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BASIC CALCIUM PHOSPHATE CRYSTALS STIMULATE CELL PROLIFERATION AND COLLAGENASE MESSAGE ACCUMULATION IN CULTURED ADULT ARTICULAR CHONDROCYTES PETER G. MITCHELL, JANINE A. STRUVE, GERALDINE M. McCARTHY, and HERMAN S. CHEUNG Objective. To investigate BCP (basic calcium phosphate) crystal-stimulated mitogenesis and collagenase gene transcription in primary cultures of porcine chondrocytes. Methods. The role of protein kinases in BCP crystal4imulated DNA synthesis was investigated using thymidine incorporation, kinase inhibitors, and protein kinase C (PKC) assays. Northern blot analysis was used to determine the levels of collagenase c-fos and c-jun message. Results. BCP crystals stimulated chondrocyte proliferation in a PKC-dependent manner. Increased levels of collagenase message were preceded by an increased accumulation of c-fos, but not c-jun. Conclusion. BCP crystals could contribute to the abnormal chondrocyte proliferation and collagenase secretion observed in some rheumatic diseases.

Intraarticular deposition of basic calcium phosphate (BCP) crystals (hydroxyapatite, octacalcium phosphate, and tricalcium phosphate) and/or calcium pyrophosphate dihydrate (CPPD) crystals is associated with a number of destructive arthropathies involving cartilage degeneration (1,2). Collagenase activity has been demonstrated in the synovial fluid of patients with BCP crystal deposition disease (3,4), From the Division of Rheumatology, Department of Medicine, Medical College of Wisconsin, Milwaukee. Dr. Cheung’s work was supported by NIH grant AR-38421. Peter G . Mitchell, PhD; Janine A. Struve. BS; Geraldine M. McCarthy, MD; Herman S.Cheung, PhD. Address reprint requests to Herman S. Cheung, PhD, Division of Rheumatology, Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226. Submitted for publication September 18, 1990; accepted in revised form October 31, 1991. Arthritis and Rheumatism, Vol. 35, No. 3 (March 1992)

although Dieppe et a1 ( 5 ) were unable to show an increase in collagenase activity in synovial fluids from a series of patients with crystal deposition disease of the shoulder (“idiopathic destructive disease of the shoulder”). Both CPPD crystals (6,7) and BCP crystals (6-9) are commonly found in osteoarthritic cartilage and have occasionally been observed within chondrocytes (6,9). It has previously been demonstrated that cultured human rheumatoid synovial cells or normal canine synovial cells will endocytose BCP or CPPD crystals (10). Crystal endocytosis was followed by the release of prostaglandins and, in addition, collagenase and neutral protease activity (11,12). It has also been demonstrated that BCP crystals are mitogenic in cultures of human foreskin fibroblasts, canine synovial fibroblasts (13), and murine fibroblasts (14). Crystalstimulated mitogenesis has been postulated to be a contributing factor in the synovial hyperplasia associated with BCP or CPPD crystal deposition diseases (1 3,15). Earlier studies demonstrated that the time to peak BCP crystal-stimulated thymidine uptake in fibroblasts was delayed by 2-3 hours compared with the peak uptake time using 10% serum (13). In addition, solubilization of the crystals was necessary in order for mitogenesis to proceed (16). In fibroblasts, BCP crystals acted similarly to a competence growth factor (17), since insulin-like growth factor 1 (IGF-1) was required in order to elicit a full mitogenic response (18). BCP crystals also induced accumulation of the competence genes c-fos and c-myc, in a protein kinase C (PKCwependent manner, in BALBk-3T3 mouse fibroblasts (14). The proliferative effects of BCP crystals on

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chondrocytes have not previously been described, although it has been shown that in primary cultures of rabbit chondrocytes, phagocytosis of BCP or CPPD crystals leads t o increased secretion of collagendegrading ' a n d neutral protease activities (12). It is possible that articular cartilage deposition of calciumcontaining crystals could contribute t o aberrant proliferation of chondrocytes and/or the stimulation of chondrocyte-derived collagenase. In t h e present study, we investigated the mechanisms of BCP crystalstimulated cell proliferation and collagenase messenger RNA (mRNA) induction in adult porcine chondrocytes.

