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The mitogenic response to stimulation with basic calcium phosphate crystals is accompanied by induction and secretion of collagenase in human fibroblasts.

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1021
THE MITOGENIC RESPONSE TO STIMULATION WITH
BASIC CALCIUM PHOSPHATE CRYSTALS IS
ACCOMPANIED BY INDUCTION AND SECRETION OF
COLLAGENASE IN HUMAN FIBROBLASTS
GERALDINE M. McCARTHY, PETER G. MITCHELL, and HERMAN S. CHEUNG
Synovial fluid basic calcium phosphate (BCP)
crystals are associated with severe destructive arthropathies that are characterized by synovial proliferation
and digestion of articular collagenous structures. BCP
crystals are potent mitogens, which may account for this
proliferation. The role of collagenase in articular degradation is controversial because, despite the massive
loss of collagen, no studies have confirmed collagenolytic
activity in synovial fluid, as originally reported. We
investigated collagenase messenger RNA induction and
enzyme activity in human foreskin fibroblasts proliferating in response to stimulation with BCP crystals, and
analyzed the associated secreted proteins. Northern
blots revealed a dose-dependent accumulation of collagenase message, evident by 4 hours and continuing to at
least 36 hours, in BCP-stimulated cultures. One- and
2-dimensional polyacrylamide gel electrophoresis of
conditioned media from BCP crystal-stimulated cultures revealed the selective induction of 2 proteins with
molecular weight and PI values consistent with those of
collagenase. Increased enzyme activity was also found.
Thus, the mitogenic response of fibroblasts to BCP
crystals is accompanied by collagenase induction and
____-
From the Division of Rheumatology, Department of Medicine, Medical College of Wisconsin, Milwaukee.
Dr. Cheung’s work was supported in part by USPHS grant
AR-38421. Dr. McCarthy’s work was supported by an Arthritis
Foundation Postdoctoral Fellowship and by a grant from the Wisconsin Chapter, Arthritis Foundation.
Geraldine M. McCarthy, MB, MRCPI; Peter G . Mitchell,
PhD; Herman S. Cheung, PhD.
Address reprint requests to Geraldine M. McCarthy, MB,
MRCPI, Division of Rheumatology, Department of Medicine, Medical College of Wisconsin, 8700 West Wisconsin Avenue, Milwaukee, W1 53226.
Submitted for publication January 14, 1991; accepted in
revised form March 12. 1991.
Arthritis and Rheumatism, Vol. 34, No. 8 (August 1991)
secretion, supporting the hypothesis that they act as a
mediator of joint destruction in BCP crystal-associated
arthropathies.
Several articular conditions associated with basic calcium phosphate (BCP) crystals (e.g., hydroxyapatite, octacalcium phosphate, tricalcium phosphate)
have been described (1). BCP crystals are common in
osteoarthritic knee effusions, and their presence correlates strongly with radiographic evidence of cartilaginous degeneration (2). They are uniformly present in
Milwaukee shoulder syndrome (MSS), a destructive
arthropathy characterized by synovial proliferation
and widespread loss of intrasynovial collagenous
structures including cartilage, tendons, ligaments, and
adjacent bone (3). There is also loss of the intraarticular portion of the long head of the biceps tendon, with
preservation of the extraarticular portion and reattachment to the summit of the bicipital groove (4).
Synovial biopsies in 4 patients with MSS have
demonstrated increased numbers of villi, focal synovial lining hyperplasia, and intra- and extracellular
BCP crystal deposits (5). BCP crystals, like other
calcium-containing crystals, stimulate fibroblast mitogenesis in vitro (6), acting as a “competence” growth
factor similar to platelet-derived growth factor (7).
This mitogenic property may account for the synovial
proliferation in MSS.
