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(Received for publication, June 23, 1994; and in revised form, November 28, 1994) From the
Interleukin-1 (IL-1) is an important mediator of inflammation
and also modulates fibroblast metabolism. To assess mechanisms of
IL-1-induced signal transduction and calcium flux, early passage human
fibroblasts were loaded with fura2/AM. Cells grown on coverslips
exhibited dose-dependent
[Ca
Interleukins-1 (IL-1) ( IL-1 The detailed mechanism of action of IL-1
is unknown. Although the initial signaling step appears to involve
binding to plasma membrane receptors(18, 19) , the
mechanisms by which the occupied receptor generates intracellular
signals and the nature of these signals are not well understood. For
example, the cytoplasmic domain of IL-1 receptor (8) shows no
sequence similarity to other protein tyrosine kinase receptors such as
platelet-derived growth factor(20) . Although there have been
conflicting reports of changes in second
messengers(21, 22, 23) , there is some
evidence from early changes in protein phosphorylation (24, 25) that binding of IL-1 to its receptors induces
protein kinase activity(26) . There is also a possible
involvement of G-proteins in signal
transduction(27, 28) . Calcium is an important
second messenger that mediates a large number of cellular processes. An
increase in intracellular Ca IL-1 strongly affects periodontal
connective tissue metabolism(37, 38) . Cells from
these tissues exhibit large numbers of high affinity receptors (15) and have been used extensively to study IL-1 regulatory
mechanisms(15, 17, 39) . Therefore, we have
used human gingival fibroblasts as a model to examine the role of focal
adhesions and associated tyrosine kinases in IL-1-induced calcium
signaling in fibroblasts.
Comparison of IL-1 binding in well-spread, attached cells and in
detached cells was evaluated by affinity labeling with IL-1
In substrate-attached cells grown on glass, there was a
dose-dependent increase of [Ca
Figure 1:
Dose-response data of peak
[Ca
Figure 2:
Sample
tracings showing the [Ca
Figure 3:
Fluorescence micrographs and measurements
of [Ca
As cytoskeletal components interact with
the cytoplasmic domains of integrins at focal adhesions, cytoskeletal
organization may orchestrate signals from the extracellular matrix. To
probe the role of actin filaments in
[Ca
As
protein kinase C is also localized to focal adhesions(51) , we
employed the specific inhibitor H-7 (9 µM; 30 min; (52) ) to evaluate the role of protein kinase C in
IL-1 Immunolocalization of pp125
Figure 4:
Fluorescence micrographs and intracellular
calcium measurements showing dependence of IL-1-induced calcium flux on
focal adhesion kinases. A and B, paired fluorescence
confocal micrographs of single fibroblasts double-stained for talin (A) and for pp125
We
examined the role of focal adhesion kinases in
[Ca We used another, more
direct approach to assess the role of pp125
Figure 5:
Increased phosphorylation of
pp125
We
examined the calcium response of cells spread on fibronectin that were
preincubated with cytochalasin D and then treated with PGE To determine the relative specificity of the genistein
block on PGE
Our findings show that
the dose-dependent calcium flux induced by IL-1 was mediated through
the type I IL-1 receptor and was due predominantly to extracellular
Ca Our data show that IL-1
Tyrosine kinase activity
in focal adhesion proteins is an important signaling system for
integrin-dependent pathways(57, 61) . As shown by the
genistein blockade experiment, tyrosine kinase activity was also
essential for the IL-1-induced calcium flux, an observation that is
supported by the increased phosphorylation of pp125 Several
kinases have been identified in focal adhesions including protein
kinase C, pp60
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6042-6049
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
]
responses that
were maximal at 10
M IL-1
with time to
maximum flux of 50 s. Cells incubated with anti-Type 1-IL-1 receptor
antibody exhibited a 45 nM increase in
[Ca] above baseline
but demonstrated no calcium response after IL-1 treatment.
Incubation with EGTA (5 mM) or thapsigargin (1
µM) caused 75% and 37% reductions, respectively, in the
IL-1-induced [Ca] increase, suggesting that extracellular Ca predominates in IL-1-stimulated calcium flux. Cells in suspension
did not exhibit [Ca] responses to IL-1. The relationship between
[Ca]signaling and
focal adhesions was examined by plating cells on fibronectin or
poly-L-lysine, conditions that either permitted or blocked the
formation of focal adhesions. Cells on fibronectin exhibited
co-distribution of immunostaining for talin, vinculin, IL-1 receptor,
and focal adhesion kinase (pp125
) in focal
adhesions and demonstrated
[Ca]responses with
10M IL-1. Cells on
poly-L-lysine or cells in suspension did not exhibit
co-distribution of pp125, IL-1 receptor, and
focal adhesion proteins and did not exhibit calcium flux. The
dependence of IL-1-stimulated
[Ca] responses on
tyrosine kinases was examined first by treating cells with genistein, a
selective inhibitor of tyrosine kinases. Genistein (100
µM) completely blocked
[Ca]responses to
10M IL-1, whereas its inactive analogue
genistin was not inhibitory. Second, fibroblast lysates were
immunoprecipitated with an antiphosphotyrosine antibody and the lysates
were Western-blotted with an anti-pp125 antibody. Cells grown on fibronectin and stimulated with
IL-1 exhibited tyrosine phosphorylation of pp125 whereas untreated cells or cells grown on
poly-L-lysine and treated with IL-1 showed no reaction.
