopedics, Zhongshan Hospital, Fudan University, Shanghai, 200032, China. FAU - Hu, An-Nan AU - Hu AN AD - Department of Orthopedics, Zhongshan Hospital, Fudan University, Shanghai, 200032, China. FAU - Zou, Jia-Peng AU - Zou JP AD - Department of Orthopedics, Zhongshan Hospital, Fudan University, Shanghai, 200032, China. FAU - Li, Xi-Lei AU - Li XL AD - Department of Orthopedics, Zhongshan Hospital, Fudan University, Shanghai, 200032, China. Electronic address: li.xilei@zs-hospital.sh.cn. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20200820 PL - United States TA - Exp Cell Res JT - Experimental cell research JID - 0373226 RN - 0 (RNA, Small Interfering) RN - EC 2.7.11.1 (ROCK2 protein, rat) RN - EC 2.7.11.1 (rho-Associated Kinases) RN - EC 3.6.5.2 (RhoC protein, rat) RN - EC 3.6.5.2 (rhoC GTP-Binding Protein) SB - IM MH - Animals MH - Autophagy/*physiology MH - Bodily Secretions/metabolism MH - Exosomes/*metabolism MH - Nucleus Pulposus/*metabolism MH - RNA, Small Interfering/genetics/metabolism MH - Rats, Sprague-Dawley MH - rho-Associated Kinases/metabolism MH - rhoC GTP-Binding Protein/*genetics/metabolism OTO - NOTNLM OT - Autophagy OT - Exosome OT - Nucleus pulposus cell OT - ROCK2 OT - RhoC EDAT- 2020/08/24 06:00 MHDA- 2021/03/04 06:00 CRDT- 2020/08/24 06:00 PHST- 2020/06/13 00:00 [received] PHST- 2020/08/14 00:00 [revised] PHST- 2020/08/17 00:00 [accepted] PHST- 2020/08/24 06:00 [pubmed] PHST- 2021/03/04 06:00 [medline] PHST- 2020/08/24 06:00 [entrez] AID - S0014-4827(20)30488-2 [pii] AID - 10.1016/j.yexcr.2020.112239 [doi] PST - ppublish SO - Exp Cell Res. 2020 Oct 15;395(2):112239. doi: 10.1016/j.yexcr.2020.112239. Epub 2020 Aug 20.