1 Department of Orthopaedic Surgery and 3 Section of Rheumatology, Department of Medicine, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932; and 2 Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino 142292, Russia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The possibility that membrane depolarization of synovial
fibroblasts caused by interleukin-1 (IL-1
) was mediated by
protein kinase C (PKC) and Ca2+
influx was studied using inhibitor and activator analysis. The effect
of IL-1
was blocked by bisindolylmaleimide I, an inhibitor of PKC,
and by the Ca2+ channel blockers
nifedipine and verapamil. In other experiments, PKC was activated using
phorbol 12-myristate 13-acetate, and
Ca2+ influx was increased by means
of a Ca2+ ionophore. Simultaneous
application of phorbol ester and
Ca2+ ionophore in the absence of
IL-1
mimicked the depolarization caused by IL-1
. The results were
consistent with the hypothesis that, under the conditions studied,
activation of PKC and Ca2+ influx
are necessary and sufficient processes in the transduction of IL-1
by synovial cells leading to membrane depolarization. The
essential role of protein phosphorylation and
Ca2+ influx in the early
electrophysiological response of synovial fibroblasts to IL-1
was
therefore established. The role of IL-1
-induced depolarization in
regulating protein expression by the cells remains to be determined,
but the results reported here, taken together with observations that
protein phosphorylation and Ca2+
influx also mediate the effect of IL-1
on protease production (1, 2), suggest that electrophysiological changes are actually part of the
pathway for expression of proteases in response to IL-1
.
membrane potential; nystatin; voltage clamp
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE CYTOKINE interleukin-1 (IL-1
) is an important
regulator of synovial cells. Binding of IL-1
by a cell
surface receptor results in signal transduction leading to the
expression of many different proteins, including metalloproteinases and
IL-1
itself (5, 8, 19). Dysregulation of this response, especially chronic overproduction of metalloproteinases, can lead to the destruction of joint cartilage and other adverse changes (17, 23).
A spectrum of second messengers, enzymes, G proteins, and transcription
factors have been implicated as components of IL-1 signal transduction
in various cell types (7, 16). The wide range of reported processes and
mechanisms suggests that the consequences of ligation of IL-1 with
its receptor vary with cell type, and perhaps even within a cell type
under different study conditions.
Contemporary ideas regarding the effect of IL-1 in inexcitable cells
do not attribute a significant role to electrophysiological factors (7,
16). Nevertheless, using the perforated-patch method, we observed
changes in the current-voltage
(I-V)
characteristics of rabbit synovial fibroblasts during transduction of
IL-1
. When aggregated, the cells exhibited two states having high
and low membrane potentials
(Vm),
respectively. IL-1
caused cells to transit from the high to the low
state, particularly when the cell was voltage clamped under conditions
that favored Ca2+ influx (14).
Thus it is possible that IL-1
can affect important cellular
processes known to be influenced by
Vm, such as
transport of amino acids and exocytosis.
The aim of this study was to determine the mechanism responsible for
the previously observed joint effect of IL-1 and voltage clamp on
the Vm of
synovial cells. We hypothesized that activation of protein kinase C
(PKC) and Ca2+ influx were
necessary and sufficient processes for the effect of IL-1
, and this
was established using inhibitor and activator analysis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells. Rabbit synovial fibroblasts (HIG-82, American Type Culture Collection) were grown at 37°C with 5% CO2 in 25-ml polystyrene flasks containing F-12 medium (GIBCO BRL) with 10% fetal bovine serum and without antibiotics. For passage, confluent cultures were trypsinized (1 ml, 0.08%) for 3-5 min, after which 4 ml of medium were added and the suspended cells were centrifuged, resuspended, and then seeded (106 cells) into 4 ml of medium. For electrophysiological measurements, 105 cells were added to 35-mm petri dishes and incubated at 37°C for 24 h, after which the cells were treated for 2 min with 1 ml of 0.01% collagenase and 0.01% hyaluronidase. This step, which was mild by comparison with routine trypsinization of the cells, was necessary to obtain stable gigaseals. The cells were incubated in medium for 40-60 min at 37°C to allow recovery from the enzyme treatment. The medium was then replaced with bath solution, and all measurements were made in bath solution at 25°C, employing the cells (10-20 µm in diameter) that remained adherent to the bottom of the petri dish (most cells remained adherent following enzyme treatment).
Electrodes.
