Figure 4. Inhibition experiments and transcription analysis exclude the involvement of other chemokine receptors.
(A) Pertussis toxin (PTX, 200 ng/ml) inhibiting Gi/o-signaling of classical chemokine receptors has no effect on s-chemokine-mediated phosphorylation of kinases in U343 or A764 cells. However, in CX3CR1-expressing THP-1 cells (compare Figure 1) CX3CL1 mediated phosphorylation of Akt (stimulation with 1 nM for 20 min) can be inhibited by pre-incubation with PTX. An engineered variant of CX3CL1, the recombinant CX3CR1-antagonist F1 (100 nM, 20 min) induces also signal transduction in primary glioma cells indicating a mechanism different from CX3CR1-binding (and antagonism). Shown are representative Western blots after SDS-PAGE separation of lysates from stimulated U343 or A764 glioma cells stained for pERK 1/2 or pAkt (re-blot to non-phosphorylated kinases, control of equal loading) from 3 independent experiments, compare Figure 4—figure supplement 1. (B) The transcription profile of classical chemokine receptors and chemokine decoy receptors as determined by quantitative RT-PCR shows that the chemokine receptors CXCR3, CXCR4, CXCR6 and CX3CR1 are absent in responsive U343 and A764 and non-responsive LOX cells. However, the atypical chemokine receptor CXCR7 that is known to signal G protein-independently is expressed in responsive cell lines and absent in LOX cells. The chemokine decoy receptors D6 and CCX-CKR are expressed at comparable levels in responsive and non-responsive cells, whereas DARC is absent (n = 3 biological replicates, indicated by diamonds). Additionally, the cytomegalovirus-derived gene US28 that encodes for a putative CX3CR1 receptor could not be detected in these cell lines. The highly glycosylated protein CD44, that may putatively sequester chemokines, was detected at comparable protein levels in U343, A764 and LOX cells (n = 3). (C) To investigate the contribution of CXCR7 in s-chemokine mediated signaling U343 cells were pre-incubated with the CXCR7-antagonist CCX733 (100 nM, 2 hr) and stimulated with 1 nM of s-CXCL16 or s-CX3CL1 for 20 min. Controls ± s-chemokines were pre-incubated with 0.1% DMSO as solvent controls. The CXCR7-antagonist CCX733 does not impair s-chemokine-mediated ERK phosphorylation. Representative Western blots from 3 independent experiments. For biological replicates of western blots please refer to Figure 4—figure supplement 1.
Figure 4—figure supplement 1. Biological replicates of western blot experiments with s-chemokines and Pertussis toxin (compare Figure 4A).