Most connective tissue cells, including fibroblasts, produce low or even undetectable levels of
collagenase in monolayer culture. They can, however,
synthesize high levels of collagenase and other metalloproteinases in the presence of various growth factors
(8), mononuclear cell cytokines such as interleukin- 1
(9), and tumor necrosis factor (TNF) (lo), or chemical
1022
McCARTHY ET AL
agents such as phorbol esters (1 1). Similarly, BCP and
calcium pyrophosphate dihydrate crystals have been
shown to stimulate collagenase and neutral protease
activity by rabbit articular chondrocytes (12) and
cultured mammalian synovial cells (13).
Collagenase activity and neutral protease activity have been demonstrated in synovial fluid from the
shoulderjoints of some, but not all, patients with MSS
(14). Dieppe et a1 (15) were unable to demonstrate
collagenase activity following gel filtration of synovial
fluid from a series of patients with “idiopathic destructive disease of the shoulder,” a condition synonymous
with MSS. Analysis of synovial fluids from patients
with rheumatoid arthritis (RA) by the same authors,
using the same technique, demonstrated collagenase
activity in fewer samples than expected, although
other investigators have demonstrated the synthesis of
collagenase within RA synovium (16,17). The difficulty
in demonstrating collagenase activity in synovial fluid
ex vivo may reflect the avid binding of collagenase to
its articular substrate, collagen. Thus, collagenase
may be lost during specimen handling or may attach to
articular collagen or to the collagen fibrils present in
synovial fluid (18). The role of collagenase and other
neutral proteases in the articular degradation occurring in association with BCP crystals therefore remains controversial.
Using a model system with human foreskin
fibroblasts, we investigated induction of collagenase
messenger RNA (mRNA) and enzyme activity in
response to stimulation with BCP crystals. We demonstrated accumulation of collagenase mRNA, secretion of proteins with a molecular weight and isoelectric
point consistent with collagenase, and extracellular
collagenase activity, in response to BCP stimulation.
MATERIALS AND METHODS
Crystal synthesis. BCP crystals were synthesized by
a modification of published methods (7,19). Briefly, 15 ml of
0.2M sodium phosphate buffer, pH 6.7, was slowly added to
12.5 ml of 0.2M CaCI, and stirred for 1 hour at room
temperature. The precipitate formed was centrifuged at low
speed and then washed twice with water. Twenty microliters
of 0.5N HCI was added to the stirred precipitate suspended
in 2 ml of water, and the pH was adjusted to -8.5-10.0, with
20% NaOH. The suspended precipitate was stirred overnight
and then washed 4 times with water. The precipitate was
then washed once with absolute ethanol and once with
acetone, then dried in a vacuum desiccator. Mineral prepared by this method had a calcium/phosphate molar ratio of
1.59 and contained partially carbonate-substituted hydroxyapatite (HA) with admixed octacalcium phosphate (OCP), as
shown by Fourier transform infrared spectroscopy. The
crystals were crushed and sieved to yield 10-20-pm aggregates, which were sterilized and rendered pyrogen free by
heating at 200°C for 90 minutes. Heating to 200°C did not
alter the crystal character or the relationship of HA and
OCP, as confirmed by x-ray diffraction and Fourier transform infrared spectroscopy.
Crystals were weighed and suspended by sonication
in Dulbecco’s modified Eagle’s medium (DMEM). The
amounts of BCP crystals used were determined based on our
previous studies of dose-response relationships between
BCP crystals and the mitogenic response (6). In those
studies, maximal mitogenic responses were achieved with
BCP levels of 50-100 pg/ml. These levels are approximately
equivalent to concentrations of BCP crystals found in vivo.
Epidermal growth factor (EGF) was from Collaborative
Research (Bedford, MA).
Probes and reagents. The collagenase probe was a
2.05-kb Hind IIIISma I insert from the pC1 lase I clone (20),
obtained from the repository of human DNA probes of the
American Type Culture Collection (Rockville, MD). The
pHcGAP plasmid containing glyceraldehyde-3-phosphate
dehydrogenase (GAPD) complementary DNA (cDNA) (21)
was also from the American Type Culture Collection.