Fibroblasts electroinjected with anti-pp125monoclonal antibody showed no
[Ca] response, whereas
cells treated with an irrelevant antibody exhibited a normal
[Ca] response.
Collectively, these data indicate that fibroblasts require substrate
attachment and clustering of IL-1 receptors to focal adhesions for
IL-1-induced [Ca] responses. Calcium fluxes are mediated through tyrosine
kinases whose substrates include pp125. These
studies therefore demonstrate that activation of intracellular
signaling pathways by IL-1 is dependent on IL-1 receptor-cytoskeletal
protein interactions.
)are a group of
monocyte-derived peptides that play a pivotal role in regulating the
host response to infection and injury. Two related forms of IL-1 (
and ) exhibit 26% identity at the amino acid sequence
level(1) . These cytokines mediate many features of
inflammation such as fever, the acute phase response, leukocyte
accumulation, and bone resorption (2) as well as connective
tissue degradation and remodelling(3) . IL-1 affects collagen
synthesis by osteoblasts(4) , proteoglycan synthesis by
chondrocytes(5) , and induces proliferation (6) and
collagenase secretion in fibroblasts(7) . and
- share a common, high affinity cell surface receptor which is
thought to mediate their biological
effects(8, 9, 10) . Two types of receptors
for IL-1 have been cloned and characterized biochemically (11) but only the type 1 (80-kDa) IL-1 receptor appears to
mediate biological responses to IL-1(12) . The function of the
type II (60-kDa) IL-1 receptor is not as well understood but does not
appear to transduce signals(13) . Studies of 
I-labeled IL-1 and - binding to human
fibroblasts reveal high numbers (5,000-15,000) of IL-1 receptors
per cell(14) . Evidence from internalization and localization
studies in fibroblasts indicates that IL-1 receptors are concentrated
at focal adhesions(15) . These data suggest that IL-1 may
affect the interactions of fibroblasts with the extracellular matrix by
modulating cell-matrix interactions at focal adhesions(16) .
Indeed, IL-1 causes a transient increase in phosphorylation and
redistribution of talin by rapid post-translational
modification(17) . concentration
([Ca]) is critical for
signal transduction in many cell types(29, 30) . For
example, Ca flux has been implicated in the initial
action of another interleukin, IL-2(31) . However, there are
very few reports on IL-1 regulation of
[Ca]. IL-1 does not
appear to induce [Ca] responses in UMR-160 cells, an osteoblastic cell
line(32) , in human neutrophils(33) , in a T lymphoma
cell line(34) , or in a pre-B cell line(35) . However,
one report on foreskin fibroblasts indicated a very slow increase in
[Ca] 45-60 min
after incubation with IL-1 (36) , the physiological
significance of which is unclear.
Materials
Recombinant human IL-1 and - (40) and IL-1B-PE were obtained from R& Systems
(Minneapolis, MN). The protein was purified by sequential
chromatography (to >97% purity), and the endotoxin level was
determined to be 
0.1 ng/µg IL-1. Fura2 and fura2/AM were
obtained from Molecular Probes. Ionomycin was from Calbiochem. Mouse
monoclonal antibody to the human IL-1 type I receptor was from Genzyme.
Rabbit and mouse antifocal adhesion kinase antibodies were from UBI,
mouse monoclonal antibodies to vinculin (clone hvin-1) and talin (clone
cd4) were from Sigma and anti-human CD4 antibody was from Coulter.
TRITC- and FITC-conjugated antibodies, cytochalasin D, and PGE
were from Sigma. SM-2 Bio-Beads and electroporation cuvettes and
columns were from Bio-Rad. Protein G beads were from Pharmacia Biotech
Inc., and the ECL system was from Amersham. Genistein was from Life
Technologies, Inc., genistin was from Extrasynthase (France), and
1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) was from Toronto
Research Biochemicals.Cell Culture
Human periodontal ligament and human
gingival fibroblasts obtained as described (41) were grown in
T-75 flasks containing minimal essential medium supplemented with
antibiotics (0.17% penicillin V, 0.1% gentamycin sulfate, and 0.01
µg/ml amphotericin) and 15% fetal bovine serum. The cells were
cultured at 37 °C in a humidified atmosphere of 95% air and 5%
CO and passaged after detachment with 0.01% trypsin. Prior
to experiments with IL-1, cells were detached by gentle scraping with a
rubber policeman or by incubation for 8 min at 37 °C with 0.5 M EDTA after washing with phosphate-buffered saline. Fibroblasts
were plated on 60-mm Petri dishes 2 days before performing experiments.
Cells between 4th and 12th passages were used in all experiments.Dye Loading
Cells in suspension were loaded with 1
µM fura2/AM for 15 min at 37 °C followed by 15 min at
room temperature to obtain uniform dye loading. Visual inspection of
loaded cells by fluorescence microscopy showed even distribution of dye
throughout the cytoplasm with no evidence of discrete vesicular
labeling. The cells were washed twice to remove extracellular dye and
finally suspended in buffer containing 145 mM NaCl, 5 mM KCl, 5 mM MgCl, 10 mM glucose, 10
mM HEPES, 1 mM CaCl, with pH adjusted to
7.4. Osmolality of the buffer was measured by freezing point depression
and adjusted to 291 mosm. Attached cells were loaded with 3 µM fura2/AM at 25 °C for 35 min. CaCl was omitted
from the buffer solution where indicated.[Ca
The relative fluorescence intensity of
suspended cells was estimated by using single-wavelength excitation
(380 nm; Hitachi-F2000) or by dual wavelength excitation (355 nm/380
nm; Photon Technology International, London, ON) with emission at 510
nm. The slit widths were set at 5 and 10 nm for the Hitachi system and
2 and 5 nm for the PTI system. Estimates of
[Ca]
Measurement] independent of the precise
intracellular concentration of fura2 were calculated from emitted
fluorescence according to the equation of Grynkiewicz et al.(42) where [Ca] (in nM) = K
Sf/Sb (R - R
)/(R
- R). The K (224 nM) and
Sf/Sb ratio were calculated at 380 nM from 11 excitation wavelength scans of 1 µM fura2
free acid in buffers with [Ca] ranging from 0-39.8 µM. The maximal 346/380
ratio (R) was measured after saturation of
intracellular fura2 with Ca by adding 3 µM ionomycin to allow equilibration with extracellular calcium ions.
The minimal 346/380 ratio (R) measured during
complete disassociation of fura2 from Ca was obtained
by adding 2 mM EGTA to the bathing buffer for each measured
cell. In each experiment, background was estimated by incubation of
cells with 10 mM Mn, and this value was
subtracted from the cellular fluorescence values before the 346/380
ratios were calculated. Intracellular calcium measurements on attached
cells were obtained by a dual excitation epifluorescence illumination
system (PTI, London, Canada) using a Nikon 40 oel objective (NA
1.32). A variable aperture mask (50 µm) within the
optical path was used to restrict measurements to single cells. The
emission signal was filtered by a 510 nm long pass barrier filter and
detected by photon counting. The output of the photon counter was
digitized and recorded on the system computer.Flow Cytometry
Single cell suspensions were
prepared by treating cell cultures with 0.5 mM EDTA and
washing twice with saline buffer. The cells were subsequently stained
with IL-1 conjugated to phycoerythrin (IL-1-PE). Nonspecific
IL-1-PE staining was evaluated by preincubation with 100-fold
molar excess of unlabeled IL-1. Measurements of fluorescent
intensity for individual cells due to specific binding of IL-1-PE
were obtained by comparing cells stained with IL-1-PE and cells
stained with streptavidin-PE as controls to determine fluorescence
channel thresholds. To assess the relative abundance of IL-1 receptors,
cells in suspension were labeled with monoclonal antibody to IL-1 type
1 receptor (1:10 dilution) for 1 h followed by FITC-conjugated goat
anti-mouse (1:100 dilution). Background fluorescence of cells due to
nonspecific staining by second antibody was estimated by measuring
cells without primary antibody. Samples were analyzed on a FACSTAR Plus
flow cytometer (Becton-Dickinson) at 200 cells/s with 488 nm laser
excitation. Emission signals were obtained with a 590/30 nm band pass
filter. Ten thousand cells were analyzed in each sample, and data were
collected in list mode using logarithmic amplifiers. To eliminate
signals due to cellular debris, particles with forward light scatter
comparable to previously established threshold values for fibroblasts
were assessed.Immunofluorescence Staining
Multichamber glass
slides were coated with bovine plasma fibronectin (1 µg/ml) or
poly-L-lysine (1 mg/ml) when required and allowed to dry for 3
h under sterile conditions before plating the cells. Cells were plated
for 24 h, fixed in 3.7% buffered formaldehyde for 15 min at room
temperature, permeabilized with 0.3% Triton X-100, and thoroughly
rinsed with phosphate-buffered saline. Immunofluorescence staining for
the focal adhesion kinase (pp125
) was performed using a
rabbit antifocal adhesion kinase polyclonal antibody (1:20 dilution) or
a mouse monoclonal antifocal adhesion kinase antibody (1:10 dilution)
for 1 h at 37 °C followed by a TRITC-conjugated donkey anti-rabbit
antibody (1:100 dilution) for 1 h at 37 °C. Mouse monoclonal
anti-human talin, anti-human vinculin, anti-human IL-1 type I receptor,
or anti-pp125 antibodies were used at 1:10 or 1:20
dilutions followed by FITC-conjugated goat anti-mouse antibody (1:100
dilution). Nonspecific labeling associated with TRITC-conjugated
reagents was reduced by incubating TRITC-conjugated antibodies with gel
filtration beads followed by centrifugation, a procedure that removes
free dye. Preimmune goat and donkey sera were used to block nonspecific
staining prior to each antibody incubation. Control staining was
performed on the same slide using secondary antibody only. All slides
were counterstained with 4`,6-diamidino-2-phenylindole (1 µg/ml).
