Abstract
IntroductionBone marrow-derived stromal cells (BMSCs), also known as mesenchymal stem cells, are the focus of intensive efforts worldwide to elucidate their function and biology. Despite the importance of BMSC migration for their potential therapeutic uses, the mechanisms and signalling governing stem cell migration are still not fully elucidated.MethodsWe investigated and detailed the effects of MCP-1 activation on BMSCs by using inhibitors of G protein-coupled receptor alpha beta (GPCR αβ), ROCK (Rho-associated, coiled-coil containing protein kinase), and PI3 kinase (PI3K). The effects of MCP-1 stimulation on intracellular signalling cascades were characterised by using immunoblotting and immunofluorescence. The effectors of MCP-1-mediated migration were investigated by using migration assays (both two-dimensional and three-dimensional) in combination with inhibitors.ResultsWe established the kinetics of the MCP-1-activated signalling cascade and show that this cascade correlates with cell surface re-localisation of chemokine (C motif) receptor 2 (CCR2) (the MCP-1 receptor) to the cell periphery following MCP-1 stimulation. We show that MCP-1-initiated signalling is dependent on the activation of βγ subunits from the GPCR αβγ complex. In addition, we characterise a novel role for PI3Kγ signalling for the activation of both PAK and ERK following MCP-1 stimulation. We present evidence that the Gβγ complex is responsible for PI3K/Akt, PAK, and ERK signalling induced by MCP-1 in BMSCs. Importantly, we found that, in BMSCs, inhibition of ROCK significantly inhibits MCP-1-induced chemotactic migration, in contrast to previous reports in other systems.ConclusionsOur results indicate differential chemotactic signalling in mouse BMSCs, which has important implications for the translation of in vivo mouse model findings into human trials. We identified novel components and interactions activated by MCP-1-mediated signalling, which are important for stem cell migration. This work has identified additional potential therapeutic targets that could be manipulated to improve BMSC delivery and homing.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0125-y) contains supplementary material, which is available to authorized users.
Highlights
Bone marrow-derived stromal cells (BMSCs), known as mesenchymal stem cells, are the focus of intensive efforts worldwide to elucidate their function and biology
BMSCs migrate in response to the chemokine Monocyte chemoattractant protein 1 (MCP-1) We found that our cultured mouse BMSCs displayed three distinct populations: a tiny immotile fraction (
Investigating localisation of the extracellular MCP-1 receptor CCR2 on the surface of non-permeabilised untreated mouse BMSCs, we found that CCR2 displayed diffuse generalised cell surface staining (Fig. 3a)
Summary
Bone marrow-derived stromal cells (BMSCs), known as mesenchymal stem cells, are the focus of intensive efforts worldwide to elucidate their function and biology. It was demonstrated that Pericentrin-1 binds activated CCR2, linking it to the PI3 kinase (PI3K)-Rac-lamellipodium cascade [7]. It is unresolved whether these signalling events are likewise involved in the chemotactic response of BMSCs. RhoGTPase family members and the Rho kinase ROCK (Rhoassociated, coiled-coil containing protein kinase), which are important mediators of polarisation and migration in many cells types, remain to be confirmed in BMSCs [10,11,12,13,14,15]. Using a combination of migration assays, high-resolution imaging, and pharmacological inhibitors, we have explored the critical mediators and order of signalling in MCP-1-mediated BMSC migration
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