Time-Dependent Internalization of S100B by Mesenchymal Stem Cells via the Pathways of Clathrin- and Lipid Raft-Mediated Endocytosis.

Ying Zhang,Hao Xu,Jing Zhu,Liang Ye,Xinyuan Zhang,Bin Tan,Qin Yi,Min Xie,Liang Yan

doi:10.3389/fcell.2021.674995

Abstract

Mesenchymal stem cells (MSCs) are promising tools for cancer therapy, but there is a risk of malignant transformation in their clinical application. Our previous work revealed that the paracrine protein S100B in the glioma microenvironment induces malignant transformation of MSCs and upregulates intracellular S100B, which could affect cell homeostasis by interfering with p53. The purpose of this study was to investigate whether extracellular S100B can be internalized by MSCs and the specific endocytic pathway involved in S100B internalization. By using real-time confocal microscopy and structured illumination microscopy (SIM), we visualized the uptake of fluorescently labeled S100B protein (S100B-Alexa488) and monitored the intracellular trafficking of internalized vesicles. The results showed that S100B-Alexa488 was efficiently internalized into MSCs in a time-dependent manner and transported through endolysosomal pathways. After that, we used chemical inhibitors and RNA interference approaches to investigate possible mechanisms involved in S100B-Alexa488 uptake. The internalization of S100B-Alexa488 was inhibited by pitstop-2 or dyngo-4a treatment or RNA-mediated silencing of clathrin or dynamin, and the lipid raft-mediated endocytosis inhibitors nystatin and MβCD. In conclusion, our findings show that clathrin and lipid rafts contribute to the internalization of S100B-Alexa488, which provides promising interventions for the safe application of MSCs in glioma therapy.

Highlights

Gliomas are malignant tumors with poor prognosis and a short median survival time and constitute the majority of neoplasms in the central nervous system (Reifenberger et al, 2017)
Ligand-receptor interactions cannot explain the upregulation of endogenous S100B in Mesenchymal stem cells (MSCs); it is necessary to reveal the mechanism of endogenous S100B elevation induced by exogenous S100B
Ligand-receptor interactions cannot explain the upregulation of endogenous S100B in MSCs, which may affect cell homeostasis by inhibiting P53 transcriptional activity (Fernandez-Fernandez et al, 2005)

Summary

Introduction

Gliomas are malignant tumors with poor prognosis and a short median survival time and constitute the majority of neoplasms in the central nervous system (Reifenberger et al, 2017). Internalization of S100B by MSCs tumor similar to malignant glioma after receiving stem cell therapy (Berkowitz et al, 2016). Our previous studies found that exogenous S100B promotes the malignant transformation of MSCs, and this effect may be mediated via its receptor advanced glycation end products (RAGE) (Tan et al, 2018). Clathrin-mediated endocytosis (CME) is a classic and well-characterized endocytic pathway regulated by multiple proteins especially clathrin and dynamin (Mettlen et al, 2018). Detailed molecular mechanisms of clathrin-independent endocytosis (CIE) pathways remain largely elusive (Sorkina et al, 2018). Still, they can be subdivided depending on the involvement of dynamin (Cendrowski et al, 2016). Lipid rafts are microdomains enriched with cholesterol and sphingolipids, the common feature of many CIE routs is that they often occur at lipid rafts regions (El-Sayed and Harashima, 2013)

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