title: Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells.
abstract: BACKGROUND
  Fetal mesenchymal stem/stromal cells (MSC) represent a developmentally-advantageous cell type with translational potential.To enhance adult MSC migration, studies have focussed on the role of the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12), but more recent work implicates an intricate system of CXCR4 receptor dimerization, intracellular localization, multiple ligands, splice variants and nuclear accumulation. We investigated the intracellular localization of CXCR4 in fetal bone marrow-derived MSC and role of intracellular trafficking in CXCR4 surface expression and function.
  
  
  RESULTS
  We found that up to 4% of human fetal MSC have detectable surface-localized CXCR4. In the majority of cells, CXCR4 is located not at the cell surface, as would be required for 'sensing' migratory cues, but intracellularly. CXCR4 was identified in early endosomes, recycling endosomes, and lysosomes, indicating only a small percentage of CXCR4 travelling to the plasma membrane. Notably CXCR4 was also found in and around the nucleus, as detected with an anti-CXCR4 antibody directed specifically against CXCR4 isoform 2 differing only in N-terminal sequence. After demonstrating that endocytosis of CXCR4 is largely independent of endogenously-produced SDF-1, we next applied the cytoskeletal inhibitors blebbistatin and dynasore to inhibit endocytotic recycling. These increased the number of cells expressing surface CXCR4 by 10 and 5 fold respectively, and enhanced the number of cells migrating to SDF1 in vitro (up to 2.6 fold). These molecules had a transient effect on cell morphology and adhesion, which abated after the removal of the inhibitors, and did not alter functional stem cell properties.
  
  
  CONCLUSIONS
  We conclude that constitutive endocytosis is implicated in the regulation of CXCR4 membrane expression, and suggest a novel pharmacological strategy to enhance migration of systemically-transplanted cells.
authors.affiliation: UQ Centre for Clinical Research, The University of Queensland, Herston QLD 4029, Australia. r.pelekanos@uq.edu.au.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Pelekanos
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Ting
authors.forename: Varda S
authors.initials: VS
authors.lastname: Sardesai
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Ryan
authors.forename: Yaw-Chyn
authors.initials: YC
authors.lastname: Lim
authors.forename: Jerry K Y
authors.initials: JK
authors.lastname: Chan
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Fisk
mesh_terms: D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005260:Female; D005333:Fetus / Q000166:cytology*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D021381:Protein Transport; D019718:Receptors, CXCR4 / Q000032:analysis* / Q000378:metabolism*
chemical_list: D019718:Receptors, CXCR4