Tritiated thymidine (3H-TdR; 50 Ci/mmole) and I4Cmethylated protein standards (3 1-48 pCi/mg protein) were
from Amersham (Arlington Heights, IL). 35S-methionine
(1,140 Ci/mmole) was from DuPont NEN (Wilmington, DE).
Fetal bovine serum (FBS), Hanks’ balanced salt solution,
and DMEM were supplied by Gibco (Grand Island, NY).
Cycloheximide and most other chemicals used were from
Sigma (St. Louis, MO).
Cell culture. Human foreskin fibroblast cultures were
established from explants and transferred as previously
described (22). They were grown and maintained in DMEM
supplemented with 10% FBS containing 1% penicillin and
streptomycin. All experiments were performed on confluent
cell monolayers that had been rendered quiescent by removing the medium, washing with DMEM containing 0.5% FBS,
and subsequently incubating in this medium for 24 hours. All
cultures used were third- or fourth-passage cells.
Determination of mitogenesis by uptake of 3H-TdR.
Cells were grown to confluence in 35-mm culture plates and
rendered quiescent by incubation in 0.5% FBS for 24 hours.
3H-TdR (1 pCi/ml) was added to the wells 23 hours after
addition of 10% FBS, 100 ng/ml EGF, or BCP crystals (25
pg/cmZ)in 0.5% FBS, and pulse labeled for 1 hour. The cells
were then washed 3 times with phosphate buffered saline
(PBS), and macromolecules were precipitated with 5% trichloroacetic acid (TCA) solution. The precipitate was
washed again with PBS and dissolved in 1 ml 0.1N
NaOH/l% sodium dodecyl sulfate (SDS). Levels of TCAprecipitable 3H were determined in triplicate, using a liquid
scintillation counter (Packard Instruments, Downers Grove,
IL). In separate experiments, mitogenic stimulation of
monolayers was followed by trypsinization after 24 hours,
and cell counts were determined using a Coulter counter
(Coulter Electronics, Hialeah, FL).
Northern blot analysis. Northern blot analysis of total
cellular RNA was used to study the expression of collagenase mRNA in human foreskin fibroblasts after stimulation
BCP STIMULATION AND COLLAGENASE INDUCTION
with BCP crystals or EGF, a known inducer of collagenase
in fibroblasts. At appropriate time points after each treatment, the cell monolayers were washed twice with cold PBS.
One 100-mm plate was harvested for each analysis. Total
RNA was recovered by precipitation with 4M LiCl as
described by Cathala et a1 (23). Twenty micrograms of total
cellular RNA was fractionated on a 1.2% agaroseformaldehyde gel, ribosomal RNA was visualized with ethidium bromide, and the fractionated RNA was transferred to
nitrocellulose filters (24). Hybridization of the filters with
DNA probes labeled with 32P by the random primer method
was performed overnight at 42°C (25). Probes were labeled
to a specific activity of >5 x lo8 counts per minute/
microgram. The filters were washed at a maximal stringency
of 0.25x SSC ( l x SSC = 0.15M NaC1, 0.015M sodium
citrate) at 60°C for 30 minutes. Autoradiography was performed with Kodak XAR-5 film (Eastman Kodak, Rochester, NY), and signal intensity was quantified by densitometry, using a scanning laser densitometer (LKB Instruments,
Stockholm, Sweden).
One- and 2-dimensional gel electrophoresis. Quiescent
cell monolayers in 35-mm plates were placed in methioninedepleted medium and labeled at various time points with
35S-methionine for 2 hours, washed 3 times with fresh
medium, and chased in fresh medium for 2 hours. Proteins
secreted at various time points after stimulation were separated according to molecular weight, by l-dimensional SDSpolyacrylamide gel electrophoresis (SDS-PAGE), according
to the method of Laemmli (26). Two-dimensional mapping of
secreted proteins was performed by a modification of the
method of O'Farrell(27). Briefly, proteins were separated by
isoelectric focusing on 5% tube gels in the first dimension,
using carrier ampholytes, pH 2-1 1, and then further separated according to molecular weight on 10% polyacrylamide
slab gels in the second dimension. Gels were dried and then
exposed to Kodak XAR-5 film, with intensifying screens, at
-70°C. Densitometry was performed on bands of interest.
