Departments of 1 Orthopaedic Surgery and 2 Cellular Biology and Anatomy and 3 Center of Excellence for Arthritis and Rheumatology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130
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ABSTRACT |
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Synovial cells can form networks
connected by gap junctions. The purpose of this study was to obtain
evidence for a necessary role of gap junction intercellular
communication in protein secretion by synovial cells. We developed a
novel assay to measure the enzymatic activity of metalloproteinases
(MMPs) produced by synovial cells in response to interleukin-1
(IL-1
) and employed the assay to explore the biological function of
gap junctions. IL-1
produced a dose-dependent increase in MMP
activity that was blocked by exposure to the gap junction inhibitors
18
-glycyrrhetinic acid and octanol for as few as 50 min. The
inhibitors produced an immediate and marked reduction in intercellular
communication, as assessed by transient current analysis using the
nystatin perforated-patch method. These observations suggest that
communication through gap junctions early in IL-1
signal
transduction is critical to the process of cytokine-regulated secretion
of MMPs by synovial cells.
perforated patch; HIG-82 cells; gap junction inhibitors; collagen assay
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INTRODUCTION |
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GAP JUNCTIONS ARE PLAQUES of aqueous channels that facilitate direct electrical and metabolic connection between adjacent cells. The plaques consist of a few to many hundreds of individual channels, each capable of passing molecules with mass up to ~1 kDa (2). A channel is formed by noncovalent linkages between annular hemichannels composed of proteins of the connexin family; more than a dozen isoforms have been identified (3). Gap junctions are continuously formed, modified, and removed (12), resulting in restriction or augmentation of gap junction intercellular communication (GJIC).
We used patch clamping, dye transfer, and electron microscopy to
demonstrate the existence of functional and structural gap junction
channels in rabbit and human synovial cells in culture and in synovial
lining cells in tissue (9-11). The specific relevance of intercellular communication to synovial metabolism is unknown, but
several lines of evidence suggested to us that GJIC might be involved
in signal transduction. First, interleukin-1 (IL-1
) can stimulate
protein expression by synovial cells (7) and can
depolarize the cell membranes via a GJIC-dependent process (10). These observations raised the possibility that
depolarization (hence, GJIC) was necessary for stimulated expression.
Second, we showed that synovial lining cells could form highly coupled networks (9), and similar networks have been shown to
facilitate regulated secretion in other tissues (4, 14,
15).
The purpose of this study was to obtain evidence for a necessary role of gap junctions in the signaling cascade leading to protein production by synovial cells. This was accomplished by comparing the amount of proteinase activity produced by an established synovial cell line in response to cytokine stimulation in the presence and absence of channel inhibitors using a novel assay system.
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MATERIALS AND METHODS |
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Cells and reagents. Rabbit synovial fibroblasts (HIG-82, American Type Culture Collection, Manassas, VA) were grown at 37°C in 5% CO2 without antibiotics in 25-ml polystyrene flasks containing F-12 medium (GIBCO BRL, Grand Island, NY) with 10% FCS (growth medium). For passage of cells, confluent cultures were treated with 1 ml of 0.08% trypsin for 3-5 min; then 4 ml of medium were added, and the suspended cells were centrifuged, resuspended, and seeded (106 cells) into 4 ml of medium. The experiments were performed using 35-mm petri dishes seeded initially with 5 × 105 cells.
Metalloproteinases (MMPs) were assayed (see below) using 70-80% confluent monolayers in which the growth medium was replaced with serumless Neuman-Tytell medium (GIBCO) containing human recombinant IL-1Enzyme assay. Using collagen film as the substrate, we developed a quantitative non-radioisotope-based assay to measure the enzymatic activity of the MMPs (principally collagenase) produced by HIG-82 cells in response to various stimuli (1, 5, 7). Collagen solution (4 µl, 3.0 mg/ml in 0.012 N HCl; Collagen Biomaterials, Palo Alto, CA) was added to each well (6.4 mm diameter) of 96-well polystyrene plates and precipitated by slowly increasing the pH with 4 µl of H2O, 4 µl of bath solution, and 8 µl of 0.012 N NaOH. The plate was dried at 23°C for 24 h, washed with H2O, and dried again at 23°C, resulting in a thin (calculated thickness ~370 nm) film containing ~12 µg of collagen that was tightly bound to the bottom of the wells.
