Mouse mesoangioblasts release Hsp70 in a controlled manner through membrane vesicle shedding

Abstract

Mesoangioblaststs (Mabs) are mesodermal stem cells associated with vessels which can differentiate into different mesoderm cell types. They have the property to cross endothelial barrier and when injected into circulation they localize into damaged tissues. A6 cells, a clone of mouse Mabs, express Hsp70 protein in physiological conditions and this synthesis may be required to answer to several intra- and/or extra-cellular needs. It was found that Hsp70 may be released outside from several type of cells, but its role remains still undefined. In order to clarify the reason of Hsp70 constitutive expression, we prepared several A6 clones with different levels of Hsp70 expression by stable RNA interference. We found that knocked-down clones expressing ~50% of this protein have a slower duplication time (23.4h), while A6 cells duplicate in 16h. The slowdown of proliferation indicates that a part of hsp70 can affect, even indirectly, other proteins involved in cell duplication. Since Hsp70 might also have functions outside the cells, we explored this possibility and the potential mechanism of its release. Our results showed that A6 cells shed membrane vesicles (MV) of 1 micron in diameter, containing structural proteins, such as actin, beta tubulin and filamin, but also VE-cadherin and the lipid raft markers caveolin-1 and GM1. Our analysis also demonstrated the presence of signaling proteins in their active form (i.e. FGF-2, MMP-9 and MMP-2). All of them are key proteins in the biology of Mabs. MV also contain part of Hsp70 and to elucidate the extracellular role of this protein we studied the mechanism of its release. We found that Hsp70 release was reduced of 30% after depolymerization of actin stress fibres, while this treatment did not affect vesicle production. On the contrary non reduction was observed after microtubule destruction. This indicates the importance of microfilaments integrity in Hsp70 transport to plasma membrane and to MV. We also tested Hsp70 release in two different cases: A6 cells expressing more Hsp70 due to heat stress, or knocked-down cells with ~50% of Hsp70 expression. Surprisingly we found that in both cases the Hsp70 amount exported in the milieu by MV is the same as that released by control cells. This finding suggests a controlled Hsp70 release mechanism. In this repertory MV may be considered as a cargo for proteins involved in the biology of the Mabs and Hsp70 may have a role to accompany some of these proteins to their extracellular final destination.