“Inverse signaling” of the transmembrane chemokine CXCL16 contributes to proliferative and anti-apoptotic effects in cultured human meningioma cells
Hattermann et al. Cell Communication and Signaling
“Inverse signaling” of the transmembrane chemokine CXCL16 contributes to proliferative and anti-apoptotic effects in cultured human meningioma cells
Kirsten Hattermann 1
Kareen Bartsch 0
Henrike H. Gebhardt 1
H. Maximilian Mehdorn 0
Michael Synowitz 0
Anne Dorothée Schmitt 0
Rolf Mentlein 1
Janka Held-Feindt 0
0 Department of Neurosurgery, University of Schleswig-Holstein Medical Center , Campus Kiel, Arnold-Heller-Str.3, Building 41, 24105 Kiel , Germany
1 Department of Anatomy, University of Kiel , Otto-Hahn-Place 8, 24118 Kiel , Germany
Background: Chemokines and their receptors play a decisive role in tumor progression and metastasis. We recently found a new signaling mechanism in malignant glioma cells mediated by transmembrane chemokines that we termed “inverse signaling”. According to this hypothesis, soluble (s)-CXCL16 binds to the surface-expressed transmembrane (tm) -CXCL16, and induces signaling and different biological effects in the stimulated cells, so that the transmembrane ligand itself acts as a receptor for its soluble counterpart. Now, we hypothesized that “inverse signaling” via tm-CXCL16 might also take place in meningiomas, a completely different, benign tumor entity. Methods: We used quantitative reverse-transcription polymerase chain reaction, immunocytochemistry and western blot to detect CXCL16 and CXCR6 in human meningioma cells isolated from 28 human meningiomas. Subsequently, we stimulated cultured human tm-CXCL16-positive, CXCR6-negative meningioma cells with recombinant s-CXCL16 and analyzed binding, signaling and biological effects using RNAi silencing to verify specificity. Results: In fact, cultured human meningioma cells considerably express CXCL16, but substantially lack CXCR6, the only known CXCL16 receptor. These receptor-negative cells could bind s-CXCL16, and responded to s-CXCL16 application with activation of the intracellular kinases ERK1/2 und Akt. As a consequence, we observed increased proliferation and rescue of apoptosis of cultured meningioma cells. Since binding and signaling were abolished by siRNA silencing, we concluded that tm-CXCL16 specifically acts as a receptor for s-CXCL16 also in human meningioma cells. Conclusion: These findings underline our recent report on the mechanism of inverse signaling as a broad biological process also observable in more benign tumor cells and contributing to tumor progression.
Chemokines; Chemokine receptors; Cellular communication; Meningioma; Inverse signaling
corresponding receptors at the surface of the same or
another cell. Some tumor cells have high levels of
transmembrane ligands but do not produce the corresponding
receptors for these molecules. Serendipitously, in
malignant brain tumor cells we detected a novel, alternate
mechanism of cell communication which we term
“inverse signaling”: Here, a transmembrane ligand (namely
transmembrane chemokines) acts as a “receptor” for its
soluble counterpart. By studying now benign human
tumor cells derived from the linings of the brain and
spinal cord, so called meningiomas, Hattermann et al.
show that the soluble form of the transmembrane
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chemokine CXCL16 binds to its transmembrane
equivalent in these tumor cells, too. This interaction initiates
intracellular signaling pathways that promote cell growth
and make the meningioma cells more resistant to cell
death. Thus, Hattermann et al. showed that the “inverse
signaling” paradigm also takes place in benign tumor cells,
suggesting that it helps to fine-tune the communication
between cells as a broad biological process.
The chemokine CXCL16 (synonym SR-PSOX) was
originally discovered as a scavenger receptor for oxidized LDL
, and independently as a ligand for the CXC-chemokine
receptor CXCR6, also termed Bonzo, TYMSTR, STRL33
. CXCL16 is synthesized as a transmembrane (tm)
multi-domain molecule consisting of a chemokine domain
followed by a glycosylated mucin-like stalk, a single
transmembrane helix and an intracellular tail. However, a
soluble (s-)CXCL16 form can be generated by constitutive
or induced cleavage from the transmembrane form by the
cell-surface proteases ADAM10 and 17 (ADAM, a
disintegrin and metalloproteinase) [3, 4].
In previous investigations we and others determined
that CXCL16 is definitely involved in tumor progression
of different tumor types, e.g. gliomas, schwannomas,
lung and breast tumors [5–9]. However, apart from its
classical signaling mode in which the proteolytically
released chemokine domain would bind to and signal via
its receptor CXCR6, we recently discovered an alternate
signaling mechanism for transmembrane chemokines in
glioma cells which we termed “inverse signaling” .
According to this novel mechanism, the proteolytically
released (or also recombinant) s-chemokine binds to an
intact transmembrane chemokine specimen inducing
intracellular signaling and biological effects independently
from the expression of the receptor CXCR6. We initially
demonstrated this mechanism in cultivated malignant
human glioma cells, from which we had previously shown
to express high levels of CXCL16, but a lack of the
corresponding receptor CXCR6 .
Apart from that, we could show that solid human
meningiomas - the second most common intracranial
tumors - are also characterized by high expression levels
of CXCL16 while CXCR6 expression is more restricted
. Meningiomas develop from arachnoid cap cells,
and although malignant variants exist (atypical
meningiomas, World Health Organization, WHO grade II and
anaplastic meningiomas, WHO grade III) most
meningiomas (about 90 %) are slowly growing benign (WHO
grade I) tumors [13, 14].
