(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]
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
10
M 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
10
M 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-pp125
monoclonal 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.
Interleukins-1 (IL-1) ()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) .
IL-1 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) .
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 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.
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-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.
In substrate-attached cells grown on glass, there was a
dose-dependent increase of [Ca]
with increasing doses of IL-1
(Fig. 1). The dose of
IL-1
required to generate a maximal
[Ca
]
response was
10
M 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
10
M 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 10
M 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 10
M 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.
Figure 1:
Dose-response data of peak
[Ca]
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.
Figure 2:
Sample
tracings showing the [Ca]
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 10
M IL-1
, whereas no response
was detected in suspended cells, even when stimulated with
10
M IL-1
. B, top trace shows typical [Ca
]
response to IL-1
(10
M) 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
(10
M). 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
.
Figure 3:
Fluorescence micrographs and measurements
of [Ca]
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
(10
M) 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
(10
M) or of cells in suspension incubated
with soluble fibronectin (10 µg/ml) and stimulated with IL-1
(10
M). Note that sample tracings have been
offset vertically for clarity.
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]
signal transduction,
fibroblasts were treated with cytochalasin D (1 µM) for 10
min and then stimulated with 10
M 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.
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-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).
Immunolocalization of pp125 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).
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 (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
(10
M)
after electroporation with pp125
antibody or an
irrelevant antibody.
We
examined the role of focal adhesion kinases in
[Ca]
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
(10
M). 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).
We used another, more
direct approach to assess the role of pp125 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.
Figure 5:
Increased phosphorylation of
pp125after 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
).
We
examined the calcium response of cells spread on fibronectin that were
preincubated with cytochalasin D and then treated with PGE (10
M) 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).
To determine the relative specificity of the genistein
block on PGE-induced calcium flux, cells in suspension were
preincubated with 10, 50, or 100 µM genistein and then
stimulated with PGE
(10
M).
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).
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 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.
Our data show that IL-1-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) ).
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 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.
Several
kinases have been identified in focal adhesions including protein
kinase C, pp60, 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.