Collagenase assay. Labeled collagen was prepared
from human foreskin fibroblasts incubated in the presence of
3H-proline, as previously described (28). Briefly, fibroblasts
were labeled in 10 ml of DMEM containing 'H-proline (50
pCi/ml), L-ascorbic acid (50 pg/ml), p-aminopropionitrile
(125 pg/ml), 10% FBS, and 1% penicillin/streptomycin, for
24 hours. Labeled collagen was then purified as described
(28). Collagenase activity was determined by measuring the
release of labeled peptides from 'H-proline-labeled collagen
reconstituted fibrils, as described by Harris et a1 (29). The
'H-proline-labeled collagen was diluted with nonradioactive, pepsin-soluble calf skin collagen dissolved in collagenase buffer (50 mM Tris, 200 mM NaCl, 30 mM CaCI,, pH
7.5), to achieve a specific activity of 2 x lo5 cpm/mg. Latent
collagenase in conditioned medium was activated with
10 mM APMA. The collagenase activity was measured by
incubating 100 pl of conditioned medium with reconstituted
collagen fibrils at 27°C for 72 hours. To terminate the assay,
each tube was centrifuged at 10,OOOg for 5 minutes, and
radioactivity was determined in an aliquot of the supernatant. SDS-PAGE of the supernatant and fluorography were
then performed, according to the Laemmli method (26).
0
c
.--
1023
900
t
5
U
T
0.5%
10%
FBS
FBS
BCP
EGF
Figure 1. Mitogenic effects of basic calcium phosphate (BCP) crystals. Confluent cultures of human foreskin fibroblasts grown in
35-mm plates were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 0.5% fetal bovine serum (FBS). Twentyfour hours later, fresh DMEM containing FBS (0.5% or lo%), BCP
crystals (25 pg/cm*) or epidermal growth factor (EGF; 100 ng/ml)
was added. After 23 hours, all plates were pulse labeled with
3H-thymidine (1 pCi/ml) for 1 hour. The plates were then processed,
and 3H-thymidine incorporation was determined as described in
Materials and Methods. Results are expressed as the percent
3H-thymidine uptake relative to that seen with 0.5% FBS. Values
are the mean and SEM of triplicate determinations.
RESULTS
Mitogenic response of quiescent cell cultures to
BCP crystals, EGF, and 10% FBS. The addition of 25
pg/cm2 BCP crystals in 0.5% FBS-DMEM stimulated
a 7-fold increase in 3H-TdR incorporation compared
with that in cultures incubated with 0.5% FBS alone,
after 24 hours. The response to BCP stimulation was
similar to the response stimulated by 10% FBS and
greater than that stimulated by 100 ng/ml EGF (Figure
1). We also performed cell counts, to confirm that the
increased 3H-TdR uptake was accompanied by an
increase in cell number. The mean -+ SD number of
cells ( x 104/cm2)in control cultures incubated with
0.5% FBS was 3.25 5 0.20. The number of cells
( x 104/cm2)in cultures incubated with BCP crystals
was 4.00 0.25, a 23% increase over that in control
cultures. The cell numbers ( X 104/cm2) in cultures
incubated with 10% FBS and with EGF were 3.80 5
0.80 (a 15% increase over control) and 3.00 t 0.30,
respectively.
Time course and dose-responsiveness of BCP
crystal induction of collagenase mRNA. BCP crystals
induced significant accumulation of collagenase
mRNA after 4 hours of stimulation, and this continued
*
1024
McCARTHY ET AL
mRNA, a positive dose-responsiveness was demonstrated, with maximal accumulation seen after stimulation with 36 pg/cm2, the highest concentration
tested. Analysis of GAPD mRNA expression in all
samples tested confirmed the presence of equivalent
amounts of RNA under each condition.
