The Chemokine Receptor CXCR6 Evokes Reverse Signaling via the Transmembrane Chemokine CXCL16

Abstract

Reverse signaling is a signaling mechanism where transmembrane or membrane-bound ligands transduce signals and exert biological effects upon binding of their specific receptors, enabling a bidirectional signaling between ligand and receptor-expressing cells. In this study, we address the question of whether the transmembrane chemokine (C-X-C motif) ligand 16, CXCL16 is able to transduce reverse signaling and investigate the biological consequences. For this, we used human glioblastoma cell lines and a melanoma cell line as in vitro models to show that stimulation with recombinant C-X-C chemokine receptor 6 (CXCR6) or CXCR6-containing membrane preparations induces intracellular (reverse) signaling. Specificity was verified by RNAi experiments and by transfection with expression vectors for the intact CXCL16 and an intracellularly-truncated form of CXCL16. We showed that reverse signaling via CXCL16 promotes migration in CXCL16-expressing melanoma and glioblastoma cells, but does not affect proliferation or protection from chemically-induced apoptosis. Additionally, fast migrating cells isolated from freshly surgically-resected gliomas show a differential expression pattern for CXCL16 in comparison to slowly-migrating cells, enabling a possible functional role of the reverse signaling of the CXCL16/CXCR6 pair in human brain tumor progression in vivo.

Keywords: brain tumor; cellular communication; chemokine; chemokine receptor; glioma; reverse signaling; tumor cell migration.

Figure 1

Expression of CXCL16 and CXCR6 mRNA and protein in glioblastoma cells and stably transfected LOX melanoma cell clones by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunocytochemistry (ICC). (A) Expression of CXCL16 and CXCR6 was investigated in glioblastoma cell lines A172, LN229, T98G and U251MG (for biological independent results of A172 and T98G, compare also [13]). CXCL16 was detected at moderate to high extends, whereas CXCR6 was undetectable or yielded just background staining; (B) expression of CXCL16 was investigated in clones from natively CXCL16-negative, CXCR6-negative LOX melanoma cells. While the LOX-pcDNA clone was CXCL16 negative, the LOX-CXCL16 clone showed CXCL16 at the mRNA and protein level. A C-terminally truncated version of CXCL16 (in LOX-ΔCXCL16 cells) was also detectable at the mRNA and protein level (for verification of truncation, see [13]); (C) Expression of CXCR6 was investigated in LOX melanoma cell clones. While the LOX-pCMV clone was CXCR6 negative, a LOX-CXCR6 transfected clone yielded positive staining for CXCR6 and a specific signal at about 43 kDa in Western blot experiments. Values of qRT-PCR are shown as ΔC_T, meaning that a 3.33 higher ΔC_T indicates a 10-fold lower mRNA expression. n = 3 independent experiments; examples shown for immunocytochemistry. Scale bars indicate 20 µm, respectively.

Figure 2

Phosphorylation of the extra cellular-regulated kinases ERK1/2 upon stimulation with recombinant CXCR6 in glioblastoma cells. (A) T98G and U251MG glioblastoma cells were stimulated with 25 ng/mL recombinant (rec) CXCR6 for 10 or 15 min, respectively, and phosphorylation of ERK1/2 was investigated by Western blot; equal loading was ensured by reprobing of the membranes with antibodies for the non-phosphorylated kinase ERK2. Stimulation with recombinant CXCR6 yielded a clear phosphorylation signal for both cell lines; (B) when CXCL16 expression was reduced in T98G and U251MG cells to 30–40% by CXCL16-specific siRNA (siCXCL16) as proven by qRT-PCR and Western or dot blotting, ERK1/2 phosphorylation after 10 minutes of stimulation with recombinant CXCR6 was clearly diminished in comparison to control siRNA transfections. Examples of n = 3 independent experiments.

Figure 3

Phosphorylation of ERK1/2 upon stimulation with recombinant CXCR6 or membrane preparations of CXCR6-expressing and control LOX clones. (A) In LOX-CXCL16 clones, stimulation with 25 ng/mL recombinant (rec) CXCR6 (upper panel), as well as with membranes from CXCR6-expressing LOX cells (CXCR6 membranes, 5 µg/mL membrane preparation, middle panel) induced a robust phosphorylation of ERK1/2, while stimulation with control membranes lacking CXCR6 (pCMV membranes, lower panel) failed to activate ERK1/2; (B) in LOX-pcDNA cells that are CXCL16-negative and CXCR6-negative, stimulation with neither recombinant CXCR6, nor CXCR6 membranes, nor pCMV membranes yielded ERK1/2 phosphorylation; (C) LOX-ΔCXCL16 cells lacking the intracellular domain of the transmembrane CXCL16 also did not show any activation of the ERK1/2 signaling pathway upon stimulation with recombinant or membrane expressed CXCR6. Examples of n = 3 independent experiments.

Figure 4

Biological effects of reverse signaling via the CXCR6-CXCL16-axis. (A) To investigate effects on proliferation, DNA contents were measured in LOX-CXCL16 and as a control in LOX-pcDNA cells stimulated (or not) with 50 ng/mL recombinant (rec) CXCR6 for 24 or 48 h (upper part). Corresponding experiments were also performed with T98G glioblastoma cells (lower part); 10% fetal bovine serum (FBS) served as the positive control for proliferation. Unstimulated controls were set to 100%, respectively, and stimulation with CXCR6 did not yield any significant induction or reduction of DNA content. Mean ± SD from n = 3 independent experiments; (B) apoptosis was induced with 0.1 µg/mL camptothecin (Campto), in comparison to equal volumes of solvent control dimethylsulfoxide (DMSO) for 18 h in LOX-CXCL16, LOX-pcDNA and LOX-ΔCXCL16 cells or for 48 h in T98G glioblastoma cells, and simultaneous stimulation with 50 ng/mL recCXCR6 did not reduce cleavage of PARP (cPARP) as detected by Western blot or caspase 3/7 activity as determined by fluorimetric measurement of substrate cleavage, both indicating apoptosis. For Western blotting, equal loading was ensured by reprobing of the membrane with a glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific antibody. Examples (Western blot) or mean values (caspase activity) of n = 3 independent experiments; (C) to investigate migration, scratch assays were performed with LOX clones LOX-CXCL16, LOX-pcDNA and LOX-ΔCXCL16 or T98G glioblastoma cells stimulated with 50 ng/mL recCXCR6 or left unstimulated for controls. Scratch areas were measured at the beginning and after 8 h, and settled areas were determined as the percentage of the initial scratch area. Stimulation with 50 ng/mL CXCR6 promotes migration of LOX-CXCL16 and T98G cells, but not LOX-pcDNA or LOX-ΔCXCL16 cells. Mean ± SD from n = 4 independent experiments; exemplary images are shown with equal magnifications, respectively; scale bar indicates 50 µm; * p < 0.05, ** p < 0.01; (D) fast migrating glioblastoma cells from freshly-dissected glioblastomas mostly show higher CXCL16 mRNA expression levels than the slowly migrating cells of the same tumor preparation. ΔC_T levels are shown in a logarithmic scale (a 3.33 higher ΔC_T value indicates a 10-fold lower mRNA expression), and numbers above the brackets indicate the (linearized) x-fold expression difference between fast and slowly-migrating cells of ten different primary and secondary glioblastoma samples.