Preparations of attached cells were mounted with an anti-fade solution (43) . To immunolabel single cell suspensions, T-75 cell
culture flasks were washed with phosphate-buffered saline followed by
12-min incubation in 0.5 mM EDTA. Cells were fixed by 3.7%
formaldehyde, permeabilized in 0.02% Triton X-100, double-stained for
pp125 and IL-1 receptor or vinculin or talin as described
above, and suspended in phosphate-buffered saline.Electroporation
Mouse monoclonal anti-human
pp125 or an irrelevant antibody (CD4, human lymphocyte
surface marker) was electroinjected into cells by electroporation as
described(44) . Briefly, a Bio-Rad gene pulser with a
capacitance extender and sterile cuvettes were used for experiments. An
800-µl aliquot of 2 10
cells/ml was placed into
an electroporation cuvette (0.4-cm interelectrode distance). The cells
were electroporated in a buffer containing 1.26 mM CaCl, 5.37 mM KCl, 0.52 mM KHPO
, 0.64 mM MgCl,
0.63 mM MgSO, 85.5 mM NaCl, 5.8 mM NaHCO
, 0.50 mM NaHPO,
and 12.5 mM HEPES at a field strength of 250 V/cm and
capacitance of 500 µF. The cells were collected by centrifugation,
suspended in growth medium, allowed to attach, and subsequently loaded
with fura2/AM for [Ca] measurements. Previous detailed assessments of this protocol have
demonstrated that 10
antibody molecules per cell can be
introduced into viable cells (44) and that electroinjected
antibodies can specifically bind and inactivate specific functional
proteins(41) .Confocal Microscopy and Fluorescence
Spectrophotometry
The spatial distribution of probes for talin,
vinculin, IL-1 receptor, and pp125 in focal adhesions was
imaged in single cells on a confocal microscope (Leica CLSM). For
FITC-labeled probes, excitation was set at 488 nm and emission at 530
nm. For TRITC, excitation was set at 530 nm and emission at 620 nm.
Cells were imaged with a 63 oil immersion lens (N.A. 1.4), and
transverse optical sections (nominal thickness = 0.5 µm)
were obtained at the level of cell attachment to the substratum. -PE.
The fluorescence associated with labeled cells was measured with a
fluorescence spectrophotometer (MVP-SP, Leitz, Wetzlar, Germany)
equipped with a X63 PlanApo objective (N.A. 1.4, Leitz). Excitation
light was obtained from a 100-watt, voltage-stabilized (±1%)
mercury arc lamp and a 530/20 filter cube. Emission due to IL-1-PE
labeling was collected with an emission monochromator set to 610/3 nm.
The photomultiplier tube voltage was set to 781 V and gain to 4.
Corrections for background fluorescence intensity and dark current were
made by subtraction of separate unlabeled samples from previously
determined measurements of labeled. Each cell was measured five times,
and the average fluorescence intensity per cell was calculated.
Nonspecific binding was evaluated by incubation with streptavidin-PE
and subtraction from the signals obtained with IL-1-PE staining.Tyrosine Phosphorylation
If focal adhesion
proteins are involved in IL-1 signal transduction, we sought to
determine if IL-1 would increase the tyrosine phosphorylation of the
focal adhesion kinase, pp125. Cells were grown on 120-mm
dishes coated with either fibronectin or poly-L-lysine as
described above and depleted of fetal bovine serum overnight in regular
medium to decrease endogenous phosphorylation of pp125.
Cells were either stimulated with IL-1 (1 nM) or were
unstimulated, immediately washed twice with phosphate-buffered saline
and 100 µM NaVO, harvested with 0.8 ml of
0.25% SDS (w/v) in phosphate-buffered saline containing 100 µM NaVO, and put on ice for 10 min. The lysates were
boiled for 3 min, put on ice for 15 min, and centrifuged, and the
supernatant was collected and made up to a volume of 4 ml. Supernatants
were incubated with a rabbit polyclonal anti-phosphotyrosine antibody
(4 µg/ml), gently shaken at 4 °C for 1 h, and passed three
times through a 0.5 10 cm column (Bio-Rad) containing protein G
beads (Pharmacia). The column was washed with phosphate-buffered saline
plus 100 µM NaVO, and bound proteins were
eluted with 1 ml of 40 mM phenyl phosphate containing 100
µM NaVO, 90 mM NaCl, 0.05% SDS, and 1
mM NaF. Fractions (75-80 µl) were collected, run on
7.5% SDS-polyacrylamide gel electrophoresis, and immunoblotted. Blots
were probed with mouse monoclonal anti-human pp125 (4.2
µg/5 ml for 1 h) and detected with second antibodies (1:1000 for 1
h) and ECL supplied by Amersham.PGE
To assess whether
PGE Studies like IL-1 may require substrate attachment for calcium
flux, we conducted PGE dose-response studies. Attached or
suspended fibroblasts (5 10M cells per
ml) loaded with fura2/AM were stimulated with PGE. We also
examined if IL-1-induced calcium flux may be mediated indirectly
through PGE release. Cells were incubated with indomethacin
to block PGE release.Statistical Analysis
For
[Ca], fluorescence
spectrophotometry and flow cytometry data, means and standard errors
were computed and comparisons between two groups were evaluated with
unpaired Student's t test.
Calcium Response
Initial work with flow
cytometric analysis of IL-1-PE binding to gingival and periodontal
fibroblasts demonstrated that gingival fibroblasts exhibited 25% higher
mean fluorescence compared to periodontal ligament fibroblasts.
Therefore, subsequent work was performed on a gingival fibroblast line
only (PA-4; (41) ). Initial experiments were also performed to
compare rIL-1 and rIL-1
[Ca] responses in fibroblasts.
rIL-1 exhibited 20% higher and more consistent
[Ca] responses than rIL-1
at the same doses. Hence, for this study, all experiments used
rIL-1.] with increasing doses of IL-1 (Fig. 1). The dose of
IL-1 required to generate a maximal
[Ca] response was
10M with a time to maximum flux of 50 s (Fig. 2A). There was no detectable change in
[Ca] observed in suspended
fibroblasts (5 10 cells/ml) even at very high
concentrations (10
M) of IL-1 or
IL-1 (Fig. 2A), a thousandfold higher than the
concentration at which biological responses have been reported
previously(3, 45, 46) . Cells stimulated with
anti-type 1 IL-1 receptor antibody (1:10 dilution) exhibited a small
(45 nM) increase in [Ca] above baseline, but these cells did not respond subsequently to
10M IL-1 (Fig. 2B),
indicating that the IL-1-induced calcium flux was mediated through
the type 1 receptor. Cells incubated in 5 mM EGTA and
stimulated with 10M IL-1 exhibited a
120 ± 10 nM increase of
[Ca] above baseline (n = 3), which was a 75% reduction of the IL-1-induced
increase of [Ca] compared to
cells in medium with calcium (Fig. 2B; p <
0.01). Release of calcium from intracellular stores was examined by
preincubation with 1 µM thapsigargin for 30 min.
Thapsigargin is a tumor-promoting sesquiterpene lactone which blocks
the ATPase required for Ca uptake into intracellular
stores. Cells that were then stimulated with 10M IL-1 exhibited a 305 ± 30 nM [Ca] increase (n = 3), which was a 37% reduction (Fig. 2B; p < 0.01). When cells were incubated with thapsigargin (1
µM) for 30 min, reapplication of thapsigargin 10 min later
produced no further increase of
[Ca], indicating that this
protocol effectively depleted releasable intracellular stores. Thus, in
substrate-attached cells, IL-1-induced calcium flux originated
predominantly from Ca in the extracellular medium.
] in attached
fibroblasts after IL-1 stimulation. Cells were plated on glass and
formed focal adhesions. Cells were stimulated with either vehicle
(water) or with indicated dosages of IL-1. Figure is a log (IL-1
concentration) versus a linear
([Ca])
plot.
] response of suspended and attached fibroblasts to IL-1,
the dependence of calcium flux on external and internal calcium stores,
and signaling through the type 1 IL-1 receptor. A, ratio
fluorimetry of fura2-loaded fibroblasts was used to measure
[Ca]. Attached cells
exhibited a sharp increase of
[Ca] when stimulated
with 10M IL-1, whereas no response
was detected in suspended cells, even when stimulated with
10M IL-1. B, top trace shows typical [Ca] response to IL-1 (10M) of
attached cells pretreated with thapsigargin (1 µM) to
deplete internal calcium stores. Middle trace shows
representative response of cells incubated in buffer without calcium
ions and with 5 mM EGTA. Bottom trace shows
[Ca]response of
attached cells to anti-type 1-IL-1 receptor antibody and subsequently
stimulated with IL-1 (10M). Traces
have been offset vertically for clarification, but the actual baseline
[Ca] before each
treatment was not significantly different from that of the untreated
cells. The marker above each trace indicates the time of
addition of IL-1.
IL-1 Receptor Expression
As the absence of a
detectable calcium flux in suspended cells could be explained by
down-regulation of IL-1 receptors, flow cytometry was used to estimate
IL-1 receptor expression and whether suspended cells bound IL-1.
Cells stained with streptavidin-PE exhibited fluorescence (mean channel
number = 10.2 ± 0.3) which was not significantly
different from cells that were preincubated with unlabeled
10
M IL-1 and then stained with
10
M IL-1-PE (mean channel number
= 11.8 ± 0.3; p > 0.5). Cells stained with
10M IL-1-PE exhibited 
25-fold
higher fluorescence (mean channel number = 295.6 ± 9.8; p < 0.001). We next compared the binding of IL-1-PE to
cells in suspension or in populations of attached cells by
microfluorimetry. The mean fluorescence of attached cells was 18.0
± 0.44 fluorescence units and for suspended cells was 17.2
± 1.67 fluorescence units (p > 0.5), indicating that
IL-1 binding to cells was not affected by attachment to substrate.
Assessment of IL-1 receptor expression by flow cytometry showed that
cells stained with antibody to IL-1 type 1 receptor exhibited 12-fold
higher fluorescence (mean channel number = 117.104 ±
12.5) than cells stained with second antibody only (mean channel number
= 10.49 ± 3.25; p < 0.001), indicating the
presence of abundant type I receptors.Regulation by Extracellular Matrix
A large body of
evidence suggests that integrins, as cell adhesion receptors, can
transduce biochemical signals from the extracellular matrix to the cell
interior (47) . To examine if IL-1-induced
[Ca] signaling was dependent on
focal adhesions, cells were plated for 24 h on glass, fibronectin, or
poly-L-lysine. Immunolocalization of focal adhesions with
antivinculin antibody and imaging by confocal microscopy showed
discrete localization of focal adhesions at the substrate-cell
interface but only in cells plated on glass or fibronectin (Fig. 3A). Cells grown on poly-L-lysine
exhibited diffuse staining throughout the cell, and staining could be
visualized only when photomultiplier tube voltages were increased (Fig. 3B). Cells plated on fibronectin exhibited a
resting [Ca] = 97.6
± 5.9 nM (n = 4) and, when stimulated
with IL-1 (10M), exhibited a
four-fold increase of [Ca] (400
± 37.0 nM; n = 4; Fig. 3C) which was not significantly different from the
calcium flux for cells plated on glass (Fig. 1; p >
0.2). In contrast, cells plated on poly-L-lysine showed no
calcium flux, even at higher IL-1 concentrations (10M; Fig. 3C). Cells in suspension
incubated with soluble fibronectin (10 µg/ml) and stimulated with
IL-1 (10M) exhibited no
[Ca] response (Fig. 3C), demonstrating a requirement for substrate
attachment and not just occupation of fibronectin receptors for
promotion of an IL-1-induced calcium flux (Fig. 3C).