Facing the high CXCL16 expression levels in human
meningiomas, we raised the question if “inverse
signaling” of CXCL16 previously observed in highly malignant
glioma cell might also take place in benign meningioma
cells. Therefore, we analyzed binding, intracellular
signaling effects and different biological readouts in cultured
primary human meningioma cells upon stimulation with
s-CXCL16. This should validate our recent hypothesis on
inverse signaling in more benign brain tumor cells as a
broader biological process.
Meningioma samples were surgical dissected tissues
from the Department of Neurosurgery (Kiel, Germany)
and were obtained in accordance with the Helsinki
Declaration of 1975 with approval of the ethics committee
of the University of Kiel, Germany (file reference: D 442/
11) after written consent of donors. Tumors were
classified according to the WHO criteria into the various
subtypes of meningiomas . The diagnosis was
established by a pathologist. In this study, a total number of
28 meningiomas were included (P1 to P28). With
exception of P10, P25, P27 and P28 all meningiomas were
classified into WHO grade one, P10, P25, P27 and P28
were grad two meningiomas. If possible (enough
material available), for different investigations matched probes
of individual tumor samples were used (see Table 1).
Real-time RT-PCR (qRT-PCR)
RNA was isolated with the TRIZOL reagent (Invitrogen,
Carlsbad, CA, USA), digested by DNase, cDNA was
synthesized, and quantitative real time RT-PCR
(qRTPCR) was performed [12, 15] using TaqMan primer
probes (Applied Biosystems, Foster City, CA, USA):
hGAPDH (Hs99999905_m1), hCXCL16 (Hs00222859_m1),
hCXCR6 (Hs00174843_m1). All 28 different meningioma
samples were analyzed by qRT-PCR. The reaction was
carried out with the MyiQ™ Single Color Real-time PCR
Detection System (BIO-RAD, München, Germany) and
fluorescent data were converted into cycle threshold (CT)
measurements. ΔCT values of each sample were calculated
as CTgene of interest – CT GAPDH. Relative gene expression
was calculated with 2(normalized CT non-stimulated – normalized
CT stimulated) = n-fold of control. A ΔCT value of 3.33
corresponds to one magnitude lower gene expression compared
(glycerinaldehyde-3-phosphate-dehydrogenase). For each gene, logarithmic linear dependence of
CTvalues from the numbers of copies was verified by using
different amounts of cDNA.
It should be noted that all experiments were performed
with cells cultivated from surgical dissected tumors
described above. Primary human meningioma cells were
cultured in glutamine-supplemented Dulbecco’s
modified Eagle’s medium (DMEM) plus 20 % fetal calf serum
(FCS) as described previously . Subcultures from 2
We included 24 WHO grade I and 4 WHO grade II tumor samples and used
cultured meningioma cells from these samples for experiments as indicated
to 4 were used. Meningiomas express both mesenchymal
and epithelial markers of which EMA (epithelial
membrane antigen) and fibronectin can be detected in 90 to
100 % of all investigated solid meningiomas, depending
on marker and investigated cohort [16–18]. Thus,
identity and purity of meningioma cell cultures were proven
routinely by fibronectin (1:100; rabbit polyclonal
antihuman fibronectin; Santa Cruz Biotechnology, Santa
Cruz, CA), EMA (1:20; mouse monoclonal anti-human
EMA; DAKO, Glostrup, Denmark), and glial fibrillary
acidic protein (GFAP) (1:200; mouse monoclonal
antihuman GFAP, DAKO) immunocytochemistry staining.
The primary antibody was omitted for negative controls.
All cultured meningiomas showed a positive staining for
EMA and fibronectin, but lack GFAP expression.
For immunocytochemistry examination, cultured
primary human meningioma cells were seeded on sterile
glass cover slides (50,000 cells/well for routine and
CXCL16 staining, 16,000, 64,000 and 160,000/well to
investigate the density dependent expression of CXCR6)
and grown for two (for density dependent expression
analysis) to up to four days in 20 % FCS-supplemented
DMEM. In case of stimulations, cells were seeded on
glass cover slides (64,000/well), grown for two days and
stimulated for 24 h with 10 nM CXCL16 (PeproTech,
Hamburg, Germany). Then, cell were washed with
phosphate buffered saline (PBS) for three times and fixed
with methanol-acetone (1:1; ice-cold) for 10 min and
4 % para-formaldehyde in PBS for 30 min. Non-specific
binding was blocked with 0.5 % bovine serum albumin
(BSA)/0.5 % glycine in PBS for 60 min. Glass cover slides
were incubated with anti-CXCL16 or anti-CXCR6
primary antibody over night at 4 °C (anti-CXCL16, goat
polyclonal, 1:100 and anti-CXCR6, mouse monoclonal,
1:100, both R&D Systems, Wiesbaden, Germany). The
primary antibody was omitted for negative controls.
