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Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

ABSTRACT: Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.

SUBMITTER: Purushothaman A

PROVIDER: S-EPMC4722448 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

Purushothaman Anurag A Bandari Shyam Kumar SK Liu Jian J Mobley James A JA Brown Elizabeth E EE Sanderson Ralph D RD

The Journal of biological chemistry 20151124 4

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-c ...[more]

PMID: 26601950

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Project description:Rational: Ischemic injury of the skeletal muscle remains a serious clinical problem and currently there is no effective therapy. The aim of the present study is to determine whether human umbilical cord mesenchymal stem cells- derived exosomes (UMSC-Exo) could repair ischemic injury by releasing circular RNA. Methods and Results: To create hindlimb ischemia, we surgically ligated the left femoral artery in C57BL/6 mice. Using circRNA-seq analyses of total RNA from ischemic and control muscles, we found reduced expression of circHIPK3 in the ischemic muscle. To explore the role of circHIPK3 in ischemic injury, the mice were randomly assigned into three groups after surgery: 1) vehicle; 2) UMSC-Exo; 3) UMSC-Exo and siRNA targeting circHIPK3 (UMSC-Exo /si-circHIPK3). UMSC-Exo treatment significantly increased expression of circHIPK3 and improved blood perfusion, running distance and muscle force, which were reversed by injection of UMSC-Exo /si-circHIPK3, suggesting that UMSC-Exo improve muscle function by releasing circHIPK3. UMSC-Exo treatment also inhibited ischemia induced pyroptosis - cell death caused by inflammasome as evidenced by activation of NLRP3, cleaved caspase-1, and subsequent increase of IL-1β and IL-18, and the effects were reversed by injection UMSC-Exo /si-circHIPK3. Bioinformatic analysis identified miR-421/FOXO3a as a potential target for circHIPK3, which was confirmed by luciferase reporter assay. Knockdown of circHIPK3 in C2C12 cells resulted in increased expression of miR-421. We established an in vitro model of pyroptosis by stimulating C2C12 cells with LPS and ATP. LPS and ATP treatment resulted in reduced expression of circHIPK3 and increased expression of miR-421, which was prevented by UMSC-Exo. Western blot analysis showed reduced levels of NLRP3 and cleaved caspase-1 when cells were treated by UMSC-Exo. The expression of FOXO3a in C2C12 cells was increased in the presence of miR-421 inhibitor, and the expression was reduced when cells were treated by LPS and ATP. Importantly, the expression of FOXO3a was upregulated by UMSC-Exo but was reduced when si-circHIPK3 was present. Conclusions: Using loss/gain-of function method, we demonstrated that miR-421/FOXO3a is the direct target of circHIPK3, and UMSC-Exo prevent ischemic injury by releasing circHIPK3, which in turn down regulate miR-421, resulting in increased expression of FOXO3a, leading to inhibition of pyroptosis and release of IL-1β and IL-18.

Dataset Information

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

Publications

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

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