title: ROCK activity and the Gβγ complex mediate chemotactic migration of mouse bone marrow-derived stromal cells.
abstract: BACKGROUND
  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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, University Road, Galway, Ireland. carolineryan_@hotmail.com.|Systems Biology Ireland, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. carolineryan_@hotmail.com.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Ryan
authors.affiliation: Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, University Road, Galway, Ireland. james.brown@nuigalway.ie.|Systems Biology Ireland, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. james.brown@nuigalway.ie.|Present address: Discipline of Surgery, School of Medicine, Lambe Institute, Translational Research Facility, National University of Ireland Galway, University Road, Galway, Ireland. james.brown@nuigalway.ie.
authors.forename: James A L
authors.initials: JA
authors.lastname: Brown
authors.affiliation: Discipline of Pathology, School of Medicine, National University of Ireland Galway, University Road, Galway, Ireland. emer.bourke@nuigalway.ie.
authors.forename: Emer
authors.initials: E
authors.lastname: Bourke
authors.affiliation: Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, University Road, Galway, Ireland. aine.prendergast@hi-stem.de.|Systems Biology Ireland, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. aine.prendergast@hi-stem.de.|Present address: Hematopoietic Stem Cells and Stress' group, Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer feld 280, 69120, Heidelberg, Germany. aine.prendergast@hi-stem.de.
authors.forename: Áine M
authors.initials: ÁM
authors.lastname: Prendergast
authors.affiliation: Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, University Road, Galway, Ireland. claire.kavanagh2@gmail.com.
authors.forename: Claire
authors.initials: C
authors.lastname: Kavanagh
authors.affiliation: Department of Radiology, University of Arizona, Tucson, AZ, 85724-5067, USA. zliu@email.arizona.edu.
authors.forename: Zhonglin
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Centre for Micro and Imaging, National University of Ireland Galway, University Road, Galway, Ireland. peter.owens@nuigalway.ie.
authors.forename: Peter
authors.initials: P
authors.lastname: Owens
authors.affiliation: Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, University Road, Galway, Ireland. georgina.shaw@nuigalway.ie.
authors.forename: Georgina
authors.initials: G
authors.lastname: Shaw
authors.affiliation: Systems Biology Ireland, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. walter.kolch@ucd.ie.
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
authors.affiliation: Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, University Road, Galway, Ireland. timothy.obrien@nuigalway.ie.|Systems Biology Ireland, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. timothy.obrien@nuigalway.ie.
authors.forename: Timothy
authors.initials: T
authors.lastname: O'Brien
authors.affiliation: Regenerative Medicine Institute (REMEDI), Biosciences, National University of Ireland Galway, University Road, Galway, Ireland. frank.barry@nuigalway.ie.|Systems Biology Ireland, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. frank.barry@nuigalway.ie.
authors.forename: Frank P
authors.initials: FP
authors.lastname: Barry
mesh_terms: D000818:Animals; D000941:Antigens / Q000378:metabolism; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000502:physiology; D002633:Chemotaxis; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000502:physiology*; D008807:Mice, Inbred BALB C; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D000941:Antigens; C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D044387:GTP-Binding Protein beta Subunits; D043562:Receptors, G-Protein-Coupled; C086178:pericentrin; D019869:Phosphatidylinositol 3-Kinases; D054460:rho-Associated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases