Studies on biogenesis and function of extracellular vesicles

Abstract

Intercellular communication is essential for coordinating the biological processes in multicellular organisms. Cells achieve elaborate intercellular communication through secretion of communicators, a matter conveying the information, into extracellular milieu. It had been considered for a long-time that communicators are exclusively soluble factors such as growth factors, hormones, and cytokines. However, recent evidences suggest that extracellular vesicle (EV) is another type of communicators playing diverse roles in intercellular communication. EVs are spherical, bi-layered membrane vesicles secreted by various types of cells including tumor cells and various bioactive materials such as proteins lipids, and nucleic acids are enriched in it. EVs can act as potent communicators by protecting proteins and RNAs inside EVs, maintaining the topology of membrane proteins on EVs and increasing local concentration of molecules incorporated to EVs. EVs play important roles in biological processes such as immune responses and tumorigenesis. It is known that EVs generated by the release from the endosomal compartment are called as exosomes while EVs generated by the direct shedding from the plasma membrane are called as ectosomes. However, extracellular stimuli modulating such EV biogenesis and the specific molecular mechanisms underlying EV biogenesis are still unknown. In the first part of the study, extracellular stimuli modulating EV biogenesis were investigated. Tumor microenvironment is one of the most complicated conditions composed of diverse cells. In this condition, cells communicate with each other by collaboration between EVs and soluble factors, and there could be interplay between EVs and soluble factors such as pro-inflammatory cytokines. In line with this, I hypothesized that pro-inflammatory cytokines known to modulate tumor progression could affect EV biogenesis in tumor cells. I found that, among several pro-inflammatory cytokines, TNF-α stimulates EV biogenesis in tumor cells in vitro and in vivo. In TNF-α knockout (TNF-α KO) mice, the amount of EVs in tumor decreased along with the retardation of tumor growth and angiogenesis. In addition, by comparing with wild-type (WT) tumor derived EVs, I found that the diminished pro-angiogenic activity was exerted by TNF-α KO tumor-derived EVs which resulted from the declined amount of VEGF in EVs, due to EVs’ low binding activity to VEGF. Furthermore, the role of tumor-derived EVs in tumor growth was ascertained by demonstrating the restoration of tumor growth in TNF-α KO mice by the addition of WT tumor-derived EVs. Taken together, these results demonstrates that TNF-α stimulates both the expression of soluble factors such as VEGF and biogenesis of EVs which cooperate to exert pro-angiogenic activity of TNF-α. In the second part, since the molecular mechanisms underlying EV biogenesis are still unknown, I tried identifying the signaling molecules involved in EV biogenesis to improve the understanding of the molecular mechanisms regulating EV biogenesis. The effect of various kinds of kinase inhibitors on EV biogenesis was examined. I found that several proteins including EGFRK, SRC, GSK3β and Cdk5/p25 were involved in EV biogenesis and that they were all under EGFR signaling. In addition, I demonstrated that GSK3β regulates EV biogenesis through modulating microtubule dynamics followed by altering vesicle trafficking. In this study, I elucidated the kinase components involved in EGFR signaling-mediated EV biogenesis and the novel function of GSK3β in regulating EV biogenesis. Cell adhesion molecules (CAMs) including E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) are predominantly expressed on the cell surface of activated endothelial cells and play a pivotal role in leukocyte infiltration. It is reported that those CAMs are secreted as both EV-associated membranous form and soluble form. Until now, most studies on secreted CAMs have been achieved without distinguishing EV-associated membranous form from soluble form. However, it is necessary to discriminate these two forms considering the followings: 1) EV-associated membranous form and soluble form of secreted CAMs have distinct activity and different local concentration. 2) the analysis of EV-associated membranous form to soluble form of CAMs in blood could be a diagnostic parameter. In the last part of this study, I comprehensively studied the EV-associated form and soluble form of secreted E-selectin, ICAM-1, and VCAM-1 from activated endothelial cells. I found that E-selectin and ICAM-1 are secreted as both EV-associated membranous form and soluble form, in contrast, most of the secreted VCAM-1 is in soluble form. In addition, it was demonstrated that E-selectin-containing EVs has larger proportion than that of ICAM- or VCAM-1-containing-EVs, and single CAM-containing-EVs are more abundant than double CAMs containing-EVs. These results indicate that each CAM has the distinct pattern of secreted forms and activated endothelial cells produce different kinds of EVs having different repertoire of CAMs. This study is expected to provide new insights on the understanding of the secreted CAMs. In summary, I elucidated that TNF-α stimulates biogenesis of EVs having pro-angiogenic activity and suggest the novel underlying mechanism of tumor-promoting activity of TNF-α. In addition, I identified the kinase components of EGFR signaling involved in EV biogenesis and role of GSK3β in basal EV biogenesis via modulating microtubule dynamics. Furthermore, I thoroughly investigated the secreted forms of CAMs including E-selectin, ICAM-1, and VCAM-1. I found that the patterns of secreted forms of these CAMs are different each other and different kinds of EVs containing CAMs released from activated endothelial cells. I believed that this study could provide a new perspective on understanding biogenesis and function of EVs.