Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells

Abstract

BackgroundFetal 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.ResultsWe 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.ConclusionsWe 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.

Comprehensive Screening of Cell Surface Markers Expressed by Adult‐Derived Human Liver Stem/Progenitor Cells Harvested at Passage 5: Potential Implications for Engraftment

“…However, several groups have reported that CXCR4 expression decreases rapidly after MSCs isolation and only a very small percentage of cells or none at all express CXCR4 after a few passages [31, 32]. In fact, in vitro expansion of MSCs induces progressive internalization of CXCR4 as a way for cells to adapt to culture conditions, to the point where there is no CXCR4 remaining on their surface [12, 33–35]. Considering that ADHLSCs emerge from the parenchymal fraction of the adult liver after about one month in culture and then need several more weeks to reach passages 4 to 6 at which time they are traditionally used for experiments, we suspected that the same phenomenon may be taking place.…”

Overview and potential unifying themes of the atypical chemokine receptor family

“…2B). However, of note, constitutive trafficking is not a unique feature of scavenger ACKRs, as increasing, emerging evidence indicates that several conventional chemokine receptors also undergo constitutive internalization and recycling in specific cell types and conditions (CXCR4 [141][142][143]; CXCR3 [144]; CCR7 [145]; CCR1 [146]). Interestingly, constitutive trafficking correlates with ACKR subcellular localization, as scavenger ACKRs are mainly located in recycling endosomes [138][139][140] [unpublished results], whereas transporter and presenter ACKRs are preferentially expressed on the cell membrane [42, 62,82,128,129] (Fig.…”

Neuropilin-2 Regulates Endosome Maturation and EGFR Trafficking to Support Cancer Cell Pathobiology

“…Rapid degradation of E-cadherin, aberrant trafficking, and recycling of integrins are a few of the underlying mechanisms through which endocytosis promotes oncogenesis and metastasis. Recent evidence indicates that altered endocytosis in cancer cells also promotes aberrant tyrosine kinase and G-protein-coupled receptor signaling, either by recycling them at a faster rate through the cell surface or enabling altered receptor signaling from the endosomal compartment (27,28,46). Thus, it is important to understand the mechanisms by which the NRP2 axis regulates endocytosis in cancer cells and the effect that the NRP2 axis has on regulating endocytic trafficking of other tyrosine kinase and G-protein-coupled receptors, thereby modifying their functions.…”