The cell supernatants to be analyzed were filtered, and 1 mM p-aminophenylmercuric acetate was added to activate MMPs (1). Enzymatic reactions of the supernatants (200 µl/well) against the collagen films were carried out in quadruplicate at 24°C for various times up to 15 h. The digested portion of the film was washed away, the undigested portion was fixed for 3 h with 0.1% Coomassie brilliant blue G in 10% acetic acid and 45% methanol, and the wells were washed and dried. The undigested film was then dissolved in 50 µl of ethanol for 20 min to obtain a uniform color distribution, and the optical density (OD) of each well was read with a microplate reader (model MR 5000, Dynatec Laboratories) at 570 nm (wavelength of maximum Coomassie brilliant blue G absorbance). Four wells were averaged to characterize the MMP activity of each petri dish, and 4 petri dishes were averaged for each condition.Calculation of activity.
The density of the initial collagen film was 12 µg/3.2 · 2 · mm2 = 373 mg/m2, and its OD was 0.85 ± 0.02. The amount of
collagen degraded per unit area of film was determined from a
measurement of OD as follows: (0.85
OD)(373/0.85)
mg/m2, and MMP activity was assessed at a
standard reaction time of 13 h [34(0.85
OD)
mg · h
1 · m
2]. The
supernatants degraded
35% of collagen film thickness under the
conditions of our experiments.
Electrodes.
The nystatin perforated-patch method (10) was used to
measure the transmembrane current under voltage clamp. The method permitted use of the whole cell configuration for measuring electrical properties of the cell while preserving intracellular regulation by
preventing diffusion of small signaling molecules from the cell into
the electrode. Glass capillaries 1.0 mm in diameter were pulled in two
steps (PB-7, Narishige, East Meadow, NY) and fire polished in a
microforge (model MF-9, Narishige). The resistance of the electrodes
was 7-9 M in bath solution. The pipette solution contained (in
mM) 125 potassium aspartate (monopotassium salt), 30 KCl, 4 NaCl, and
10 HEPES-KOH, pH 7.2 (calculated osmolarity = 318 mosmol/l). The
bath solution consisted of (in mM) 145 NaCl, 5.4 KCl, 1.5 CaCl2, 1.0 MgCl2, 5.0 HEPES-NaOH, and 5.0 glucose, pH 7.3 (calculated osmolarity = 328 mosmol/l). Because
nystatin interfered with giga-seal formation, the tip of the pipette
was filled with a nystatin-free solution before the addition of pipette solution containing nystatin (0.3 µg/ml). The giga-seal was formed during the time needed for the nystatin to diffuse to the tip of the micropipette.
Electrical measurements.
Giga-seals (~10 G) were formed under negative pressure (5-10
cmH2O), typically within 0.5-5 min; the success rate
was >50%. After giga-seal formation, nystatin channels formed within
5-15 min; the resistance of the perforated-patch membrane was
40 ± 20 M
. Giga-seals and nystatin pores usually remained
stable for hours.
Northern blot. Total cellular RNA was obtained by TriPure isolation (Roche, Indianapolis, IN). Aliquots (10 µg) of RNA were heat denatured and then size separated in a 1% agarose gel containing 2.2 M formaldehyde. Ethidium bromide was used to stain for the 28S and 18S ribosomal RNA subunit bands to check RNA integrity and loading. The RNA was transferred to a nylon membrane (Schleicher and Schuell, Keene, NH); after uniform transfer was assessed by visual inspection of the ribosomal RNA bands, the RNA was cross-linked to the membrane (Stratalinker, Stratagene, La Jolla, CA).
The membranes were treated for 3 h at 42°C with hybridization buffer [50% formamide, 5× SSC (3 M NaCl, 0.3 M sodium citrate), 10× Denhardt's solution, 1% SDS, 100 µg/ml salmon sperm DNA] before hybridization at 42°C for 16 h with 32P-labeled complementary DNA fragments of pro-MMP-1. The membranes were then washed in 2× SSC-0.1% SDS for 5 min at 25°C and in 0.1× SSC-0.1% SDS for 15 min at 68°C and autoradiographed (Eastman Kodak, Rochester, NY) at ![]() |
RESULTS |
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MMP assay.
Synovial cells incubated for 48 h in the presence of IL-1
released MMPs into the supernatant. The amount of collagen degraded was
linearly proportional to the reaction time, as evaluated using our
photometric method (Fig. 1); on the basis
of this result, we chose 13 h as the standard reaction time. This
choice ensured that only a part of the collagen film would be degraded
under the conditions studied and that the enzyme reactions would occur with constant speed. The accuracy and sensitivity of the photometric method were compared with the corresponding published values found using a radioisotope-based method (5), with PMA as the MMP stimulus. PMA (0.01-100 ng/ml) elicited dose-dependent production of MMPs, with saturation at ~10 ng/ml (data not shown); half-maximum activity occurred at 1.7 ± 0.5 ng/ml, and the standard error of measurement was ~7% of maximum MMP activity. Effectively the same results were obtained using radioisotopes (5).