Effect of inhibition of protein synthesis. Since it
has been demonstrated that protein synthesis is required for accumulation of collagenase mRNA in
response to other stimuli (30), we studied the effect
Figure 2. Basic calcium phosphate (BCP) crystal-induced accurnulation of collagenase messenger RNA (mRNA): time course. Confluent cultures of human foreskin fibroblasts in 100-mm plates were
incubated in Dulbecco’s modified Eagle’s medium (DMEM) and
0.5% fetal bovine serum (FBS) for 24 hours. At the indicated time
points prior to harvesting, plates were stimulated with BCP crystals
(B; 18 pg/cm2). Plates were also stimulated in parallel with epidermal growth factor (E; 100 ng/ml). Control fibroblast cultures, with
no addition to the FBS-DMEM, were harvested after 36 hours.
Total RNA was isolated after harvesting, and Northern blot analysis
was performed using a specific complementary DNA (cDNA) probe
for collagenase (Coll), followed by autoradiography. The collagenase probe was then removed by washing, and the blot was
reprobed with glyceraldehyde-3-phosphatedehydrogenase (GAPD)
cDNA, to ensure analysis of equivalent quantities of total mRNA
under each condition. The positions of the 28s and 18s ribosomal
bands are shown.
up to at least 36 hours (Figure 2). At each time point,
accumulation of collagenase mRNA in response to
BCP crystals was greater, by at least 50%, than
accumulation in response to EGF, as assessed by
densitometry . Collagenase mRNA expression in human foreskin fibroblasts after 24 hours of stimulation
with various concentrations of BCP crystals is illustrated in Figure 3. While BCP concentrations as low as
1.8 pg/cm2 resulted in significant accumulation of
Figure 3. BCP crystal-induced accumulation of collagenase
mRNA: dose-response. Confluent cultures of human foreskin fibroblasts in 100-mm plates were incubated in DMEM and 0.5% FBS for
24 hours, and were then stimulated with BCP crystals in the
quantities shown. Control cultures were stimulated with 10% FBS
or left unstimulated. All plates were harvested after 24 hours, and
total RNA was isolated. Northern blot analysis was performed using
a specific cDNA probe for collagenase, followed by autoradiography. The collagenase probe was then removed by washing, and the
blot was reprobed with GAPD cDNA, to ensure analysis of equivalent quantities of total mRNA under each condition. The positions
of the 18s and 28s ribosomal bands are shown. See Figure 2 for
definitions.
BCP STIMULATION AND COLLAGENASE INDUCTION
1025
of inhibition of protein synthesis by cycloheximide.
The inhibition of protein synthesis did not alter levels
of collagenase mRNA in unstimulated cells, but it did
counteract the accumulation of collagenase mRNA in
response to BCP crystals when the inhibitor and the
crystals were added simultaneously. To determine the
critical period of protein synthesis required for collagenase mRNA induction, cycloheximide (10 pg/ml)
was added to human foreskin fibroblast cultures at
various times after the addition of BCP crystals. When
cycloheximide was added at 2 hours or 3 hours after
addition of BCP, the induction of collagenase mRNA
was inhibited by 80%. Even when the addition of BCP
crystals preceded the addition of cycloheximide by 6
hours, there was still -60% inhibition of collagenase
mRNA accumulation noted 24 hours after stimulation
with BCP (Figure 4).