After washing steps with PBS, glass cover slides were
incubated with the Alexa Fluor 555-coupled secondary
antibody (red, 1:1,500, donkey anti-goat or anti-mouse
IgG, Invitrogen, Life Technologies, Karlsruhe, Germany)
for 1 h at 37 °C in darkness. After washing with PBS,
nuclei were stained with 4′,6-diamidino-2-phenylindole
(DAPI; Molecular Probes/Invitrogen; 1:30,000, 30 min
room temperature), washed with PBS (3×) and finally
distilled water. After embedding in Immu-Mount
(Shandon, Pittsburgh, PA, USA) digital photography was
performed using a Zeiss fluorescence microscope and
Zeiss camera (Zeiss, Jena, Germany). As a positive control
for CXCR6 immunoreactivity, we used LOX melanoma
cells transfected with an expression vector for CXCR6
(OriGene, Rockville, MD). Native LOX melanoma cells
were a kind gift of Professor Udo Schumacher, University
Cultured primary human meningioma cells were grown
for up to four days on sterile glass cover slides in 20 %
FCS-supplemented DMEM, washed with PBS for three
times and incubated for 15 min on ice. Meningioma
cells were incubated with Cy3-labeled CXCL16 (2 μl
diluted in 50 μl PBS) or Cy3-labeled lactalbumin (2 μl
diluted in 50 μl PBS) for 60 min on ice. For labeling of
CXCL16 and lactalbumin, 2 μg protein were incubated
with a 4-fold excess of monoreactive Cy3-NHS ester (GE
Healthcare, Freiburg, Germany) in 0.2 M NaHCO3
buffer, pH 8.4 (total reaction volume 90 μl). After a
washing step with PBS, cells were fixed in
methanolacetone (1:1; ice-cold) for 10 min, and washed for
three times with PBS. Nuclei were stained with DAPI
(see above), and after embedding in Immu-Mount
(Shandon) digital photography was performed using a
Zeiss fluorescence microscope and Zeiss camera
Primary human meningioma cells (1.5 × 105) were grown
for two days in 20 % FCS-supplemented DMEM, washed
in DMEM with 0.5 % FCS for three times (20 min,
respectively) and stimulated for 10 min, 20 min and
40 min in the same medium with 10 nM CXCL16
(PeproTech) or for 10 min with epidermal growth factor
(EGF; 10 ng/ml; Pepro Tech) or for 20 min with
antiCXCL16 (1 μg/ml; R&D Systems), respectively. In
parallel, control cells were kept without stimulation, these
were also used to confirm lack of CXCR6 expression on
protein level by western blot . Cells were harvested
with 1 ml lysis buffer [50 mM TRIS, 100 mM NaCl,
2 mM EDTA, 1 % Triton-X-100, and 1 mM sodium
vanadate, 1× Halt™ Phosphatase Inhibitor Cocktail (Thermo
Scientific, Bonn, Germany)], 3 μg of protein per sample
was loaded on 10 % SDS-polyacrylamide gels for
electrophoresis and then transferred to a polyvinylidene
difluoride membrane (Hybond™-P PVDF membrane,
GE Healthcare, Freiburg, Germany). As a positive control
for CXCR6, 50 ng/lane recombinant human CXCR6
(Biozol, Eching, Germany) was applied in a separate lane. To
verify CXCL16 expression in membrane isolates, and to
exclude that CXCL16 stimulation might induce CXCR6
expression, meningioma cells were grown until
confluency, stimulated with 10 nM CXCL16 or not, and cells
were harvested and lysed in 5 mH Hepes, pH 7.4. Then,
10 vol.% 200 mM Hepes, 1.4 mM NaCl pH 7.4 was added
to each sample, detritus was removed by centrifugation
(8 min, 800 xg), and membranes were isolated by
centrifugation for 60 min at 14 000 xg. Membrane preparations
were solubilized in 20 mM Hepes, 0.14 mM NaCl, pH 7.4,
and protein amounts of 5 μg/lane were applied to
electrophoresis and blotting as described above. The
polyvinylidene difluoride membranes were blocked with
5 % BSA/TBST and incubated with primary antibodies
against phospho-p42/44 MAPK (Cell Signaling, Beverly,
MA, 1:1,000), phospho-Akt (Cell Signaling, 1:250),
CXCR6 (Acris, Hiddenhausen, Germany, 1:250) or
CXCL16 (PeproTech, 1:250) at 4 °C overnight. The
membranes were incubated with the secondary antibody
(1:30,000, donkey anti-rabbit IgG-HRP, Santa Cruz
Biotechnology) for 1 h at room temperature, and horseradish
peroxidase activity was detected by applying an ECL
Advance Western Blotting Detection Kit (GE Healthcare)
followed by exposure of the membranes to a sheet of
autoradiography film (Hyperfilm™ECL™, GE Healthcare). Equal
protein loading was confirmed by either reprobing the
membranes with anti-ERK-2 (1:200, Santa Cruz
Biotechnology), anti-Akt (1:500, Cell Signaling) or anti-Caveolin-1
(1:200, Santa Cruz Biotechnology) after antibody stripping
for 30 min using Reblot Stripping Solution (Millipore,
Temecula, CA, USA), or by performing second
SDSpolyacrylamide gel with same probes in parallel.
Primary human meningioma cells were plated into 96-well
dishes (1,000 cells/ well), grown for two days in 20 %
FCSsupplemented DMEM, and stimulated in the same medium
with 10 nM CXCL16 (Pepro Tech), 1 μg/ml anti-CXCL16
(R&D Systems) or 10 ng/ml EGF for up to 24 h. In parallel,
control wells were kept without stimulation. Proliferation
was determined by the measurement of tetrazolium salt
WST-1 cleavage (Roche, Mannheim, Germany) and
normalized to non-stimulated control (4 individual wells for
Caspase-3 activity assay
Primary human meningioma cells were plated into
96-well dishes (10,000 cells/ well), grown for two days
in 20 % FCS-supplemented DMEM, washed in 37
°Cthermostatted 0.5 % FCS-supplemented DMEM for three
times (20 min, respectively), and stimulated in the same
medium with 50 μg/ml camptothecin (Sigma Aldrich,
Steinheim, Germany), 10 nM CXCL16 (Pepro Tech) or
with combination of both for up to 24 h. In parallel,
control wells without/with stimulation in DMSO were
used. For detection of active caspase-3 amounts, samples
were washed in PBS and incubated in 100 μl
Homogeneous Caspase 3/7 substrate (Apo-ONE® Homogeneous
Caspase-3/7 Assay; Promega, Madison, USA) for 30 min
according to the manufacturer’s instruction and as
described before . The amounts of active caspase-3 were
determined in relation to a caspase 7 standard (Enzo Life
Science, Lörrach, Germany), and camptothecin-stimulated
wells were set 100 %.
After cultivation of primary human meningioma cells
in DMEM plus 20 % FCS in 6-well dishes (150,000
cells/well; for Western Blot experiments) or on sterile
glass cover slides (15,000 cells/well; for binding
experiments) for 24 h, cells were transfected with siCXCL16
RNA (CXCL16 siRNA ID: s33808; 50 pmol/well; Life
Technologies, Darmstadt, Germany) dissolved in a
mixture of Opti-MEM Medium and lipofectamine
(Life technologies) for 6 h. In parallel a transfection
with silencer select negative control siRNA (Life
technologies) was performed under same conditions.
After transfection cell culture medium was changed
and meningioma cells were cultured for another 24 h
in DMEM plus 20 % FCS. Then, cells were washed
20 min for three times, respectively, with DMEM plus
0.5 % FCS and afterwards stimulated for 15 min with
or without recombinant CXCL16 (10 nM; PeproTech)
dissolved in DMEM plus 0.5 % FCS, lysed and applied
for Western Blot experiments as described above. For
binding experiments primary human meningioma cells
were washed with PBS for three times, incubated for
15 min on ice, and stained with Cy3-labeled CXCL16
(2 μl diluted in 50 μl PBS) or Cy3-labeled lactalbumin
(2 μl diluted in 50 μl PBS) for 60 min on ice. Fixation
of cells and counterstaining of nuclei were performed
as described above.
For controlling the knockdown efficiency, RNA of
transfected cells were purified in parallel with the
PicoPure RNA Isolation Kit (MDS Analytical Technologies,
Sunnyvale, CA) according to the manufacturer’s
instructions, and qRT-PCR using TaqMan primer probes (Applied
Biosystems): hGAPDH (Hs99999905_m1) and hCXCL16
(Hs00222859_m1) was performed as described above.
Cultured human meningioma cells express CXCL16, but
We initially measured CXCL16 and CXCR6 mRNA
(Fig. 1a) expression in cultured meningioma cells used
in our experimental settings. In comparison to CXCL16,
which was clearly detectable in all investigated samples,
CXCR6 transcription was (nearly) undetectable. To
precisely identify the primary cultures as meningioma
cells, each culture was routinely immunostained for
epithelial membrane antigen (EMA), fibronectin and
glial fibrillary acidic protein (GFAP). Examples of this
routine staining are shown in Fig. 1b. Only cultures
showing positive immunostaining for EMA and
fibronectin combined with negative staining for GFAP were
used for experiments. On protein level, we could detect
CXCL16 expression by immunocytochemistry and
western blots of meningioma cell membrane
preparations (Fig. 1c) yielding specific bands for the
transmembrane (tm-)CXCL16. In contrast, CXCR6 could
not be detected on protein level by western blot and
immunocytochemistry (Fig. 1d and e), and its
expression was neither provoked by stimulation with 10 nM
CXCL16 nor by cultivation of different cell densities
ranging from 16,000 to 160,000 cells per well, which
represent the cell densities seeded for subsequent
experiments (Fig. 1e). Thus, we concluded that human
meningioma cells cultivated from different patients
samples substantially express CXCL16 expression while
CXCR6 is absent.
Cultured primary human meningioma cells bind s-CXCL16
and show subsequently activated intracellular signaling
We measured in a next step if cultured
CXCR6negative human primary meningioma cells were able to
bind s-CXCL16 and transduce signaling effects. In fact,
Cy3-labeled s-CXCL16 was found to bind to the
meningioma cells (Fig. 2a), whereas Cy3-labeled
lactalbumin as a negative control did not yield any staining.
Next to binding, s-CXCL16 was able to induce
phosphorylation and thereby activation of both p42/44
extracellular mitogen-activated kinase (ERK1/2) and
Akt in a time-dependent manner (Fig. 2b; exemplary
results are shown). In all independent experiments, we
observed an activation of the respective kinases
between 10 and 40 min. However, since primary cultures
were obtained from various patients, the time course
and intensity of activation differed between cultures.
Stimulation with epidermal growth factor (EGF) served
as a positive control.
Additionally, in accordance with our recent findings in
gliomas , application of a specific CXCL16 antibody
(αCXCL16) which may also induce the intrinsic activity
of tm-CXCL16, resulted in phosphorylation of both Akt
and ERK1/2 (Fig. 2b, right side) after 20 min.
Soluble s-CXCL16 induces proliferation and rescue from
To understand which biological consequences were
induced after s-CXCL16 induced “inverse signaling”, we
investigated proliferation effects and whether s-CXCL16
could prevent apoptosis in CXCR6-negative but
tmCXCL16-positive cultured primary human meningioma
cells (Fig. 3).
Indeed, after 24 h stimulation time with s-CXCL16,
proliferation was induced up to 117 % (P1 and P3;
control = 100 %) and 118 % (P4; control = 100 %; Fig. 3a).
Comparable results were detectable after application of
the specific CXCL16 antibody (αCXCL16). Valuating
these results, one should keep in mind that cultured
human meningioma cells are large and slow-growing cells.
Thus, regarding the fact that the positive control yielded
proliferation of meningioma cells within the same range
(P1 = 130 %; P3 = 132 %; P4 = 126 %; control = 100 %),
CXCL16 stimulation clearly yielded proliferative effects
in these cells.
Additionally, CXCL16 was able to reduce caspase-3
activity after camptothecin treatment in CXCR6-negative
but tm-CXCL16-positive cultured primary human
meningioma cells (Fig. 3b). In detail, for sample P6
camptothecin-induced caspase-3 activity (DMSO as
control = 0 %; camptothecin dissolved in DMSO =
100 %) was reduced up to 51 %, for P7 up to 65 %, and
for P8 up to 84 % after s-CXCL16 application.
Fig. 1 (See legend on next page.)
(See figure on previous page.)
Fig. 1 Expression of CXCL16 and its receptor CXCR6 in cells cultivated from human meningiomas. a Transcription of CXCL16 and its receptor
CXCR6 in human meningioma primary cultures was determined by qRT-PCR (each triangle indicates an individual patient’s sample). Meningioma
primary cultures show high mRNA levels of CXCL16, whereas the expression of CXCR6 is hardly detectable or completely lost. b The identity and
purity of meningioma cultures was assured for every culture by immunocytochemistry on EMA, fibronectin and GFAP, only cultures with positive
reactivity for EMA and fibronectin and negative staining for GFAP were used for further experiments (exemplary data). c Expression of surface
tm-CXCL16 on protein level was confirmed by immunocytochemistry using a CXCL16 specific antibody and a red labeled secondary antibody,
nuclei were counterstained with DAPI. Furthermore, membrane expression of CXCL16 was proven by western blot using membrane preparations
isolated from meningioma cells. Caveolin-1 (Cav-1) served as membrane-specific loading control. d Lack of CXCR6 expression was confirmed on
protein level by western blot. In parallel to subsequent experiments, cell lysates were analyzed using a CXCR6-specific antibody. Whereas the
positive control, human recombinant CXCR6, was sensitively detected (50 ng/lane), cultured meningioma cells did not show any CXCR6
expression. e CXCR6 expression was neither induced by CXCL16 stimulation nor by different cell densities. Stimulation with 10 nM
recombinant human CXCL16 for 24 h, the maximum stimulation time for subsequent experiments, did not yield an induction of CXCR6
expression as shown by ICC and western blot. Additionally, different cell densities in a range from 16,000 to 160,000 cell/well (representing
the cell densities seeded for subsequent experiments) did not influence on the expression of CXCR6 as shown by ICC. Exemplary data of
technical (P3 a and b different cultivation passages) and biological replicates are shown. LOX melanoma cells which were transfected with an
expression vector for human CXCR6 served as a positive control to confirm the specificity of the CXCR6 antibody in ICC, recombinant human
CXCR6 served as positive control for western blot experiments
CXCL16 knockdown showed specificity of results
In a next step, we aimed to show that tm-CXCL16
expression is mandatory to the signaling mediated by
s-CXCL16, to further support the hypothesis of
“inverse signaling” in human meningioma cells. In our
recent investigations in cultured human glioma cells
Fig. 2 CXCL16 binds to meningioma cells and activates intracellular
signaling pathways. a Cy3-labeled s-CXCL16 binds to primary human
meningioma cells, while after incubation with Cy3-labeled lactalbumin
binding could not be observed (equal exposure times, representative
examples). b Stimulation of cultured primary meningioma cells with
10 nM s-CXCL16 (left) yields a time-dependent activation of the signal
kinases ERK1/2 and Akt as detected by western blot using antibodies
for the phosphorylated kinases (pAkt and pERK). Equal protein loading
was confirmed by detection of the non-phosphorylated kinases (Akt
and ERK2), respectively. This effect was also observed when a
CXCL16targeting antibody (right, αCXCL16, 1 μg/ml) was used for stimulations.
EGF served as positive control, shown are representative examples of
at least 3 independent experiments with cultures from different donors
we proved the specificity of this effect by several
different approaches . Due to limited material and
characteristics of cultured meningioma cells (e.g. decreased
proliferation and increased susceptibility to apoptosis
after transfection) we chose binding experiments and
kinase activation as model experiments to prove the
specificity of the results by CXCL16 knockdown.
Results are shown in Fig. 4.
Binding of Cy3-labeled s-CXCL16 to tm-CXCL16
expressed by cultured human meningioma cells was
almost completely abolished after siCXCL16 knockdown
in the tumor cells (Fig. 4a, upper part). In contrast,
control siRNA transfected meningioma cells were still
able to bind s-CXCL16. Cy3-labeled lactalbumin served
as a negative control for testing unspecific binding
background. The expression of CXCL16 as determined
by qRT-PCR was reduced to 6.7 % (P15) and to 46 %
(P16; Fig. 4b).
Additionally, in relation to meningioma cells
transfected with control siRNA, siCXCL16 transfected ones
showed reduced phosphorylation and thereby activation
of ERK1/2 after s-CXCL16 treatment (exemplary results
in Fig. 4a, lower part). These results are sustained by the
reduction of CXCL16 mRNA expression to 11.5 % (P9)
and 28.7 % (P10; Fig. 4b, bottom; control siRNA = 100 %)
as determined by qRT-PCR.
Summarized, in cultured primary human meningioma
cells the classical CXCL16 receptor CXCR6 is lost, but
nevertheless CXCL16 is still able to bind to and
transduce signals into the cells resulting in increased
proliferation and rescue of apoptosis of the cells. The binding
and subsequent cellular activation clearly depends on
the expression of tm-CXCL16 as shown by siRNA
knockdown. Thus, “inverse signaling” of the
transmembrane chemokine CXCL16 occurs in cultured primary
human meningioma cells, and is involved in progression
of human meningiomas.
Fig. 3 s-CXCL16-mediated effects on proliferation and rescue from
apoptosis. a 10 nM s-CXCL16 and 1 μg/ml αCXCL16 promote the
proliferation of slowly growing human meningioma cells as measured
by MTT activity assay. EGF (10 ng/ml) served as positive control.
Control values were set 100 %, and shown are three independent
experiments with cultures from different donors, with mean ± SD
from 4 technical replicates. b When apoptosis was induced in
human primary meningioma cells by 50 μg/ml camptothecin
(Campto = 100 %), additional incubation with 10 nM CXCL16
could drastically reduce the caspase-3 activity. Shown are three
independent experiments with cultures from different donors
We recently discovered in highly malignant glioma cells
a novel form of para- or autocrine signaling mechanism
for transmembrane chemokines which we termed
“inverse signaling” . This new signaling concept
anticipates that the proteolytically released chemokine domain
(s-chemokine) specifically binds to its transmembrane
counterpart resulting in activation of intracellular
kinases and induction of proliferation and anti-apoptosis.
Thus, in this novel signaling concept, the
transmembrane ligands act as receptors for their soluble
counterparts. This signaling concept war sustained by various
silencing and transfection experiments. It requires di-/
multimerization of the tm-chemokine, which can also be
induced by di-, but not by monovalent (Fab-fragments)
antibodies. We could also show that the intracellular
domain (that contains motifs for binding adapter proteins)
is essential for signaling. In the present investigation we
now describe that cultured primary human meningioma
cells exhibited high CXCL16 expression in vitro, whereas
the CXCL16-specific chemokine receptor CXCR6 was
mostly absent. Facing the discrepancy between
tm-chemokine expression and the lack of the corresponding
receptor also in meningioma cells, we hypothesized
that the concept of inverse signaling as a broad
biological concept may be extended further to more benign
The expression of the chemokine CXCL16 has been
reported previously for monocytes/macrophages, B
cells, dendritic cells, keratinocytes and endothelial
cells [1, 5, 19, 20] while the receptor CXCR6 has
been detected quite selectively on activated T cells,
NK cells and bone marrow plasma cells [2, 20, 21].
Apart from its physiological expression, CXCL16 is
pathologically expressed in cancer cells of different
origin including malignant glioma cells and reactive
astrocytes in situ and in vitro [5–7, 11, 22].
However, although cultured human meningioma cells
lack the CXCL16-specific receptor CXCR6, stimulation
with s-CXCL16 was able to transduce intracellular
signaling effects. The recombinant s-CXCL16 was able to
bind to the cell surface and to induce phosphorylation
and thereby activation of the kinases ERK and Akt in a
time dependent manner. These results are in a way
comparable with previous ones which described
CXCL16mediated ERK activation in schwannoma cells [6, 23], or
Akt activation in human aortic smooth muscle cells .
However, as the human cultured meningioma cells lack
the corresponding receptor CXCR6, a classical receptor
mediated signaling could be excluded. To investigate the
relevance of tm-CXCL16 in the observed signaling
process, we performed siCXCL16 knockdown
experiments and were able to show that binding to and
induction of intracellular signals clearly depends on the
expression of tm-CXCL16.
In general, the activation of the ERK signal transduction
pathway often results in elevated cell growth, and the Akt
pathway in anti-apoptotic mechanisms of tumor cells.
Therefore, we wanted to know whether stimulation with
s-CXCL16 could induce these biological responses in
cultured primary human meningioma cells. In fact, we could
show that both effects – activation of proliferation and
rescue of apoptosis – were triggered by s-CXCL16 in
cultured CXCR6-negative, but tm-CXCL16-positive
meningioma cells. Additionally, for a more general view on the
biological role and regulation of the “inverse signaling” of
CXCL16 one should keep in mind that tm-chemokines
Fig. 4 Binding of s-CXCL16 and signaling upon stimulation with s-CXCL16 depends on the tm-CXCL16 level. Expression of the tm-CXCL16 was
reduced in human meningioma cells by siRNA targeting CXCL16 (for controls, an unspecific control siRNA was used). a Knockdown of CXCL16
yielded a drastically reduced binding signal of Cy3-labeled s-CXCL16 (upper part). Additionally, we observed reduced activation of the ERK signaling
pathway as determined by Western blot on kinase phosphorylation (lower part). b The efficient knockdown of CXCL16 was determined by qRT-PCR
(bottom right). Shown are 2 independent experiments with meningioma primary cultures from different donors
like CXCL16 need to be cleaved by the cell surface
proteases ADAM10 and ADAM17 in order to
produce their soluble counterparts . Regarding the
biological role of CXCL16 in tumors, contrasting its
already mentioned effects in glioma and schwannoma
cells [5, 6, 21], high CXCL16 expression correlates
with good prognosis and increased levels of
tumorinfiltrating lymphocytes in renal cancer , while in
human rectal cancer a downregulation of CXCL16 has
been reported . However, pro-tumorigenic effects of
CXCL16 have been reported for several other tumors, e.g.