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IL-1-induced secretion.
Proteinase activity in the supernatants from cells treated with IL-1
was detected after an incubation period of ~24 h. Subsequently, the
extent of substrate degradation was dependent on IL-1
concentration (Fig. 2). To determine whether MMP
production required the continuous presence of IL-1
in the medium,
enzyme activity was measured during 0-48 h of incubation with
IL-1
(100 pg/ml); then the medium was replaced with fresh medium
containing no IL-1
, and the measurements were continued. MMP
production dropped to baseline after removal of the IL-1
and did not
rise during the subsequent 96 h, suggesting that the continuous
presence of IL-1
was required for MMP production.
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Effect of gap junction inhibitors on secretion.
The role of GJIC in the stimulated production of MMPs was studied using
the gap junction inhibitors GR and octanol. GR was added to the medium
in the petri dish at time 0, IL-1 (100 pg/ml final
concentration) was added 10 min later, and MMP activity was measured as
a function of incubation time. Addition of GR resulted in a
dose-dependent inhibition of IL-1
-stimulated MMP production (Fig.
3). Half-maximum inhibition occurred at
7.0 ± 0.6 µM GR. GR (5 and 10 µM) alone (without IL-1
) did
not induce MMP production (<0.7
mg · h
1 · m
2, not shown).
The effect of 1 mM octanol on IL-1
-stimulated production of MMPs was
similar to that of GR (Fig. 4). Octanol
alone did not induce MMP production (not shown).
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Effect on secretion of impeding conductance early in transduction.
In separate experiments, GR and octanol were added at time 0 to the medium in dishes containing cells, and IL-1 (100 pg/ml) was
added 10 min later. After another 40 min, the medium was replaced with
medium containing IL-1
but no inhibitor, and the cells were incubated for up to 48 h. We found that the MMP activity of the supernatant was significantly reduced by the brief treatment with the
inhibitors (Fig. 5); 50 min of treatment
with GR (10 µM) or octanol (1 mM) alone did not induce MMP production
(not shown).
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Generalized inhibition of MMP release.
To evaluate whether the decrease in MMP activity caused by the
disruption of GJIC was specific for IL-1 signaling, we investigated whether disruption of GJIC also interfered with PMA-stimulated MMP
release. The effect of the gap junction inhibitors on MMPs was
essentially the same when PMA was used as the stimulus (Figs. 3, 4, and
6), indicating that the relationship
between GJIC and MMP activity did not depend on the specific details of
IL-1
signaling.
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Effect of inhibitors and IL-1 on gap junction channel
conductance.
The effect of GR and octanol on intercellular communication was
assessed using transient current analysis (10). For
aggregated cells in the medium, the time constant after application of
a voltage step was 20 ± 5 ms (Fig.
7, curve 1), indicating that the cells were initially connected by gap junctions (10).
In the presence of GR or octanol, however, the time constant quickly decreased to 3.8 ± 1 ms. At 50 min after addition of the
inhibitor, the bath solution was replaced with inhibitor-free solution,
and the measurements were repeated, with results identical to those in
curve 1, Fig. 7, A and B (not
shown).
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Northern blot.
The cells synthesized inducible but not constitutive MMP-1 mRNA in
response to treatment with IL-1 (Fig.
9, lanes 2 and 1, respectively). Addition of the gap junction inhibitors had no significant effect on the total amount of cytokine-induced cellular mRNA for MMP-1 (Fig. 9, lanes 3 and 4).
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DISCUSSION |
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The effect of PMA on MMP production by HIG-82 cells was the same
as that described previously (5). The result can therefore be viewed as a positive control for our assay method. Using our assay
method, we found that addition of IL-1 to the cell medium resulted
in a dose-dependent increase of MMP activity (Fig. 2). The IL-1
concentrations (1 and 100 pg/ml) were comparable to those found in
marrow plasma (200-300 pg/ml) (8) and synovial fluid
(150 pg/ml) (13), suggesting that the processes underlying the observed secretion were physiologically relevant. Previous electrophysiological studies indicated that the first steps of IL-1
signal transduction in HIG-82 cells occurred within 10 min (10). Thus the delay in MMP production (Fig. 2) did not
imply that the initial steps of IL-1
signal transduction were slow processes.
Removing IL-1 from the medium caused termination of production of
the enzymes that degraded the collagen film, indicating that IL-1
was not simply a trigger that initiated a signal cascade leading to
secretion. Rather, the continuous presence of IL-1
in the medium was
required for MMP production. This conclusion is in general agreement
with our earlier data showing that IL-1
-induced depolarization was
reversed when the cytokine was removed from the medium
(10).
