Meccanismo di rilascio di Hsp70 tramite vescicole di membrana e suo ruolo nella migrazione di cellule staminali di topo

Mouse mesoangioblast are vessel-derived stem cells which are able to differentiate in most mesodermal tissues, and have also the ability to migrate and cross the endothelial barrier. In our previous work we have demonstrated that these cells release in the extracellular medium membrane vesicles containing matrix metalloproteinases MMP-2 and MMP-9. We have also demonstrated that they express Hsp70 under basal growth condition. According to literature data, the aim of this study was to investigate the possible role of Hsp70 on MMPs regulation. To do this we compared mesoangioblasts A6 cell clone with two other cell clones: NM3, partially knockdown for Hsp70 expression and a cell clone overexpressing it, named T. We first studied Hsp70 localization inside A6 cells and we demonstrated that it was localized in lipid raft microdomain as a transmembrane protein. We have also demonstrated that it was released in the extracellular milieu through membrane vesicles. To check whether or not MMP-2 and Hsp70 were related at transcriptional level we analyzed by qRT-PCR the MMP-2 mRNA content of the three cell lines. We found that in T cells there was an increase in MMP-2 mRNA level, whereas in NM3 cells there was a reduction in its level. This result was confirmed by transfection assays with a plasmid containing MMP-2 promoter region. We performed western blot analysis on vesicles obtained from each cell line to determinate the Hsp70 level that they release and we found different level of this protein inside them. This result suggested us that MMP-2 level regulation it depends on the Hsp70 extracellular level. This result also confirm our hypothesis that membrane vesicles are vehicles for Hsp70 release in the extracellular environment. To study the role of the different Hsp70 levels on cell migration we performed scratch test and transwell assays, to evaluate its effects on ECM invasion. Both experiments demonstrated that Hsp70 silenced clone cells have a lower migrating capability than A6 cells. On the contrary, the overexpressing cells show a higher capability in matrix degradation. We have also performed transwell assays with various inhibitors, to study Hsp70 involvement and with two different MMPs inhibitor to check which gelatinases is the major responsible for their migration capability. We demonstrated the presence of Hsp70 specific receptors, TLR-2 and 4, in the three cell clones and we studied the Hsp70 receptors involvement in cell migration by transwell assays with 2 different inhibitors: RAP, for CD91 and a neutralizing antibody for TLR-2. Transwell assays with both inhibitors showed a decrease of migrating capability. Literature data indicated that NF-kB also promotes the transcription of MMP2/MMP9 and that Hsp70 is involved in the regulation of NF-kB pathway. By immunoprecipitation assays we found that there was coimmunoprecipitation between Hsp70 and NF-kB, however not with the inhibitor IkB. So we analyzed whether Hsp70 levels affect NF-kB quantity in three cell clones. Western blot analysis showed an increase of NF-kB level in overexperssing cell clone and a decrease in silenced clone (knocked down cell clone). To confirm this result we performed qRT-PCR and we found that there is a relationship between Hsp70 level and NF-kB mRNA quantity, as we demonstrated for the protein level. We studied the NF-kB pathways involvement in cell migration by transwell assays with two different inhibitors: the first one, JSH-23, for NF-kB, and the second one, Wedelolactone, for the kinase IKK. After both treatments cells showed a lower migrating capability than each controls, results that confirm the NF-kB role on MMP-2 transcription and then on mesoangioblasts migration. Thus we hypothesize that membrane vesicle are vehicles for the Hsp70 and that the extracellular Hsp70 can regulate MMP-2 function, by specific receptor, as TLR-2 and CD91. These receptors are able to activate NF-kB pathway, the transcription factor responsible for the transcription of MMP-2. So the Hsp70 overexpressing clone releases higher Hsp70 quantity in the extracellular milieu, than A6, and for this reason it shows a higher migrating capability; on the contrary the partially silenced clone releases lower Hsp70 quantity and shows also a lower migrating capability