breast cancer [9, 27], lung cancer, and is discussed as a
promotor of inflammation-associated cancer in general
, which is underlined by our own findings, that
microglia/macrophages isolated from human glioma
samples show high expression levels of CXCL16 themselves
. Thus, the inverse signaling mechanism of CXCL16
we here describe for meningiomas may occur in a variety
of benign and malignant tumors, modulating amongst
others the interplay between tumor and immune cells and
influencing on tumor progression. Targeting the tumor
promoting effects of CXCL16 might be a promising
therapy amendment e.g. for atypic meningiomas and other
tumors with limited treatment options.
Summarized, in the present investigations we were able
to show that “inverse signaling” takes place in cultured
primary human meningioma cells resulting in increased
proliferation and rescue of apoptosis of these cells.
Thereby, we verified this new mechanism beside its
appearance in malignant glioma cells  in a different
system, and showed that “inverse signaling” seems to be
a broad biological process as it is also observable in
more benign tumor cells.
ADAM: A disintegrin and metalloproteinase; CT: Cycle of threshold;
DMEM: Dulbecco’s modified Eagle’s medium; EGF: Epidermal growth
factor; EMA: Epithelial membrane antigen; FCS: Fetal calf serum;
GAPDH: Glycerinaldehyde-3-phosphate-dehydrogenase; GFAP: Glial
fibrillary acidic protein; ICC: Immunocytochemistry; qRT-PCR: Quantitative
reverse-transcription polymerase chain reaction; s: Soluble; tm: Transmembrane;
WHO: World Health Organization
This work was supported by the University of Kiel, the “Deutsche
Forschungsgemeinschaft” (HE 3400/5-1; ME 758/10-1), and by the popgen 2.0
network [(P2N; supported by a grant from the German Ministry for Education
and Research (01EY1103)]. The funding bodies did not have any influence on
the design of the study, the collection, analysis and interpretation of data or in
the writing of the manuscript.
Availability of data and material
The datasets generated and/or analysed during the current study are available
from the corresponding author on reasonable request.
KH, JHF, RM: Conception and design of the study, interpretation of data,
writing of the manuscript. KB, HG: Performance of experiments, data analysis.
ADS, HMM, MS: Acquisition of patients’ material and data. All authors have
significantly contributed to the content of the manuscript including carefully
and critically revising and approved the final manuscript version.
Ethics approval and consent to participate
All human materials were obtained in accordance with the Helsinki
Declaration of 1975 with approval of the ethics committee of the University
of Kiel, Germany (file reference: D 442/11) after written consent of donors.
1. Shimaoka T , Kume N , Minami M , Hayashida K , Kataoka H , Kita T , Yonehara S. Molecular cloning of a novel scavenger receptor for oxidized low density lipoprotein, SR-PSOX, on macrophages . J Biol Chem . 2000 ; 275 : 40663 - 6 .
2. Matloubian M , David A , Engel S , Ryan JE , Cyster JG. A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo . Nat Immunol . 2000 ; 1 : 298 - 304 .
3. Abel S , Hundhausen C , Mentlein R , Schulte A , Berkhout TA , Broadway N , et al. The transmembrane CXC-chemokine ligand 16 is induced by IFN-gamma and TNF-alpha and shed by the activity of the disintegrin-like metalloproteinase ADAM10 . J Immunol . 2004 ; 172 : 6362 - 72 .
4. Gough PJ , Garton KJ , Wille PT , Rychlewski M , Dempsey PJ , Raines EW . A disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of CXC chemokine ligand 16 . J Immunol. 2004 ; 172 : 3678 - 85 .
5. Ludwig A , Schulte A , Schnack C , Hundhausen C , Reiss K , Brodway N , Held-Feindt J , Mentlein R. Enhanced expression and shedding of the transmembrane chemokine CXCL16 by reactive astrocytes and glioma cells . J Neurochem . 2005 ; 93 : 1293 - 303 .
6. Held-Feindt J , Rehmke B , Mentlein R , Hattermann K , Knerlich F , Hugo H-H , et al. Overexpression of CXCL16 and its receptor CXCR6/Bonzo promotes growth of human schwannomas . Glia . 2008 ; 56 : 764 - 74 .
7. Hattermann K , Ludwig A , Gieselmann V , Held-Feindt J , Mentlein R. The chemokine CXCL16 induces migration and invasion of glial precursor cells via its receptor CXCR6 / Bonzo . Mol Cell Neurosci . 2008 ; 39 : 133 - 41 .
8. Hu W , Liu Y , Zhou W , Si L , Ren L. CXCL16 and CXCR6 are coexpressed in human lung cancer in vivo and mediate the invasion of lung cancer cell lines in vitro . PLoS One . 2014 ; 9 : e99056 .
