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. 2017 Jul;31(7):3084-3097.
doi: 10.1096/fj.201700013R. Epub 2017 Mar 30.

Epithelial Chemokine CXCL14 Synergizes With CXCL12 via Allosteric Modulation of CXCR4

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Free PMC article

Epithelial Chemokine CXCL14 Synergizes With CXCL12 via Allosteric Modulation of CXCR4

Paul J Collins et al. FASEB J. .
Free PMC article

Abstract

The chemokine receptor, CXC chemokine receptor 4 (CXCR4), is selective for CXC chemokine ligand 12 (CXCL12), is broadly expressed in blood and tissue cells, and is essential during embryogenesis and hematopoiesis. CXCL14 is a homeostatic chemokine with unknown receptor selectivity and preferential expression in peripheral tissues. Here, we demonstrate that CXCL14 synergized with CXCL12 in the induction of chemokine responses in primary human lymphoid cells and cell lines that express CXCR4. Combining subactive concentrations of CXCL12 with 100-300 nM CXCL14 resulted in chemotaxis responses that exceeded maximal responses that were obtained with CXCL12 alone. CXCL14 did not activate CXCR4-expressing cells (i.e., failed to trigger chemotaxis and Ca2+ mobilization, as well as signaling via ERK1/2 and the small GTPase Rac1); however, CXCL14 bound to CXCR4 with high affinity, induced redistribution of cell-surface CXCR4, and enhanced HIV-1 infection by >3-fold. We postulate that CXCL14 is a positive allosteric modulator of CXCR4 that enhances the potency of CXCR4 ligands. Our findings provide new insights that will inform the development of novel therapeutics that target CXCR4 in a range of diseases, including cancer, autoimmunity, and HIV.-Collins, P. J., McCully, M. L., Martínez-Muñoz, L., Santiago, C., Wheeldon, J., Caucheteux, S., Thelen, S., Cecchinato, V., Laufer, J. M., Purvanov, V., Monneau, Y. R., Lortat-Jacob, H., Legler, D. F., Uguccioni, M., Thelen, M., Piguet, V., Mellado, M., Moser, B. Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4.

Keywords: CXCR4; allosteric receptor modulation; signal transduction; synergism.