One- and 2-dimensional electrophoresis analysis
of secreted proteins from BCP crystal-stimulated fibroblasts. The selective induction by BCP crystals and
EGF of 2 proteins with approximate molecular weights
of 50 kd and 55 kd, consistent with that of collagenase
(31), is demonstrated in Figure 5. These proteins were
first evident 8 hours after stimulation and were demonstrable at least through 36 hours (not shown). They
BCP -
+
-
CHX
-
+
+
0
+
2
+
3
+
6
1
I
+
2
I
Hours After BCP
Figure 4. Time-dependent effects of inhibition of protein synthesis
on collagenase messenger RNA (mRNA) accumulation. RNA was
isolated, after 24 hours, from untreated human foreskin fibroblasts,
from human foreskin fibroblasts treated with cycloheximide (CHX;
10 pggiml) alone, and from human foreskin fibroblasts treated with
basic calcium phosphate (BCP) crystals (18 pg/cmZ),with or without
CHX. CHX was added either simultaneously with BCP crystals or
at the indicated time point after the addition of BCP crystals.
Northern blot analysis for RNA was performed using a specific
complementary DNA probe for collagenase, and collagenase mRNA
was quantitated by densitometry. Results are expressed as the
percent of maximal expression.
Figure 5. One-dimensional sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) analysis of secreted proteins from
basic calcium phosphate (BCP)-stimulated human foreskin fibroblasts. Confluent, quiescent human foreskin fibroblasts in 35-nim
plates were stimulated with BCP crystals (100 pg/ml) or with
epidermal growth factor (EGF; 100 ng/ml) in 0.5% fetal bovine
serum (FBS); fibroblasts in 0.5% FBS alone were used as a control.
After 20 hours, cultures were placed in methionine-depleted medium, labeled with "S-methionine (25 pCi/ml) for 2 hours, and
chased in fresh medium for a further 2 hours. Proteins secreted in
the culture media under these 3 experimental conditions were then
analyzed by SDS-PAGE. Lane A, Control. Lane B, BCP crystalstimulated cultures. Lane C, EGF-stimulated cultures. Molecular
weight markers are shown at the left; proteins identified at -50 kd
and -55 kd are indicated by arrows.
were not apparent in the conditioned media of unstimulated control cultures. Further analysis of these
-50-kd and -55-kd proteins by 2-dimensional gel
electrophoresis demonstrated apparent pl values of
1026
McCARTHY ET AL
Figure 6 . Two-dimensional gel electrophoresis analysis of secreted proteins from basic calcium phosphate (BCP)stimulated human foreskin fibroblasts. Confluent, quiescent human foreskin fibroblasts in 35-mm plates were stimulated
with BCP crystals in 0.5% fetal bovine serum (FBS); fibroblasts in 0.5% FBS alone were used as a control. After 20 hours,
cultures were placed in methionine-depleted medium, labeled with ”S-methionine (25 pCiiml) for 2 hours, and chased in
fresh medium for a further 2 hours. Proteins secreted in the culture media of control and stimulated cells were analyzed
according to isoelectric point in the first dimension, and then according to molecular weight in the second dimension. A,
Control cultures. B, BCP crystal-stimulated cultures. Arrows indicate pl values of 6.9 and 6.7, respectively, for the -50-kd
protein and the -55-kd protein.
6.9 and 6.7, respectively, consistent with those previously reported for collagenase (31) (Figure 6).
The kinetics of collagenase mRNA accumulation relative to secretion of the proteins is illustrated in
Figure 7. Significant collagenase mRNA accumulation
in response to BCP crystals was first apparent 4 hours
after stimulation, followed by the appearance of the
secreted proteins by 8 hours after stimulation.
Collagenase activity. The presence of collagenase activity in the APMA-activated conditioned media from cells stimulated with BCP crystals was confirmed by SDS-PAGE of collagen “split products,”
following incubation of conditioned media with 3Hproline-labeled collagen gel. Figure 8 shows the A and
B collagen fragments resulting from collagen digestion
by collagenase.
DISCUSSION
We have demonstrated a significant in vitro
induction of collagenase mRNA expression accompanying the mitogenic response to stimulation with BCP.
