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. 2014 Feb;65(2):121-5.
doi: 10.1016/j.cyto.2013.12.008. Epub 2013 Dec 24.

CXC chemokine receptor 4 signaling upon co-activation with stromal cell-derived factor-1α and ubiquitin

Abhishek Tripathi  1 , Jeffrey D Davis  1 , Daniel M Staren  1 , Brian F Volkman  2 , Matthias Majetschak  3
Affiliations

CXC chemokine receptor 4 signaling upon co-activation with stromal cell-derived factor-1α and ubiquitin

Abhishek Tripathi et al. Cytokine. 2014 Feb.

Abstract

Recently, we reported that extracellular ubiquitin functions as another agonist of CXC chemokine receptor (CXCR)4. Whereas the cognate CXCR4 ligand, stromal cell-derived factor (SDF)-1α, is also a CXCR7 agonist, ubiquitin does not bind to CXCR7. Because both ligands are present in the extracellular environment, co-activation of CXCR4 appears to be physiologically relevant. CXCR4 mediated effects of ubiquitin, however, are not well understood and consequences of co-activation of CXCR4 with both ligands are unknown. Utilizing proximity ligation assays and flow cytometry, we detected CXCR4, but not CXCR7, on the cell surface of THP-1 cells, which suggests that confounding effects of CXCR7 are unlikely. Time course and magnitude of reduction of cell surface CXCR4 expression were comparable after stimulation of THP-1 cells with both ligands. SDF-1α was more efficacious than ubiquitin to mobilize Ca(2+). Co-stimulation of THP-1 cells with both ligands resulted in synergistic effects on Ca(2+) fluxes at suboptimal ligand concentrations. Homologous desensitization of Ca(2+) fluxes was detectable with both ligands. SDF-1α pre-stimulation desensitized ubiquitin induced Ca(2+) fluxes, but not vice versa. Effects of SDF-1α and ubiquitin on cAMP levels, Akt and ERK1/2 phosphorylation and chemotactic responses were additive. The chemotactic activities of ubiquitin and SDF-1α were sensitive to AMD3100, pertussis toxin, U73122, LY94002 and U0126. These data suggest that CXCR4 activation with SDF-1α and ubiquitin results in partially synergistic effects on cellular signaling events and in differential effects on receptor desensitization. The ligand ratio that is present in the extracellular environment may contribute to the regulation of CXCR4 mediated functions.

Keywords: CXCL12; CXCR7; Calcium; Chemotaxis; Cyclic AMP.

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Figures

Fig. 1
Fig. 1
(A) In situ proximity ligation assays (PLA) to detect CXCR4 and CXCR7 on THP-1 cells. The Duolink In Situ Detection Reagent Red was used to visualize CXCR4 (top row) and CXCR7 (center row). Bottom row: control, no primary antibody. From left to right: phase contrast, PLA – red, DAPI, merged PLA/DAPI and merged phase contrast/PLA/DAPI (merge). (B) In situ proximity ligation assays (PLA) to detect CXCR4 and CXCR7 on A7r5 cells. The Duolink In Situ Detection Reagent Green was used to visualize CXCR4 (top row) and CXCR7 (center row). Bottom row: control, no primary antibody. Merged phase contrast/PLA/DAPI images (merge) are shown. (C) Detection of CXCR4 and CXCR7 on THP-1 (top) and A7r5 cells (bottom) by FACS analyses. Thick red lines: cells labeled with rabbit anti-CXCR4/FITC-conjugated anti-rabbit goat IgG. Thick blue lines: cells labeled with rabbit anti-CXCR7/FITC-conjugated anti-rabbit goat IgG. Thin gray lines: control – cells labeled with rabbit anti-IgG/FITC-conjugated anti-rabbit goat IgG. Gray: unstained cells. (D) CXCR4 expression on THP-1 cells 15 min after treatment with 100 nM of the CXCR4 ligands or control (PBS) at 37 °C. Red thick line: cells labeled with rabbit anti-CXCR4/FITC-conjugated anti-rabbit goat IgG, control. Green thick line: cells labeled with rabbit anti-CXCR4/FITC-conjugated anti-rabbit goat IgG, SDF-1α treatment. Blue thick line: cells labeled with rabbit anti-CXCR4/FITC-conjugated anti-rabbit goat IgG, ubiquitin treatment. Thin gray line: cells stained with rabbit anti-IgG/FITC-conjugated anti-rabbit goat IgG, PBS treatment. Gray: unstained cells. (E) Quantification of CXCR4 expression on THP-1 cells after ligand treatment by FACS analyses, as in D. Cell surface CXCR4 expression is expressed as % of untreated control (n = 4–6). Open squares: 100 nM SDF-1α treatment. Gray circles: 100 nM ubiquitin treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
(A and B) Intracellular Ca2+ fluxes in THP-1 cells after stimulation with equimolar concentrations of SDF-1α (gray squares), ubiquitin (open squares) and SDF-1α plus ubiquitin 1:1 (mol/mol) (black squares). (A) Ligand concentration 10–20 nM; n = 7 (4 experiments with 10 nM and 3 experiments with 20 nM ligand concentration) with 5–10 replicates per experiment and condition. (B) Ligand concentration 200 nM; n = 3 with 5–10 replicates per experiment and condition. The arrow indicates the time point when SDF-1α/ubiquitin were added. RFU: relative fluorescence units. (C) Intracellular Ca2+ fluxes in THP-1 cells after repetitive stimulation with 10 nM SDF-1α or ubiquitin (n = 3–4 with 5–10 replicates per experiment and condition). From left to right: initial stimulation with SDF-1α (1→SDF-1α); initial stimulation with ubiquitin (1→Ub); subsequent stimulation with SDF-1α after initial stimulation with SDF-1α (gray circles; 1→SDF-1α 2→SDF-1α) or ubiquitin (black circles; 1→Ub 2→SDF-1α); subsequent stimulation with ubiquitin after initial stimulation with ubiquitin (gray circles; 1→Ub 2→Ub) or SDF-1α (black circles; 1→SDF-1α 2→Ub). The arrows indicate the time points when SDF-1α/ubiquitin were added. (D) Reduction of cAMP levels in forskolin stimulated THP-1 cells by 200 pM SDF-1α, 200 pM ubiquitin (Ub) or 100 pM SDF-1α plus 100 pM ubiquitin; n = 3. Data are expressed as % of untreated cells (=100%). (E) Top: Western blot analyses of Akt and ERK1/2 phosphorylation after stimulation (15 min, 37 °C) of THP-1 cells with 200 nM ubiquitin (Ub), SDF-1α or 100 nM of both ligands. Bottom: Quantification of the chemiluminescence signals after cell stimulation, as shown in the top panel; n = 3. Data are expressed as % of untreated cells (=100%). (F) Migration of THP-1 cells towards 10 nM of SDF-1α, 10 nM of ubiquitin (Ub) or 10 nM of both ligands. CI: chemotactic index. (G) Pharmacological inhibition of SDF-1α (left) and ubiquitin (right) induced chemotaxis in THP-1 cells. AMD3100: chemotaxis in the presence of 10 µM AMD3100. Cells were pre-incubated with all other inhibitors (pertussis toxin (PTx)-100 ng/mL, 2 h; U73122-5 µM, 30 min; LY294002-50 µM, 1 h; U0126-10 µM, 30 min; trichostatin A (TSA)-20 µM, 18 h) at 37 °C, washed and then used for filter migration assays. TSA was used as a negative control. Data are expressed as % control (no inhibitor = 100%). * p < 0.05 versus control.
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