] to illustrate
the dependence of IL-1-induced calcium flux on focal adhesions. A and B, fluorescence confocal micrographs of fibroblasts
stained for vinculin when grown on either fibronectin (A) or
on poly-L-lysine (B). Note that vinculin labeling is
concentrated at the cell attachment sites on fibronectin, whereas on
poly-L-lysine there is diffuse, uniform staining throughout
the cell. In the cells plated on poly-L-lysine, the
photomultiplier tube voltages of the confocal microscope were sharply
increased so that vinculin staining could be visualized. C,
representative tracings demonstrating
[Ca]responses to
IL-1 (10M) of attached fibroblasts
plated on fibronectin (1 µg/ml) or on poly-L-lysine (1
mg/ml). Sample tracings show responses of cells treated with
cytochalasin D (1 µM) and then stimulated with IL-1
(10M) or of cells in suspension incubated
with soluble fibronectin (10 µg/ml) and stimulated with IL-1
(10M). Note that sample tracings have been
offset vertically for clarity.
] signal transduction,
fibroblasts were treated with cytochalasin D (1 µM) for 10
min and then stimulated with 10M IL-1
after the cytochalasin was washed out with fresh buffer. This protocol
is known to completely disrupt cortical actin filaments in fibroblasts (48) . Cytochalasin D-treated cells exhibited no
[Ca] response to IL-1 (Fig. 3C) indicating that calcium flux may be mediated
through filamentous actin inserting into focal adhesions.Role of Focal Adhesion Kinases
As IL-1 receptors
are clustered at focal adhesions in substrate-attached
cells(15) , we reasoned that IL-1 signal transduction occurs
through focal adhesions and possibly involves focal adhesion-associated
kinases(49) . First we used genistein as a selective inhibitor
of tyrosine kinases as it does not inhibit other kinases such as
protein kinase A and protein kinase C (50) . Incubation with
genistein (100 µM; 10 min) followed by stimulation with
10M IL-1 showed complete inhibition
of the calcium response. An inactive analogue of genistein that lacks
anti-tyrosine kinase activity, genistin (100 µM), did not
markedly inhibit IL-1-induced
[Ca] responses (Table 1).
In Western blots of cell lysates that were probed with a rabbit
anti-phosphotyrosine polyclonal antibody, there was complete blockade
of activity with 100 and 50 µM genistein but not with the
genistin, indicating that this protocol was indeed effective.
-induced calcium flux. Cells treated with H-7 showed a 37%
reduction of [Ca] responses to
IL-1 compared to untreated controls, indicating that the IL-1-induced
calcium flux is mediated partly through protein kinase C (Table 1). and
vinculin, or talin or IL-1 receptor showed co-distribution of
probes in focal adhesions of double-labeled cells. In well-spread
fibroblasts, confocal optical sections showed bright staining of
arrowhead-shaped structures reminiscent of focal adhesions at the
substratum-cell interface (Fig. 4, A-D). However,
we were unable to detect co-distribution of pp125,
vinculin, or IL-1 receptor in suspended cells (not shown).
(B)
demonstrating co-distribution at the focal adhesions. C and D, paired confocal micrographs of fibroblasts double-stained
for IL-1 receptor (C) and for pp125(D) showing co-distribution of receptor and focal
adhesion kinase in the focal adhesions. E, confocal micrograph
of a fibroblast stained with FITC-labeled goat anti-mouse antibody
after electroporation in the presence of pp125 monoclonal antibody. The micrograph demonstrates specific
binding of the antibody to focal adhesions and shows that cells
electroinjected with pp125 antibody are fully
capable of attaching and spreading. F,
[Ca] responses of
attached fibroblasts to IL-1 (10M)
after electroporation with pp125 antibody or an
irrelevant antibody.
] signal transduction
directly by electroporating cells in the presence of monoclonal
pp125 antibody (250 µg/ml) or in controls
electroporated with an irrelevant antibody that does not bind to any
known fibroblast antigenic determinants (anti-CD4, a human lymphocyte
surface marker; 250 µg/ml). Cells electroinjected with
pp125 antibody were fixed and stained with FITC-labeled
goat anti-mouse antibody to determine if the electroporation protocol
resulted in antibody binding to pp125. Optical sectioning
with the confocal microscope showed discrete localization of staining
in focal adhesions (Fig. 4E). Cells electroporated with
anti-pp125 or with the irrelevant antibody showed similar
patterns of spreading on glass and fibronectin. The surface areas of
attachment to the substrate as measured by confocal microscopy were not
detectably different, indicating that the pp125 antibody
did not interfere with cell attachment, and immunoprecipitates of
tyrosine-phosphorylated proteins that were immunoblotted with
pp125 antibodies showed inhibition of phosphorylation in
cells electropored with pp125 antibodies. In separate
experiments, electroporated cells were allowed to attach, loaded with
fura2/AM, and subsequently challenged with rIL-1 (10M). Cells electroporated with antibody to pp125 and stimulated with IL-1 exhibited no significant change in
[Ca] (resting
[Ca] = 102.6 ±
4.3 nM; n = 4; IL-1-stimulated
[Ca] = 121.5 ±
6.8 nM; n = 4) whereas cells electroporated
with the irrelevant antibody showed a 3.5-fold higher
[Ca] response (Fig. 4F; resting
[Ca] = 105.6 ±
4.3 nM; n = 4; IL-1-stimulated
[Ca] = 348.3 ±
36.5 nM; n = 4). in IL-1
signal transduction by immunoprecipitating cells with an
antiphosphotyrosine antibody and then probing the cell lysates with a
monoclonal antibody to pp125. Cells grown on
poly-L-lysine did not exhibit tyrosine phosphorylation of
pp125 either before or after IL-1 stimulation (Fig. 5). In contrast, cells grown on fibronectin exhibited
increased phosphorylation after IL-1 stimulation, indicating that
pp125 is itself phosphorylated after IL-1 binds to its
receptor but only if it is aggregated into focal adhesions. The exact
time for detection of pp125 phosphorylation after IL-1
stimulation was difficult to assess because of the cell preparation
steps required for preparation of cell lysates but we estimate it to be
less than 1 min.