This was followed by the secretion of proteins that had
molecular weights and isoelectric points consistent
with those of collagenase. Furthermore, activated
conditioned media containing these secreted proteins
exhibited enhanced collagen breakdown, consistent
with collagenase activity.
Aggregates of BCP crystals have been found in
synovial fluid in association with different patterns of
arthritis, including acute gout-like attacks, erosive
polyarticular disease, osteoarthritis of the knee, and
MSS ( I ) . The role of BCP crystals in MSS remains
BCP STIMULATION AND COLLAGENASE INDUCTION
1027
unclear. Although synthetic BCP crystals are phlogistic when injected into experimental animals (32,33),
synovial fluid leukocyte counts have been found to be
no higher in human synovial fluids that contained BCP
crystals than in those that did not (34). Very low
synovial fluid leukocyte counts are a characteristic
feature of MSS, and it is unlikely that leukocytes are
an important cause of joint damage in this condition.
Whether proteolytic enzymes such as collagenase are
responsible for joint destruction in MSS has been a
subject of controversy. Although 15 of 29 patients with
MSS have had collagenase activity demonstrated in at
least 1 synovial fluid sample (14), this feature of the
disease is inconsistent.
We have shown that, in vitro, BCP crystals
consistently induce collagenase mRNA accumulation
in human foreskin fibroblasts, in a dose-responsive
manner. This accumulation is greater than that stimulated by EGF, a known inducer of collagenase (35).
Elevated mRNA levels persist for at least 36 hours
after stimulation. We do not know whether this accumulation is due to a change in mRNA stability, an
increase in mRNA transcription, or a combination of
both.
The transcription of collagenase mRNA was
inhibited by cycloheximide, suggesting that protein
synthesis is required for transcriptional activation.
x
c
.v)
C
Q,
U
4
8
12
16
24
36
Hours of incubation
Figure 7. Kinetics of collagenase messenger RNA (mRNA) accumulation and protein secretion. Collagenase mRNA accumulation
(0)in human foreskin fibroblasts at various time points following
stimulation with basic calcium phosphate (BCP) crystals was demonstrated by Northern blot analysis and quantitated by densitometry
of the autoradiographs. The secretion of a 50-kd protein (0)at
various time points following stimulation with BCP crystals was
demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitated similarly. Results are expressed as the
percent of maximal optical density obtained.
Figure 8. Collagenase activity in human foreskin fibroblastconditioned media. Confluent cultures of human foreskin fibroblasts
in 35-mm plates were stimulated with basic calcium phosphate
(BCP) crystals (25 pg/cmz) in 0.5% fetal bovine serum (FBS);
fibroblasts in 0.5% FBS alone were used as a control. After 48
hours, the medium was collected and 100-pl aliquots were incubated
with labeled collagen as described in Materials and Methods.
Collagen “split products” were then separated on a 10% polyacrylamide gel. The gel was dried, and autoradiography was performed
for 1 week. Lane 1, Control culture. Lane 2, BCP-stimulated
culture. A = collagen fragment a l ; B = collagen fragment a2; C =
collagen fragment aIA; D = collagen fragment a2A; E = collagen
fragment aIB; F = collagen fragment a2B.
Other investigators have shown that the collagenase
promoter contains a cis element, termed the TPA
response element (TRE), which is involved in in-
1028
creased transcription in response to T N F (36) and
phorbol esters (1 1). Increased transcription modulated
through the TRE depends on the transcription factor
AP-1, a heterodimer composed of the protein products
of fos and jun, both primary response genes which
interact to stimulate transcription of AP- 1-responsive
genes (37). Thus, inhibitors of protein synthesis might
interfere with this mechanism of transcriptional activation. T N F induces transient transcription of c-fos in
fibroblasts, with maximal stimulation after 1 hour.
Induction of c-jun by T N F is more prolonged, lasting
at least 6 hours.