MATERIALS AND METHODS BCP crystals were synthesized as described by Bett et al(l9). X-ray diffraction and chemical analysis of similarly synthesized crystals indicated that the crystals were almost entirely hydroxyapatite (12). 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. Platelet-derived growth factor (PDGF) was prepared from clinically unusable human platelets, by chromatography on CM Sephadex, blue Sepharose, and Bio-Gel 100, as described by Antoniades et al (20). Human platelet-poor plasma and human serum were prepared from freshly drawn blood as described by Pledger et al (17). Epidermal growth factor (EGF) was obtained from Collaborative Research (Bedford, MA). The c-fos probe was a 1.3-kilobase Pst I v - j b fragment from the pfos-l plasmid, supplied by I. Verma (Salk Institute, San Diego, CA) (21). Collagenase message was detected using a human Hind IIUSrnu I fragment from the pCllase 1 plasmid (22), obtained from the repository of human DNA probes at American Type Culture Collection (Rockville, MD). The c-jun probe was a 0.9-kb Bum HIIPst I insert from the RSV-cJ plasmid (23). supplied by Dr. Michael Karin (University of California, San Diego, CA). The a-tubulin probe, used as a control, was a Pst I insert of the PILaTl plasmid (24), from Dr. Philip Sharp (Massachusetts Institute of Technology, Cambridge, MA). Tritiated thymidine (50 Ci/mmole) and 'H-proline (35 Ci/mmole) were from Amersham (Arlington Heights, IL); ?labeled ',P-ATP (4,500 cihmole) and a-labeled "P-dATP (3,000 cilmmole) were from ICN Biochemicals (Irvine, CAI. Fetal bovine serum (FBS), Hanks' buffered saline solution, Dulbecco's modified Eagle's medium (DMEM), and serumless media (Neuman and Tytell) were supplied by Gibco (Grand Island, NY). Inhibitors H7 and HA1004 (25) were obtained from Seikagaku America (St. Petersburg, FL); staurosporine (26) was from Kamiya Biomedical (Thousand Oaks, CA). Type I1 collagenase, trypsin, phosphatidylserine, I ,3-diolein, histone (type 111-S), 12-o-tetradecanoylphorbol 13-acetate (TPA), and all other chemicals used were from Sigma (St. Louis, MO). Cell culture. Chondrocytes were prepared by digestion of cartilage (12) from adult porcine knee joints. The cells

were plated at 4 x 10'/cm2 in 10% FBS, allowed to attach, and incubated at 37°C in a humidified atmosphere containing 10% CO,. Cultures were routinely re-fed with 10% FBS in DMEM on days 2 and 4 post-plating; these were replaced with 0.5% lactalbumin hydrolysate (LAH) or serumless media on day 6. Plates were used for experiments 24 hours later. Chondrocytes plated under these conditions continued to secrete type I1 collagen, as described previously (27,28). DNA synthesis assay and cell counting. Quiescent cultures in 24-well plates were stimulated in DMEM containing low concentrations of platelet-poor plasma (0.5-1%) in the presence of I pCi/ml 'H-thymidine, and then incubated for 48 hours. For time-course experiments, 300 pl of IM ascorbic acid was added to individual wells at specific intervals. At the conclusion of the experiments, cultures were washed twice with phosphate buffered saline (PBS), extracted once with cold 10% trichloroacetic acid (TCA) for 10 minutes, washed with PBS, and the cell layer dissolved in 0.1M NaOH/I% sodium dodecyl sulfate (SDS). Aliquots were counted in a scintillation counter. For cell counts, chondrocytes in 10% FBS were plated at 3.4 x 10' cells/cm2 in 60-mm plates. After 2 days, the plates were washed and the medium changed to 0.5% LAH. Twenty-four hours later, the medium was changed to 1% platelet-poor plasma, with or without 50 pg/cm2 BCP crystals. After 60 hours, cultures were treated with 0.1% trypsinkollagenase and the cells released. Cell counts were performed using a Coulter counter. Preparation of RNA and Northern blot analysis. Cell cultures were scraped from 60-mm plates, washed in PBS, and disrupted in guanidinium isothiocyanate. RNA was precipitated in 4M LiCl and processed as described by Cathala et a1 (29). RNA (5 pgAane) was electrophoresed on 0.66M formaldehyde/l.2% agarose gels and transferred to nitrocellulose as described previously (30). Filters were hybridized with probes labeled to specific activities of greater than 5 x 10' counts per minute/pg, using the random primer method (31). Blots were washed twice with t x SSC/O.l% SDS (1 x SSC = 0.15M NaCl and 0.015M sodium citrate) at room temperature, followed by 2 washes in 0 . 2 5 ~SSC/O.I% SDS at 55°C. After washing, the blots were exposed to film, with enhancers, for 1-4 days at -70°C. Densitometry was performed using a scanning laser densitorneter (LKB, Cambridge, England). Probes were stripped from blots by heating for 10 minutes in 0.1 x SSC/O.5% SDS at 95°C. Protein k i n e C assay. PKC activity in membrane fractions was extracted using a technique similar to that described by Brenner et al(32). PKC activity was assayed in the following buffer: 10 mM Tris HCI, pH 7.5, 10 mM MgCI,, 0.5 mg/ml histone 111-S, 125 p M ATP, and 5 X lo6 cpm y3*P-ATP (4,500 Ci/mmole). Each sample was assayed in the presence of calcium (2 mM), phosphatidylserine (100 pg/ml), and diolein (10 pdml). The samples were also assayed in the presence of 1 mM EDTA, without calcium, phosphatidylserine, and diolein. The difference between the findings under these 2 conditions is reported as specific PKC activity. Reaction mixtures were incubated at 30°C for 5 minutes, after which 1 ml of 15% TCA was added and the samples were filtered through 0.45-pm filters. The filters