after IL-1 stimulation of fibroblasts on
fibronectin but not poly-L-lysine substrates. Fibroblasts were
grown on fibronectin (F, FC) or
poly-L-lysine (P, PC), depleted of fetal
bovine serum overnight to reduce endogenous phosphorylation of
pp125 and either stimulated (F, P) or not stimulated (FC, PC) with IL-1 (1
nM). Cell lysates were immunoprecipitated with
antiphosphotyrosine antibodies, and two different column fractions
(P, PC, F, FC or
P, PC, F, FC were
blotted and probed with
anti-pp125).
Comparison with PGE
As IL-1induced calcium flux in fibroblasts appeared to be
dependent on substrate attachment, we asked if other agents may also
require cell attachment in order to induce a calcium flux. PGE-induced Calcium
Flux was used as a model cytokine to stimulate calcium flux in
attached cells or in cell suspensions. Cells were incubated with
PGE at doses between 10
and
10M. For cells in suspension, maximal
[Ca] responses were obtained at
2.5 10M PGE with time
to maximum flux of 40 s whereas for substrate-attached cells, the
maximal response was obtained at 1.5 10M PGE with time to maximum flux of 20 s. As
IL-1 stimulates PGE release from fibroblasts(28) ,
we determined if the IL-1-induced calcium flux may be mediated through
PGE. Cells were pretreated with 10
M indomethacin to block PGE release. Previous studies on
periodontal fibroblasts have shown that this protocol abrogates
PGE secretion(53) . Indomethacin-treated cells
exhibited normal IL-1-induced calcium responses (resting
[Ca] = 106.9 ±
11.2 nM; n = 4; IL-1-stimulated
[Ca] = 345.5 ±
29.2 nM; n = 4) indicating that the IL-1
response was not likely mediated through PGE. (10M) to determine if the disruption
of actin in focal adhesions and stress fibers would also inhibit
calcium flux. In contrast to IL-1, cells exhibited
[Ca] responses albeit at an
attenuated amplitude ([Ca] baseline = 115 ± 7.3 nM; stimulated with
PGE = 282 ± 35.5 nM; pretreated with
cytochalasin and stimulated with PGE = 168 ±
32.1 nm).-induced calcium flux, cells in suspension were
preincubated with 10, 50, or 100 µM genistein and then
stimulated with PGE (10M).
Genistein reduced but did not completely inhibit the calcium response,
even at 100 µM ([Ca] values: at 10 µM genistein, baseline = 83
± 4.0 nM, stimulated = 211 ± 20.2
nM; at 50 µM genistein, baseline = 87
± 7.8 nM, stimulated = 140 ± 15.0
nM; at 100 µM genistein, baseline = 87
± 5.8 nM, stimulated = 132 ± 9.4
nM).
Calcium Flux
We have demonstrated an absolute
requirement of substrate attachment and focal adhesion formation for
IL-1-induced calcium flux in fibroblasts. A thousandfold higher
than the required dose of IL-1 for maximal Ca response in attached cells failed to induce calcium flux in cell
suspensions. This result was not simply due to dye leakage or cell
death as the cells responded to low doses of ionomycin and also
exhibited calcium fluxes to PGE. Thus, the methods employed
to detect changes in [Ca] were
of adequate sensitivity. The failure to induce calcium flux in
suspended cells was also not because of stripping receptors from the
cell surface as studies with flow cytometry and microfluorimetry
indicated the presence of IL-1 receptors and specific IL-1 binding
in detached and attached fibroblasts. Notably, previous reports have
also failed to show IL-1- or IL-1-induced calcium flux in
suspended UMR-106 cells(32) , T lymphoma cells(34) ,
pre-B cells(35) , or in human neutrophils(33) .
However, Bouchelouche et al.(36) reported a very
delayed (45 min) calcium flux in response to rIL-1 and -
treatment of fibroblasts. These results were not suggestive of a
classical receptor-mediated calcium response. and to a lesser extent originated from
intracellular stores. These data indicate that IL-1 may regulate a
Ca-permeable ion channel and to a lesser extent may
activate inositol 1,4,5-trisphosphate-dependent Ca release from internal stores. In T lymphoma cells, both external
and internal calcium sources contributed significantly to calcium flux
after IL-1 treatment but only when cells were preincubated with
phytohemagglutinin (34) and there was no calcium flux with
IL-1 alone. In view of these reports, it is conceivable that,
depending on the type of cell attachment to the substrate, IL-1 might
differentially activate target cells depending on the predominant
intracellular signaling pathway for the particular cell
type(16) . In the cells studied here, IL-1 alone was able to
trigger a classical, receptor-dependent calcium flux within 50 s of
incubation.-stimulated
[Ca] responses and
phosphorylation of pp125 in attached fibroblasts were
dependent on the previous formation of focal adhesions (Fig. 5).