Whether TRE is important in the BCP induction
of collagenase accumulation is unknown. However,
we have previously demonstrated that BCP crystals
induce the transient accumulation of c-fos in BALB/
c-3T3 fibroblasts (38), and we have recently found that
BCP crystals induce the accumulation of c-jun in
human foreskin and NIH/3T3 fibroblasts (McCarthy
GM, Mitchell PG, Cheung HS: unpublished observations). Since inhibition of protein synthesis blocked
the BCP crystal stimulation of collagenase mRNA
accumulation, it is possible that the synthesis of a
transactivating complex containing fos and jun is also
important in the BCP crystal induction of collagenase
transcription.
Analysis of conditioned media of fibroblasts
stimulated with BCP crystals and with E G F showed
the induction of proteins that had both a molecular
weight and an isoelectric point consistent with those
previously reported for collagenase (3 1). These proteins were not found in the conditioned media of
unstimulated control cultures. Since E G F has been
shown to stimulate collagenase secretion by fibroblasts, and since proteins secreted in response to BCP
and E G F were of identical molecular weight, BCP
most probably induces collagenase secretion, as well
as mRNA induction. The kinetics of the induction of
collagenase mRNA and the appearance of the secreted
protein support this concept. Significant collagenase
mRNA accumulation in response to BCP was first
apparent 4 hours after stimulation, followed by the
appearance of the secreted proteins 8 hours after
stimulation.
Significant collagenase activity was demonstrated in the activated conditioned media of fibroblasts stimulated with BCP crystals, as indicated by
the demonstration of “split products” of radiolabeled
type I collagen after exposure to conditioned media.
No such products were found after incubation of
radiolabeled collagen with conditioned media from
McCARTHY ET AL
unstimulated cultures. This contrasts with the inconsistent demonstration of collagenase activity in synovial fluids of patients with MSS. This inconsistency
among MSS patients may be a consequence of the
rapid binding of collagenase in vivo to its substrate
collagen, which is abundant in both joint fluid and in
tissue contiguous to the fluid. Alternatively, it may
result from the presence in synovial fluid of specific
inhibitors, such as tissue inhibitor of metalloproteinases (15), or nonspecific inhibitors, such as azmacroglobulin (18). Collagenase has been postulated
to be one of the mediators of joint destruction in RA
(15). However, many investigators have had difficulty
demonstrating collagenase activity consistently when
performing enzyme assays on the synovial fluid of
patients with active RA (15,39). The same situation
may apply in patients with MSS.
These findings support our hypothesis that the
mitogenic response to BCP crystals is accompanied by
collagenase induction and secretion. This may underly
the noninflammatory digestion of joint structures
found in association with the presence of BCP crystals
and accompanying synovial proliferation in MSS.
ACKNOWLEDGMENTS
We gratefully acknowledge the helpful comments of
Drs. D. J. McCarty and L. M. Ryan.
REFERENCES
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2.
3.
4.
5.
6.
(apatite, octacalcium phosphate, tricalcium phosphate)
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Halverson PB, McCarty DJ: Identification of hydroxyapatite crystals in synovial fluid. Arthritis Rheum 22:
389-395, 1979
McCarty DJ, Halverson PB, Carrera GF, Brewer BJ,
Kozin FK: “Milwaukee shoulder”-association
of microspheroids containing hydroxyapatite crystals, active
collagenase, and neutral protease with rotator cuff defects. I. Clinical aspects. Arthritis Rheum 24:464-473,
1981
McCarty DJ: Robert Adams rheumatic arthritis of the
shoulder: “Milwaukee shoulder” revisited. J Rheumatol
16~668-670,1989
Halverson PB, Garancis JC, McCarty DJ: Histopathologic and ultrastructural studies of Milwaukee shoulder
syndrome: a basic calcium phosphate crystal arthropathy. Ann Rheum Dis 43:734-741, 1984
Cheung HS, Story MT, McCarty DJ: Mitogenic effects
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crystals, induction, phosphate, accompanied, human, stimulating, collagenase, response, basic, secretion, calcium, mitogenic, fibroblasts
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