9. Fang Y , Henderson FC Jr, Yi Q , Lei Q , Li Y , Chen N. Chemokine CXCL16 expression suppresses migration and invasiveness and induces apoptosis in breast cancer cells . Mediators Inflamm . 2014 ; 2014 : 478641 .
10. Hattermann K , Gebhardt H , Krossa S , Ludwig A , Lucius R , Held-Feindt J , Mentlein R. Transmembrane chemokines act as receptors in a novel mechanism termed inverse signaling . eLIFE . 2016 ; 5 : e10820 .
11. Hattermann K , Held-Feindt J , Ludwig A , Mentlein R. The CXCL16-CXCR6 axis in glial tumors . J Neuroimmunol . 2013 ; 260 : 47 - 54 .
12. Li G , Hattermann K , Mentlein R , Mehdorn HM , Held-Feindt J. The transmembrane chemokines CXCL16 and CX3CL1 and their receptors are expressed in human meningiomas . Oncol Rep . 2013 ; 29 : 563 - 70 .
13. Louis DN , Scheithauer BW , Budka H , von Deimling A , Kepes JJ . Meningiomas, in World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of the Nervous System . In: Kleihues P and Cavenee WK, editors. Lyon: IARC Press ; 2000 . p. 176 - 184 .
14. Whittle IR , Smith C , Navoo P , Collie D. Meningiomas . Lancet. 2004 ; 363 : 1535 - 43 .
15. Tong Y , Mentlein R , Buhl R , Hugo H-H , Mehdorn HM , Held-Feindt J. Overexpression of midkine contributes to anti-apoptotic effects in human meningiomas . J Neurochem . 2007 ; 100 : 1097 - 107 .
16. Ng HK , Wong AT . Expression of epithelial and extracellular matrix protein markers in meningiomas . Histopathology . 1993 ; 22 : 113 - 25 .
17. Das A , Tan WL , Smith DR. Expression of extracellular matrix markers in benign meningiomas . Neuropathology . 2003 ; 23 : 275 - 81 .
18. Hitchcock E. Morris CS Immunocytochemistry of intracranial meningiomas . J Neurooncol . 1987 ; 5 : 357 - 68 .
19. van der Voort R , van Lieshout AW , Toonen LW , Sloetjes AW , van den Berg WB , Figdor CG , et al. Elevated CXCL16 expression by synovial macrophages recruits memory T cells into rheumatoid joints . Arthritis Rheum . 2005 ; 52 : 1381 - 91 .
20. Hase K , Murakami T , Takatsu H , Shimaoka T , Iimura M , Hamura K , et al. The membrane-bound chemokine CXCL16 expressed on follicle-associated epithelium and M cells mediates lympho-epithelial interaction in GALT . J Immunol. 2006 ; 176 : 43 - 51 .
21. Kim CH , Kunkel EJ , Boisvert J , Johnston B , Campbell JJ , Genovese MC , et al. Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with extralymphoid tissue homing potential . J Clin Invest . 2001 ; 107 : 595 - 601 .
22. Ludwig A , Mentlein R. Glial cross-talk by transmembrane chemokines CX3CL1 and CXCL16 . J Neuroimmunol . 2008 ; 198 : 92 - 7 .
23. Rangwala R , Banine F , Borg J-P , Sherman LS . Erbin regulates mitogen-activated protein (MAP) kinase activation and MAP kinase-dependent interactions between merlin and adherens junction complexes in Schwann cells . J Biol Chem . 2005 ; 280 : 11700 - 97 .
24. Chandrasekar B , Bysani S , Mummidi S. CXCL16 signals via Gi , phosphatidylinositol 3 -kinase, Akt, IκB kinase, and nuclear factor-κB and induces cell-cell adhesion and aortic smooth muscle cell proliferation . J Biol Chem . 2004 ; 279 : 3188 - 96 .
25. Gutwein P , Schramme A , Sinke N , Abdel-Bakky MS , Voss B , Obermüller N , et al. Tumoural CXCL16 expression is a novel prognostic marker of longer survival times in renal cell cancer patients . Eur J Cancer . 2009 ; 45 : 478 - 89 .
26. Wågsäter D , Hugander A , Dimberg J. Expression of CXCL16 in human rectal cancer . Int J Mol Med . 2004 ; 14 : 65 - 9 .
27. Xiao G , Wang X , Wang J , Zu L , Cheng G , Hao M , Sun X , Xue Y , Lu J , Wang J. CXCL16/CXCR6 chemokine signaling mediates breast cancer progression by pERK1/2-dependent mechanisms . Oncotarget . 2015 ; 6 : 14165 - 78 .
28. Darash-Yahana M , Gillespie JW , Hewitt SM , Chen YY , Maeda S , Stein I , Singh SP , Bedolla RB , Peled A , Troyer DA , Pikarsky E , Karin M , Farber JM . The chemokine CXCL16 and its receptor, CXCR6, as markers and promoters of inflammationassociated cancers . PLoS One . 2009 ; 4 : e6695 .
29. Hattermann K , Sebens S , Helm O , Schmitt AD , Mentlein R , Mehdorn HM , Held-Feindt J. Chemokine expression profile of freshly isolated human glioblastoma-associated macrophages/microglia . Oncol Rep . 2014 ; 32 : 270 - 6 .