ROCK activity and the Gß¿ complex mediate chemotactic migration of mouse bone marrow-derived stromal cells

Caroline M Ryan, James AL Brown, Emer Bourke, Áine M Prendergast, Claire Kavanagh, Zhonglin Liu, Peter Owens, Georgina Shaw, Walter Kolch, Timothy O¿Brien, Frank P Barry, Ryan, Caroline M, Brown, James A L, Bourke, Emer, Prendergast, Áine M, Kavanagh, Claire, Liu, Zhonglin, Owens, Peter, Shaw, Georgina, Kolch, Walter, O'Brien, Timothy, Barry, Frank P, Caroline M. Ryan, James A. L. Brown, Áine M. Prendergast, Timothy O’Brien, Frank P. Barry, Ryan, Caroline M., Brown, James A. L., Prendergast, Áine M., Barry, Frank P.

Bone 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. We investigated and detailed the effects of MCP-1 activation on BMSC using inhibitors of GPCR αβ, ROCK and PI3K. The effects of MCP-1 stimulation on intracellular signalling cascades were characterised using immunoblotting and immunofluorescence. The effectors of MCP-1 mediated migration were investigated using migration assays (both 2D and 3D), in combination with inhibitors. We established the kinetics of the MCP-1 activated signalling cascade and show this cascade correlates with cell surface re-localisation of 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 characterize 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 signaling induced by MCP-1 in BMSCs. Importantly, we found that in BMSC inhibition of ROCK significantly inhibits MCP-1 induced chemotactic migration, in contrast to previous reports in other systems. Our results indicate differential chemotactic signalling in mouse BMSC, 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.