The nystatin perforated-patch method (9) was used to measure the
transmembrane current under voltage clamp. The perforated-patch method
was employed because it permitted use of the whole cell configuration
to measure electrical properties of the cell while preventing diffusion
of small signaling molecules from the cell into the electrode, thereby
preserving intracellular regulation. Glass capillaries 1.0 mm in
diameter were pulled in two steps (PB-7, Narishige) and fire polished
in a microforge (MF-9, Narishige). The resistance of the electrodes was
7-9 M in bath solution. The pipette salt solution was (in mM)
125 monopotassium aspartate, 30 KCl, 4 NaCl, and 10 HEPES-KOH (pH 7.2;
318 mosmol/l, calculated). The composition of the bath solution was (in
mM) 145 NaCl, 5.4 KCl, 1.5 CaCl2,
1.0 MgCl2, 5.0 HEPES-NaOH, and 5.0 glucose (pH 7.3; 328 mosmol/l, calculated). Because nystatin interfered
with gigaseal formation, the tip of the pipette was filled with a
nystatin-free solution before the addition of pipette solution
containing 0.3 µg/ml nystatin. The gigaseal was formed during the
time needed for the nystatin to diffuse to the tip of the micropipette
(9).
Inhibitors and activators.
The dish containing the clamped cell was perfused with bath solution
containing the agent under study. The agents used were human
recombinant IL-1 (Sigma no. I 4019), phorbol 12-myristate 13-acetate
(PMA; Sigma no. P 8139), bisindolylmaleimide I (Calbiochem no. 203290),
A 23187 Ca2+ ionophore (Sigma no.
C 7522), verapamil (Sigma no. V 4629), and nifedipine (Sigma no. N
7634). IL-1
was added into solution in the presence of 0.1% bovine
albumin (Sigma no. A 2153) as carrier protein. Control experiments
showed that 0.1% bovine albumin did not influence the
I-V
characteristics of the cells. PMA, bisindolylmaleimide I, A-23187
Ca2+ ionophore, verapamil, and
nifedipine were dissolved in DMSO (Sigma no. D 5879) and then added to
the bath solution. Final concentration of DMSO did not exceed 0.3%;
control experiments showed that it did not influence the
I-V
characteristics of the cells.
Electrical measurements.
Gigaseals (~10 G) were formed under negative pressure (5-10
cm H2O), typically within
0.5-5 min; the success rate was >50%. After gigaseal formation,
the negative pressure was removed and the nystatin channels formed
within 5-15 min; the resistance of the perforated-patch membrane
was 40 ± 20 M
. Gigaseals and nystatin pores usually remained
stable for hours.
Procedure.
The influence of different agents on the IL-1-induced changes in the
I-V
curves of synovial fibroblasts was studied, and the responsible
mechanisms were evaluated using substances having known physiochemical
properties. In each experiment, the
I-V
curve was measured initially in bath solution and then measured again after perfusion of the dish with bath solution containing the agent or
combination of agents under study.
I-V
curves obtained before and after addition of the test agent were
compared to determine the influence of the agent. Each measurement was
repeated five times using five cells in five different dishes.
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of IL-1.
The effect of IL-1
on the
I-V
curve of cells having a high
Vm is shown in
Fig. 1. After the initial (control) measurements in bath solution
(Fig. 1A), the cells
were held at
30 mV for 15 min before the addition of IL-1
(1 ng/ml of bath solution), whereupon the
I-V
curves were measured again after 5 min (Fig. 1D). The results of normalizing
each control curve to the mean and the means ± SE of these curves
are shown in Fig. 1, B and C, respectively. The corresponding
results following addition of IL-1
are shown in Fig. 1,
E and
F. IL-1
caused an increase in
inward current and a shift in the reversal potential (Table 1), confirming our previous results (14).
|
Effect of IL-1 in presence of PMA.
PMA, an activator of PKC, mimicked the effect of IL-1
on induction
of metalloproteinase synthesis by HIG-82 cells at 0.15 µM (1),
possibly indicating that protein phosphorylation by PKC was responsible
for the membrane depolarization caused by IL-1
. To test this
hypothesis, we studied the effect of PMA on Vm using the same
protocol as for IL-1
. Control measurements showed that 1.5 µM PMA
did not change the
I-V
curve or the Vm (Table 1). Thus activation of PKC by PMA did not produce membrane depolarization.
|
Effect of IL-1 in presence of bisindolylmaleimide I.
Although PKC alone was not responsible for changing the
I-V
curves following addition of IL-1
, PKC could have functioned with
other cell systems to produce membrane depolarization. To test this
hypothesis, we studied the effect of IL-1
in the presence of
bisindolylmaleimide I, a protein kinase inhibitor that is specific for
PKC and that inhibits the activity of all four PKC subtypes with
similar potency (21). Addition of 3 µM bisindolylmaleimide I alone to
the bath solution did not change the
I-V
curve or the Vm
of the cells (Table 1).
|
Effect of PMA in presence of
Ca2+ ionophore.
Previous evidence suggested that the system responsible for
intracellular Ca2+ homeostasis was
the system that worked with PKC to mediate the effect of IL-1 (7,
18). If so, then addition of PMA and Ca2+ ionophore together should
produce membrane depolarization. Addition of 10 µM
Ca2+ ionophore did not alter the
Vm (Table 1).
|
Effect of IL-1 in presence of nifedipine and
verapamil.
To clarify the role of Ca2+ in the
effect of IL-1
on
Vm, we used the
Ca2+ channel blockers nifedipine
and verapamil. Neither 50 µM nifedipine nor 10 µM verapamil altered
the Vm.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein expression by synovial cells in culture in response to IL-1
typically occurs after exposure for 1-2 days (5, 8, 19), and there
is some evidence suggesting that PKC and
Ca2+ are involved somewhere in the
signaling pathway (1). Before our work (14), the initial steps of
IL-1
transduction in synoviocytes were unstudied. We found that
IL-1
induced relatively fast (~5 min) switching of
electrophysiological states of synovial fibroblasts, leading to
transitions to a state of low
Vm.
IL-1 activates different signaling pathways in different cell types,
and only some of the pathways involve PKC and
Ca2+ (7, 16). The hypothesis of
this study was that one particular pathway of the large number of
theoretically possible pathways was responsible for the early
electrophysiological response of synovial fibroblasts to IL-1
. As
hypothesized, we found that activation of PKC and
Ca2+ influx were necessary and
sufficient processes in the transduction of IL-1
by synovial cells,
under the conditions studied, to cause membrane depolarization.
Membrane depolarization occurred soon after ligation of IL-1 by its
receptor; within 5 min after addition of IL-1
, the mean reversal
potential of the cells decreased from
69 to
29 mV (Fig. 1, Table 1). Bisindolylmaleimide I alone had no effect on
Vm, indicating
that activation of PKC was not necessary to maintain a high
resting potential. However, the IL-1
-induced decrease in
Vm was blocked by
bisindolylmaleimide I (Fig. 3), thereby implicating participation of
PKC in the effect of IL-1
.
The Ca2+ channel blockers
nifedipine and verapamil had no effect on
Vm, indicating
that passage of Ca2+ from the bath
solution into the cell through voltage-gated ion channels was not
necessary for maintenance of the cell resting potential. However,
nifedipine (Fig. 5) and verapamil (Fig. 6) blocked the action of
IL-1, indicating that Ca2+
influx was an essential step in the signal pathway of IL-1
leading to membrane depolarization.
The question of whether PKC activation and
Ca2+ influx were sufficient to
produce depolarization was studied by exposing the cells to PMA and
Ca2+ ionophore in the absence of
IL-1. Neither agent alone affected the
Vm or
I-V
characteristics of the cells. Thus neither activation of PKC alone nor
Ca2+ influx alone was sufficient
to mimic the effect of IL-1
on
Vm. Jointly,
however, the agents produced a membrane depolarization that was
indistinguishable from the effect of IL-1
(Fig. 4).
It could be argued that PKC in the synovial cells was self-activating
and that the basic role of IL-1 was to increase
Ca2+ influx, which then permitted
the already activated PKC to catalyze processes that led to
depolarization. However, this view is inconsistent with the observation
that Ca2+ ionophore alone did not
mimic the effect of IL-1
(Table 1). A similar argument in favor of
an effect of IL-1
on PKC activation with spontaneously open
Ca2+ channels is inconsistent with
the evidence that PMA alone did not mimic the effect of IL-1
(Table
1). It can be concluded, therefore, that the PKC was not
self-activating and that Ca2+
channels were not spontaneously open but rather that both processes were activated by IL-
.
Changes in Ca2+ influx and in intracellular Ca2+ have previously been implicated in the regulation of protease production by synovial cells (10, 22), but a clear picture of the role of PKC in the IL-1 signaling pathway in synovial cells has not emerged from previous PKC inhibitor studies. IL-1-induced expression of proteases (3), protease inhibitor (6), IL-1 (24), and arachidonic acid (4) was suppressed by PKC inhibitors. In other studies, however, different effects were seen. The inhibitors were associated with increased expression of PGE2 (20), a biphasic effect on IL-8 production (12), and no effect on proteases (11). One possible explanation for the range of observed responses is the occurrence of nonspecific reactions induced by the various PKC inhibitors. For example, IL-1 can induce expression of PGE2 (5), which is an agonist of cAMP (15); cAMP can downregulate stromelysin transcription (13). Thus the observation that staurosporine antagonized stromelysin production (3) could possibly have been due to a PKC-independent mechanism (upregulation by staurosporine of IL-1-induced PGE2, leading to increased levels of cAMP that inhibited stromelysin). The point is that inhibitor analysis is best in relatively simple cases in which the system has few different intermediate states in its signaling pathway. It is logically difficult to identify pathways when observations are made many hours after addition of the inhibitor because alternative and indirect signaling pathways potentially could increase the probability of an unspecified interaction with the inhibitor. This consideration underscores the importance of electrophysiological measurements in the study of IL-1 transduction because the short time scale of the measurements tends to exclude long-term processes, thereby making inhibitor analysis a more probative instrument.
IL-1 is a potent inducer of metalloproteinases by synovial cells, and
this process probably plays an important role in the pathophysiology of
joints (5, 8, 17, 19). Attempts at therapy based on reducing the levels
of metalloproteinases in diseased joints do not appear to be successful
(17), suggesting that blocking the disease earlier in its development
might be a more effective strategy. This consideration provided the
impetus for our study of the early electrical events associated with
binding of IL-1 by synovial cells.
We conclude that the depolarization that occurs as an early consequence
of transduction of IL-1 by voltage-clamped HIG-82 cells is caused by
activation of PKC and Ca2+ influx.
The role of the IL-1
-induced depolarization in regulating protein
expression by the cells remains to be determined, but the results
reported here, taken together with observations that activation of PKC
and Ca2+ influx also mediate the
effect of IL-1
on protease production (1, 2), suggest that
electrophysiological changes are actually part of the pathway for
expression of proteases in response to IL-1
.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported in part by the Center for Excellence in Arthritis and Rheumatology of the Louisiana State Medical Center and by the Hamilton Foundation.
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: A. A. Marino, Dept. of Orthopaedic Surgery, Louisiana State University Medical Center, PO Box 33932, Shreveport, LA 71130-3932.
Received 2 July 1998; accepted in final form 13 September 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Baratz, M. E.,
H. I. Georgescu,
and
C. N. Evans.
Studies on the autocrine activation of a synovial cell line.
J. Orthop. Res.
9:
651-657,
1991[Medline].
2.
Brinkerhoff, C. E.,
R. H. Gross,
H. Nagase,
L. Sheldon,
R. C. Jackson,
and
E. D. Harris.
Increased level of translatable collagenase messenger ribonucleic acid in rabbit synovial fibroblasts treated with phorbol myristate acetate or crystals of monosodium urate monohydrate.
Biochemistry
21:
2674-2679,
1982[Medline].
3.
Case, J. P.,
R. Lafyatis,
G. K. Kumkumian,
E. F. Remmers,
and
R. L. Wilder.
IL-1 regulation of transin/stromelysin transcription in rheumatoid synovial fibroblasts appears to involve two antagonistic transduction pathways, an inhibitory, prostaglandin-dependent pathway mediated by cAMP, and a stimulatory, protein kinase C-dependent pathway.
J. Immunol.
145:
3755-3761,
1990
4.
Cisar, L. A.,
R. J. Schimmel,
and
E. Mochan.
Interleukin-1 stimulation of arachidonic acid release from human synovial fibroblasts: blockade by inhibitors of protein kinases and protein synthesis.
Cell. Signal.
3:
189-199,
1991[Medline].
5.
Dayer, J.-M.,
B. de Rochemonteix,
B. Burrus,
S. Demczuk,
and
C. A. Dinarello.
Human recombinant interleukin 1 stimulates collagenase and prostaglandin E2 production by human synovial cells.
J. Clin. Invest.
77:
645-648,
1986[Medline].
6.
DiBattista, J. A.,
J. P. Pelletier,
M. Zafarullah,
K. Iwata,
and
J. Martel-Pelletier.
Interleukin-1 induction of tissue inhibitor of metalloproteinase (TIMP-1) is functionally antagonized by prostaglandin E2 in human synovial fibroblasts.
J. Cell. Biochem.
57:
619-629,
1995[Medline].
7.
Dinarello, C. A.
The interleukin-1 family: 10 years of discovery.
FASEB J.
8:
1314-1325,
1994
8.