Attached fibroblasts on poly-L-lysine could not form focal
adhesions, did not exhibit calcium fluxes, and did not demonstrate
increased phosphorylation of pp125 after IL-1 treatment,
whereas cells on glass or fibronectin-coated glass did form focal
adhesions, did exhibit calcium flux, and did exhibit increased
phosphorylation of pp125. Immunolocalization studies with
antibody to type I IL-1 receptor and affinity labeling with
IL-1-PE demonstrated that the receptors were present at focal
adhesions and that there was avid binding of IL-1 to receptors in both
attached and suspended cells. These findings are consistent with
reports that human gingival fibroblasts have high numbers (11,000
± 100) of receptors per cell which bind IL-1 with high
affinity (10
± 10
M
; (16) ) and that 70% of
radiolabeled IL-1 localizes to focal
adhesions(15, 39) . As IL-1 receptors are concentrated
in focal adhesions, it is conceivable that signal transduction is not
so much dependent on the absolute number of receptors per cell but
rather upon the concentration of receptors to localized regions where
receptor density is high. The actual mechanism for signal transduction
through the receptor is not known, but previous data have shown that
IL-1 binding induces phosphorylation via a protein serine/threonine
kinase(19, 25) . Consequently, it is possible that
upon IL-1 binding to its receptor, the receptor phosphorylates
calcium-permeable ion channels on serine and threonine residues and
alters channel-opening probability, a phenomenon that has been
described in other ion channels (for review, see (54) ).Focal Adhesions
Increasing evidence has shown that
the integrin family of cell adhesion receptors can transduce signals
from the extracellular matrix to the cell interior(47) . There
are also convincing data to show that cytokines can regulate
intracellular metabolism by inducing integrin clustering after ligand
binding, which in turn regulates the assembly of cytoplasmic plaques
and stress fibers (55, 56, 57) . Our data
indicated that the formation of focal adhesions can also regulate a
separate cell signaling system, the IL-1 receptor. Thus, fibroblasts
plated on fibronectin or on glass and stimulated with IL-1
exhibited elevations of [Ca],
whereas cells on poly-L-lysine showed no response. Further,
preincubation of suspended cells with soluble fibronectin was not
sufficient to permit IL-1induced calcium flux, apparently because
spreading and focal adhesion formation do not occur in suspended cells.
This finding is consistent with integrin-mediated cell spreading and
regulation of [Ca] in human
endothelial cells (58) , and the generation of spontaneous or
chemoattractant-triggered [Ca] elevations in neutrophils adherent to fibronectin-coated
surfaces(59) . Selective disruption of actin filaments with
cytochalasin D completely inhibited the ability of ILl-1 to
increase [Ca], consistent with
the inhibition of bombesininduced signaling in Swiss 3T3 cells by
cytochalasin(60) . Thus, substrate attachment, focal adhesion
formation, and actin filaments were necessary conditions for
IL-1-induced calcium flux in fibroblasts. Notably, depolymerization of
actin filaments by cytochalasin D also reduced the amplitude of
[Ca] elevations after PGE treatment, but there was not the complete inhibition as seen with
IL-1. Thus, actin filaments are important for function of
ligand-activated calcium-permeable channels, but the degree of
sensitivity appears to be ligand-specific. after
IL-1 stimulation. However, the IL-1 receptor contains no amino acid
sequences in the cytoplasmic domain that are suggestive of tyrosine
kinase activity (16) nor do the cytoplasmic tails of the
or chains of the integrins exhibit such sequences(62) .
Therefore, the increased phosphorylation of pp125 that we
observed is probably not a result of activated IL-1 receptor directly
phosphorylating pp125 but instead may be a reflection of
either intermediate kinase cascades or autophosphorylation.
, and pp125. We
immunolocalized vinculin, talin, IL-1 receptors, and pp125 to common sites, indicating that these proteins are concentrated
in focal adhesions and may be involved in IL-1-induced signal
transduction. Plating cells on poly-L-lysine prevented the
localization of pp125 to the focal adhesions, an
observation consistent with the inhibition of pp125 phosphorylation when NIH 3T3 cells were plated on
poly-L-lysine(63) . Electroinjection of a blocking
antibody to the pp125 completely inhibited calcium flux,
suggesting that the pp125 is an essential component of
the IL-1-induced calcium flux. In contrast, inhibition of protein
kinase C by H-7 pretreatment of cells only partly inhibited
IL-1-induced calcium flux, indicating that this enzyme is not
absolutely essential for the IL-1 signaling pathway. These data suggest
that pp125 phosphorylation is an important component of
IL-1-induced signal transduction and help to explain the dependence of
IL-1-induced calcium flux on focal adhesion formation. Further, these
findings in fibroblasts are consistent with data on ion channels in
carbachol-stimulated cardiac muscle and neurons indicating that calcium
flux is dependent on tyrosine kinases (54) and that
bombesin-stimulated signaling in Swiss 3T3 cells is mediated through
pp125(60) . Collectively, the data support the
notion that IL-1 signal transduction in fibroblasts is dependent on the
nature of the substrate and of the cellular attachments to the
substrate and also suggest a mechanism by which fibroblasts can only
respond to certain agonists when the conditions of their matrix
attachment are appropriate. Thus, degradation of matrix proteins in
inflammatory lesions may lead to significant alterations in cellular
attachments and in the responses of cells to cytokines like IL-1.
)
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