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.

Figure 1

A low number of human fetal bone marrow MSC (fMSC) display surface CXCR4. Three different anti-CXCR4 antibody clones were used 12G5, ab2074 and 4417 as detailed in the methods, with fluorophore labelled secondary antibodies used where necessary. A) CXCR4 surface expression (fluorescence intensity vs. forward scatter) by flow cytometry shown as dot plots. B) After permeabilisation, the majority of cells show intracellular stores of CXCR4. The horizontal gates delineate the position of relevant isotype control antibody. Human adult bone marrow MSC also show low expression of CXCR4 on the cell surface (C) with large intracellular stores of this receptor (D). E) Each of the anti-CXCR4 antibodies are able to detect >80% cells with surface expression of CXCR4 on HeLa cells. F) Both the anti-CXCR4 antibodies 12G5 and ab2074 are able to detect >80% cells with surface expression of CXCR4 on THP-1 human monocytic leukemia cells.

Figure 2

Subcellular localization of CXCR4 expression in MSC. A) Incubation of non-permeabilised fMSC with the anti-CXCR4 antibody (clone 4417) shows a distinct plasma membrane labelling of a small percentage of cells (a representative image of positive cell in the centre is shown). B) When incubated with permeabilised fMSC, the anti-CXCR4 (4417) antibody labels endosomal-like structures in a majority of cells. These CXCR4 positive vesicles have an arrangement along the cytoskeleton (upper inset) and also perinuclear accumulation (lower inset) with light nuclear staining. C) Negative control for ab 4417, using the same imaging settings. D-F) Immunofluorescence staining of fMSC with anti-CXCR4 clone ab2074 (red) strong nuclear localization of CXCR4, with diffuse, punctate cytoplasmic staining. CXCR4 colocalises with the endocytotic markers Rab5 (D) and Rab11 (E) and lysosomal marker Lamp1 (F, all green). Lamp1 displays a distinct peri-nuclear location, with larger sized vesicles. Nuclei, counterstained with DAPI (x40 magnification). G) The Duolink II proximity ligation assay (PLA) shows colocalisation of CXCR4 (ab2074) with all three Rab5, Rab11 and Lamp1 positive compartments. Each red spot corresponds to a molecular interaction (x20 magnification). H) The positive control experiment is two different antibodies to the Growth Hormone Receptor, where the bound antibodies are in close proximity to each other. Negative controls have one (#1) or both (#2) primary antibodies omitted from the PLA procedure.

Figure 3

Surface CXCR4 expression increases after treatment with endocytosis inhibitors. A) Treatment of fMSC for 24 hr with a neutralizing antibody against SDF-1 at a range of antibody concentrations. fMSC show only low level increase of CXCR4 expression (MFI calculated from flow cytometry data). B) Treatment of fMSC with the endocytosis inhibitors, blebbistatin or dynasore increases surface expression of CXCR4. Cells were treated with vehicle (0 μM) or 20–100 μM blebbistatin or dynasore for 60 min before surface expression was determined by flow cytometry (expressed as% total cells expressing surface CXCR4 ± SD). C) Kinetics of CXCR4 exocytosis in fMSC after treatment with endocytosis inhibitors. Cells were treated with 80 μM blebbistatin or dynasore then fixed and stained with anti-CXCR4 (12G5) at 0, 15, 30, 60, 90 and 120 min time points. D) MSC were incubated with vehicle or 80 μM blebbistatin or dynasore for 60 min. Fetal MSC were stained with isotype control (upper panel) or anti-CXCR4 (12G5, lower panel). (E) Inhibitor treated adult bone marrow MSC anti-CXCR4 (ab2074). Percentage of cells positive for CXCR4 expression over isotype control is indicated. Dynasore and Blebbistatin inhibit SDF-1 induced endocytosis of CXCR4 in THP-1 cells. F) In the untreated state, anti-CXCR4 antibody 12G5 detected >90% cells with surface expression of CXCR4 on THP-1 monocytic leukemia cells. G) Stimulation of THP-1 cells with 1 mg/ml of SDF-1 resulted in down-regulation of surface expression of CXCR4 while co-treatment with either blebbistatin (H) or dynasore (I) showed reduced CXCR4 endocytosis. The position of the isotype control is indicated by the gates (fluorescence intensity vs. forward scatter).

Figure 4

Endocytosis inhibitors disrupt the actin cytoskeleton of fMSC. The morphology of the actin cytoskeleton was visualized with Alexa 568-conjugated phalloidin after incubation for 1 hr in serum-free (treatment) media, or treatment media + vehicle, blebbistatin or dynasore. A) Cells at high confluence are less prone to cytoskeletal change, whereas cells at lower confluence are impacted more by inhibitors (B and C shows enlargement of key regions of B). D) A schematic illustration of C, showing actin filaments as white lines, the outline of each cell as a red line, and membrane ruffles as blue lines or spots. A normal, filamentous actin cytoskeleton is seen in fMSC with treatment media. Treatment with vehicle (DMSO) has some impact on cytoskeletal integrity, with the partial loss of long actin filaments in most cells. Blebbistatin and dynasore treatment dramatically disrupts the actin cytoskeleton in most cells. The cells develop membrane ruffles, neural-like projections and begin detaching from the culture dish (x40 magnification).

Figure 5

Blebbistatin does not dramatically alter the morphology of CXCR4 positive endosomes. The cellular distribution CXCR4 without (A) and with blebbistatin treatment (B). C) Rab5 distribution with blebbistatin (80 μM, 1 hr). There was little change in the size or distribution of the CXCR4+ or endocytotic vesicles, despite a change in actin cytoskeleton stained with phalloidin (red, A vs. B and C). However, there was a qualitative increase in the amount of staining endosomes trapped throughout the cytoplasm in cells treated with blebbistatin (A vs. B).

Figure 6

Effect of endocytosis inhibitors on fMSC migration, attachment, chemotaxis and differentiation. A and B) Scratch wound assay was carried out in a 96 well plate using the Incucyte live cell imaging system. A confluent monolayer of fMSC was wounded and treated for 1 hr with serum-free treatment media, or treatment media with DMSO vehicle, 80 μM blebbistatin or dynasore. Images were taken 4 hourly, with representative images of media and blebbistatin treated cells at 0, 6 and 12 hr intervals shown in A. The grey overlay is the automatically generated wound outline at 0 hr. B) Quantitative analysis of percentage wound confluence (N = 3 donors, replicates of 5) show no statistically significant difference in ability to migrate into the wound zone for any cell treatment. C) fMSC were prestained with CFSE, then treated with either blebbistatin or dynasore before being placed into fibronectin or collagen I coated wells of a 96 well plate. Cell adhesion was determined by fluorescence intensity after a 1 hr incubation and removal of non-adherent cells. D) Untreated or inhibitor-treated cells were placed in the upper well of a Transwell plate. Cells were incubated for 4 hr at 37°C and migration determined at a range of concentrations of SDF-1 by fluorescence intensity in the bottom well was higher (p < 0.01** for both) in inhibitor-treated cells. fMSC were treated with either vehicle, 80 μM of blebbistatin or dynasore for 2 hours and the media then replaced with either (E) osteogenic- or (F) adipogenic-induction media. After 3 weeks culture, differentiation was determined by Alizarin Red (osteogenesis) or Oil Red O (adipogenesis) staining. Undifferentiated fMSC cultured in normal growth media are shown on the right.