Duqesnoy, B.,
and
P. Degand.
Stimulation of the secretion of latent cysteine proteinase activity by tumor necrosis factor and interleukin-1.
Arthritis Rheum.
36:
772-780,
1993[Medline].
9.
Horn, R.,
and
A. Marty.
Muscarinic activation of ionic currents measured by a new whole-cell recording method.
J. Gen. Physiol.
92:
145-159,
1988[Abstract].
10.
Howarth, D.,
K. P. H. Pritzker,
T. F. Cruz,
and
R. A. Kandel.
Calcium ionophore A23187 stimulates production of a 144 kDa gelatinase.
J. Rheumatol.
20:
97-101,
1993[Medline].
11.
Hulkower, K. I.,
R. Gagi-Eisenberg,
L. M. Traub,
H. I. Georgescu,
and
C. H. Evans.
Synovial protein kinase C and its apparent insensitivity to interleukin-1.
Eur. J. Biochem.
209:
81-88,
1992[Abstract].
12.
Jordan, N. J.,
M. L. Watson,
T. Yoshimura,
and
J. Westwick.
Differential effects of protein kinase C inhibitors on chemokine production in human synovial fibroblasts.
Br. J. Pharmacol.
117:
1245-1253,
1996[Abstract].
13.
Kerr, L. D.,
N. E. Oashaw,
and
L. M. Matrisian.
Transforming growth factor beta-1 and cAMP inhibit transcription of epidermal growth factor- and oncogene-induced transin RNA.
J. Biol. Chem.
263:
16999-17005,
1988
14.
Kolomytkin, O. V.,
A. A. Marino,
K. K. Sadasivan,
R. E. Wolf,
and
J. A. Albright.
Interleukin-1 switches electrophysiological states of synovial fibroblasts.
Am. J. Physiol.
273 (Regulatory Integrative Comp. Physiol. 42):
R1822-R1828,
1997
15.
Kumkumian, G. K.,
R. Lafyatis,
E. F. Remmers,
J. P. Case,
S.-J. Kim,
and
R. L. Wilder.
Platelet-derived growth factor and IL-1 interactions in rheumatoid arthritis: regulation of synoviocyte proliferation, prostaglandin production, and collagenase transcription.
J. Immunol.
143:
833-837,
1989
16.
O'Neill, L. A. J.
Towards an understanding of signal transduction pathways for interleukin-1.
Biochim. Biophys. Acta
1266:
31-44,
1995[Medline].
17.
Pelletier, J.-P., R. McCollum, J.-M. Cloutier, and J. Martel-Pelletier. Synthesis of metalloproteases and interleukin 6 (IL-6) in human osteoarthritic synovial membrane is an IL-1 mediated
process. J. Rheumatol. 22, Suppl. 43: 109-114, 1995.
18.
Putney, J. W.
Excitement about calcium signaling in inexcitable cells.
Science
262:
676-678,
1993[Medline].
19.
Stefanovic-Racic, M.,
J. Stadler,
H. I. Georgescu,
and
C. H. Evan.
Nitric oxide synthesis and its regulation by rabbit synoviocytes.
J. Rheumatol.
21:
1892-1898,
1994[Medline].
20.
Taylor, D. J.,
J. M. Evanson,
and
D. A. Woolley.
Contrasting effects of protein kinase C inhibitor, staurosporine, on cytokine and phorbol ester stimulation of fructose 2,6-bisphosphate and prostaglandin E production in fibroblasts in vitro.
Biochem. J.
269:
573-577,
1990[Medline].
21.
Toullec, D.,
P. Pianetti,
H. Coste,
P. Bellevergue,
T. Grand-Perret,
M. Ajakane,
V. Baudet,
P. Boissin,
E. Boursier,
F. Loriolle,
L. Duhamel,
D. Charon,
and
J. Kirilovsky.
The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.
J. Biol. Chem.
266:
15771-15781,
1991
22.
Unemori, E. N.,
and
Z. Werb.
Collagenase expression and endogenous activation in rabbit synovial fibroblasts stimulated by the calcium ionophore A23187.
J. Biol. Chem.
263:
16252-16259,
1988
23.
Vincenti, M. P.,
I. M. Clark,
and
C. E. Brinckerhoff.
Using inhibitors of metalloproteinases to treat arthritis. Easier said than done.
Arthritis Rheum.
54:
417-423,
1995.
24.
Watanabe, K.,
H. Hayashi,
and
Y. Mori.
Effect of benzylidene derivative, a novel antirheumatic agent on IL-1 production.
Pharmacol. Res.
28:
59-72,
1993[Medline].