Synovial cells are connected to each other by an extensive network of
gap junctions (9), and it is possible that such networks mediate some cellular processes. We examined whether IL-1-induced MMP production might be an example and found that the stimulated secretion was eliminated by each of two chemically disparate gap junction inhibitors (Figs. 3 and 4). They rapidly blocked GJIC, and the
effect was reversible after treatment for 50 min (Fig. 7). This result
confirmed previous data from others (2, 4, 6) that for
treatment times <1 h the inhibitors worked via the same mechanism,
namely, reversible inhibition of GJIC, and did not change other cell
properties. Exposure to GR at concentrations higher than that used in
our study (30 µM) for a longer period (4 h) reduced connexin43
expression and promoted disassembly of gap junction plaques
(6). Because the cells in our study were incubated in the
presence of GR for 48 h (Figs. 3 and 4), we considered the
possibility of an effect on MMP expression that did not involve gap
junction communication. This was accomplished by exposing the cells to
the inhibitors only for the period of time during which their effects
on GJIC were reversible. Application of GR or octanol for only 50 min
caused significant inhibition of IL-1
-induced MMP production (Fig.
5), thereby showing the importance of gap junction communication in
signal transduction and the temporal localization of GJIC in the early
stages of transduction.
We showed previously that gap junction channel conductance between
HIG-82 cells could be calculated from an induced transient cellular
current by fitting the current to the sum of two exponentials and using
the time constant of the slower exponential as a measure of integral
cell conductance (10). The time constant was ~0.2 ms for
isolated cells, but cells connected by gap junctions exhibited much
longer time constants. We found that the time constant for aggregated
cells initially was 20 ± 5 ms (Fig. 8). After application of
IL-1, the time constant was unchanged after 30 min and decreased only slightly during the 1st h, indicating that the cytokine did not
significantly influence channel conductance. With GR and octanol, in
contrast, the time constant decreased almost immediately to 3.8 ± 1 ms, indicating a dramatic decrease in channel conductance. When the
inhibitors were removed, the time constants returned to their initial
values. This result provided a possible explanation for the effect of
the inhibitors in the MMP assay (Fig. 5). The gap junction channels
were closed at the time IL-1
was added and remained closed for
another 40 min, after which the channels were opened as a consequence
of removal of the blocker from the medium. Nevertheless, the period of
inhibition was enough to significantly inhibit MMP production in the
presence of IL-1
, suggesting that GJIC in the early stages of
IL-1
signal transduction facilitated signal amplification.
GJIC was necessary for IL-1-induced protease production, as
determined from the results of treatment with GJIC inhibitors (Figs.
3-5). We measured the effect of treatment with GJIC inhibitors on
the amount of MMP-1 mRNA to address the question of the level of
regulation of induced expression that was affected by inhibitors. The
data in Fig. 9 suggest that the differences in MMP-1 mRNA in cells
treated with the two inhibitors were too small to explain the effect of
the inhibitors on protease production. Consequently, we believe that it
is likely that the effect was produced by changes at some other level
of regulation, for example, secretion or translation. Future studies
involving other approaches are necessary to resolve the issue.
On the basis of our previous electrophysiological studies, we can
suggest several mechanisms by which intercellular gap junction communication could lead to signal amplification. We found that IL-1
switched the cell membrane potential from
63 to
30 mV and that
preexposure of the cells to low membrane potential (
30 mV) using
voltage clamp increased the sensitivity of the cells to IL-1
,
probably by opening Ca2+ channels and increasing
intracellular Ca2+ concentration (10, 11). It
is possible that the change of membrane potential could spread between
cells connected by gap junction channels, because they have high
conductance for monovalent ions, thereby increasing the sensitivity of
adjacent cells to IL-1
. The diffusion of second messengers through
gap junction channels is an alternative mechanism for signal
amplification. This argument requires a nonuniform distribution of at
least one element in the signaling cascade, and we previously presented evidence that intracellular Ca2+ concentration could be
such an element (10).
Perspectives
Overproduction of MMPs in response to inflammatory cytokines is probably an important cause of arthritis. A possible strategy for treating arthritis involves the inhibition of IL-1 ![]() |
ACKNOWLEDGEMENTS |
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This work was supported in part by the Center for Excellence in Arthritis and Rheumatology, Louisiana State Medical Center, and by Orthopedic Specialists of Louisiana.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. A. Marino, Dept. of Orthopaedic Surgery, LSU Health Sciences Center, PO Box 33932, Shreveport, LA 71130-3932 (E-mail: amarino{at}lsuhsc.edu).
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. Section 1734 solely to indicate this fact.
First published January 30, 2002;10.1152/ajpcell.01166.2000
Received 18 August 2000; accepted in final form 3 January 2002.
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