Extracellular Vesicle microRNAs Contribute to the Osteogenic Inhibition of Mesenchymal Stem Cells in Multiple Myeloma.

Osteolytic bone disease is the major complication associated with the progression of multiple myeloma (MM). Recently, extracellular vesicles (EVs) have emerged as mediators of MM-associated bone disease by inhibiting the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Here, we investigated a correlation between the EV-mediated osteogenic inhibition and MM vesicle content, focusing on miRNAs. By the use of a MicroRNA Card, we identified a pool of miRNAs, highly expressed in EVs, from MM cell line (MM1.S EVs), expression of which was confirmed in EVs from bone marrow (BM) plasma of patients affected by smoldering myeloma (SMM) and MM. Notably,we found that miR-129-5p, which targets different osteoblast (OBs) differentiation markers, is enriched in MM-EVs compared to SMM-EVs, thus suggesting a selective packaging correlated with pathological grade. We found that miR-129-5p can be transported to hMSCs by MM-EVs and, by the use of miRNA mimics, we investigated its role in recipient cells. Our data demonstrated that the increase of miR-129-5p levels in hMSCs under osteoblastic differentiation stimuli inhibited the expression of the transcription factor Sp1, previously described as a positive modulator of osteoblastic differentiation, and of its target the Alkaline phosphatase (ALPL), thus identifying miR-129-5p among the players of vesicle-mediated bone disease.

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer.

In this study, we report how the cholera toxin (CT) A subunit (CTA), the enzyme moiety responsible for signaling alteration in host cells, enters the exosomal pathway, secretes extracellularly, transmits itself to a cell population. The first evidence for long-term transmission of CT's toxic effect via extracellular vesicles was obtained in Chinese hamster ovary (CHO) cells. To follow the CT intracellular route towards exosome secretion, we used a novel strategy for generating metabolically-labeled fluorescent exosomes that can be counted by flow cytometry assay (FACS) and characterized. Our results clearly show the association of CT with exosomes, together with the heat shock protein 90 (HSP90) and Protein Disulfide Isomerase (PDI) molecules, proteins required for translocation of CTA across the ER membrane into the cytoplasm. Confocal microscopy showed direct internalization of CT containing fluorescent exo into CHO cells coupled with morphological changes in the recipient cells that are characteristic of CT action. Moreover, Me665 cells treated with CT-containing exosomes showed an increase in Adenosine 3',5'-Cyclic Monophosphate (cAMP) level, reaching levels comparable to those seen in cells exposed directly to CT. Our results prompt the idea that CT can exploit an exosome-mediated cell communication pathway to extend its pathophysiological action beyond an initial host cell, into a multitude of cells. This finding could have implications for cholera disease pathogenesis and epidemiology.