Figures

Figure 1.
Figure 1.
CXCL14 synergizes with CXCL12 in the induction of chemotactic migration of CXCR4-expressing cells. A) Chemotactic migration of 300.19 CXCR4+ cells toward CXCL14 (black bars), CXCL12 (white bars), or CXCL14 (100–1000 nM) in combination with a fixed concentration (1 nM) of CXCL12 (red bars). Migration is expressed as chemotactic index and means + sem of 2–8 independent experiments. *P < 0.05, ***P < 0.001 compared with migration in the absence of chemokine (0 nM; 1-way ANOVA plus Bonferroni posttest). B) Migration of 300.19 CXCR4+ cells toward CXCL12 alone (filled circles) or CXCL12 plus a fixed concentration (300 nM) of CXCL14 (open squares); mean ± sem of 3 independent experiments is shown. *P < 0.05 (100 nM CXCL12 vs. 100 nM CXCL12 + 300 nM CXCL14); 2-way ANOVA plus Bonferroni posttest.
Figure 2.
Figure 2.
CXCL14 synergizes with CXCL12 in chemotaxis of primary blood lymphocytes and NK cells. Chemotactic responses of freshly isolated human PBMCs toward CXCL14 (black bars), CXCL12 (white bars), or CXCL14 (100–1000 nM) plus a fixed concentration (1 or 10 nM) of CXCL12 (red bars). Input and migrated cells were counted by flow cytometry, with gating on surface markers CD3, CD19, and CD56 to distinguish T cells (top row), B cells (middle row), and NK cells (bottom row), respectively. Migration is expressed as a percentage of input of each cell type and means + sem of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with all other groups (1-way ANOVA plus Bonferroni posttest).
Figure 3.
Figure 3.
Synergy between CXCL14 and CXCL12 is completely abolished by blockade of CXCR4. PBMCs were pretreated with 0–10 μM AMD3100 before use in chemotaxis assays, as described above. A) Migration of T, B, and NK cells in response to the combination of 1 nM CXCL12 plus 300 nM CXCL14 is shown. Migration of each cell type in the absence of AMD3100 treatment (0 µM) is given as 100%. Data are means + sem of 4 blood donors across 2 independent experiments. ***P < 0.001 compared with no AMD3100 treatment, using a 1-way ANOVA plus Bonferroni posttest. B) Migration of monocytes toward 1 µM CXCL14 following either no treatment (−) or pretreatment with 10 µM AMD3100 (+). Migration is given as percent of total input monocytes.
Figure 4.
Figure 4.
CXCL14 synergized with CXCL12 in Ca2+ mobilization and Rac1 activation responses but not in ERK1/2 phosphorylation responses. A) Real-time changes in [Ca2+]i concentrations in CXCR4-transfected cells in response to indicated chemokines. All experiments were performed at least twice. B) Polarization of primary T cells was visualized by staining of F-actin and Rac1-GTP followed by confocal microscopy. C) Rac1 activation was measured in flow cytometry by determining mean fluorescent intensity of Rac1-GTP staining in primary T cells by using an Ab that recognizes active GTP-bound Rac1. For each analysis, 10,000 cells were measured. Data are means + sem of 5 experiments derived from individual donors. D) CXCR4-transfected cells were treated with chemokines as shown and then processed for Western blot analysis. ERK1/2 phosphorylation was determined with a phospho-ERK1/2–specific Ab. pERK, phosphorylated ERK1/2; tERK, total ERK1/2 protein. Data are representative of 4 experiments with similar results. ***P < 0.001.
Figure 5.
Figure 5.
Primary and tertiary structure comparison between CXCL14 and CXCL12. A) Ribbon diagrams of CXCL12 alone (left; UniProt P48061) and in combination with CXCL14 (right; UniProt O95715). Step 1 and step 2 indicate regions that are shown to be involved in binding to CXCR4 (step 1) and induction of signal transduction (step 2). Diagrams were computed by using PyMOL. B) Single amino acid sequence alignment of mature human CXCL12 and CXCL14. Position of conserved Cys residues are shown by vertical bars; yellow box highlights the extended 40s-loop region in CXCL14.
Figure 6.
Figure 6.
CXCL14 induces redistribution and conformational changes of cell surface CXCR4. A) Internalization of CXCR4 after incubation of 300.19 CXCR4+ cells with chemokines was determined by flow cytometry. Data from 2 independent experiments are shown; 100% CXCR4 expression refers to staining of untreated 300.19 CXCR4+ cells with anti-CXCR4 [mean fluorescence intensity (MFI), 9977 ± 1235), whereas 0% CXCR4 expression refers to staining of parental (untransfected) 300.19 cells (MFI, 148 ± 17). B) HEK293T cells were transiently cotransfected at fixed ratio with CXCR4-CFP and CXCR4-YFP to determine the effect of CXCL12 (1 or 100 nM), 300 nM CXCL14, and 1 nM CXCL12 + 300 nM CXCL14. Cells were treated with chemokines (or PBS as negative control) for 30 min at 37°C. C) HEK293T cells were transiently cotransfected with CCR2-CFP and CCR2-YFP at a fixed ratio. Cells were treated with CCL2 (0.1 or 100 nM), 300 nM CXCL14, or 0.1 nM CCL2 + 300 nM CXCL14 for 30 min at 37°C, and FRET efficiency was determined as in panel B. For data in panels B and C, statistics were calculated by using a 1-way ANOVA plus nonparametric Kruskall-Wallis test and Dunn’s multiple comparison test. N.s., not significant. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 7.
Figure 7.
Synergistic effect of CXCL14 on HIV-1 infection. A) Entry of CXCR4-tropic HIV-1 particles pNL4.3 into TZM-bl cells that coexpressed CD4, CXCR4, and CCR5 in the presence of increasing concentrations of CXCL12. Viral infection is represented by expression of a luciferase reporter and is normalized to infection in the absence of CXCL12 (medium only = 100% infection). Data are means + sem of 3 independent experiments. *P < 0.05, **P < 0.01 compared with 0 nM CXCL12 (1-way ANOVA plus Bonferroni posttest). B) Entry of pNL4.3 into TZM-bl cells in the presence of 0–1000 nM CXCL14 alone (black bars) or CXCL14 in combination with 0.1 nM CXCL12 (open bars); 100% luminescence corresponds to luciferase reporter activity in the absence of chemokines. Data are means + sem of 3 independent experiments. C) Entry of CCR5-tropic HIV-1 particles pR8Bal into TZM-bl cells in the presence of 0–100 nM CXCL12 or 0–1000 nM CXCL14 as indicated. Data are means + sem of 3 independent experiments. D) Entry of pR8Bal into GHOST cells that coexpress CD4 and CCR5 (but not CXCR4) as well as green fluorescent protein (GFP) under the control of viral long terminal repeat promoter; 100% GFP+ cells refers to fluorescence signals obtained after viral infection of GHOST cells in the absence of chemokines. Ns, not significant. Data are means + sem of 3 independent experiments.
Figure 8.
Figure 8.
CXCL14 is a PAM of CXCR4. The model explains how CXCL14 is able to synergize with CXCL12 in the induction of CXCR4-mediated chemokine responses. The pool of cell-surface CXCR4 consists of a combination of individual CXCR4 conformation states, some of which are empty receptors in monomeric, dimeric, or oligomeric arrangements, whereas other conformational states are influenced by ligand binding (shown here by shift from black to yellow conformation upon CXCL14 binding). Binding of CXCL14 to CXCR4 by itself does not generate chemokine responses. Instead, CXCL14 binding induces allosteric changes in partner molecules that are present in CXCR4 homodimers or oligomers, thereby lowering the threshold of receptor activation by the functional ligand CXCL12 (shown here by shift from black to blue conformation in the partner molecule). As a result, subactive concentrations of CXCL12 become active, which leads to G-protein signaling and cellular responses.

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