BCP-STIMULATED MITOGENESIS AND COLLAGENASE GENE TRANSCRIPTION 345 were washed with 10% TCA, dried, and the radioactivity counted in a scintillation counter.

25

20

\

RESULTS Stimulation of DNA synthesis and cell division by BCP crystals. BCP crystals were able to stimulate the incorporation of thymidine into cultures of quiescent porcine chondrocytes, in a dose-responsive manner (Figure 1). The effect was maximal at a concentration of 25 pgkm', and in this experiment, the level of incorporation was -3 times the level achieved with 1% platelet-poor plasma alone. No BCP stimulation of DNA synthesis was observed in the absence of plateletpoor plasma (results not shown). There were quantitative differences between experiments in the amount of 'H-thymidine incorporation above background. However, in all experiments the results were qualitatively similar. The stimulation of DNA synthesis was not a nonspecific effect of particulates, since another particulate (diamond dust), which was sieved to a 400 [

350

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100 50

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Figure 2. Time course of BCP crystal-stimulated DNA synthesis in porcine chondrocytes. Confluent cultures of chondrocytes, in 24well plates, were incubated in DMEM containing 0.5% lactalbumin hydrolysate. Forty-eight hours later, fresh DMEM containing 0.5% platelet-poor plasma was added, and cultures were stimulated with either platelet-derived growth factor (5 units/ml) (0)or BCP crystals (50 &cm2) (A). At &hour intervals beginning 12 hours after stimulation, I pCi/ml 3H-thymidine was added. After 2 hours of labeling, 300 pl of 1M ascorbic acid was added to each of the wells. 'H-thymidine incorporation at time points up to 52 hours was determined as described in Materials and Methods. Values are the mean f 1 SD of triplicate determinations. A = control. See Figure 1 for other definitions.

T

lii,1 HS

0

12

50

75

100

conc. BCP or DD (wicm2)

Figure 1. DNA synthesis in porcine chondrocytes stimulated with basic calcium phosphate (BCP) crystals. Confluent cultures of chondrocytes, in 24-well plates, were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 0.5% lactalbumin hydrolysate. Forty-eight hours later, fresh DMEM containing 1% platelet-poor plasma, 1 &i/ml 'H-thymidine, and BCP crystals (open bars) or diamond dust (DD) (closed bars) in amounts ranging from 0 to 100 pg/cm2 were added. As a positive control, some plates were stimulated with 10% human serum (HS).After 48 hours, the plates were processed and 'H-thymidine incorporation was determined as described in Materials and Methods. Results are expressed as the percent 'H-thymidine uptake stimulated relative to control (1% platelet-poor plasma alone). Values are the mean and 1 SD of triplicate determinations.

similar particle size, had no stimulatory effect on thymidine uptake by these cells, using the same range of concentrations (Figure 1). The observed increase in 'H-thymidine uptake stimulated by BCP crystals was associated with an increase in cell number. After 2.5 days of treatment with BCP crystals at 50 pg/cm2, the cell density was 4.03 2 0.16 x 10'/cm2, while the cell density in control plates was 3.66 x 105/cm2 (means & 1 SD, from 7 plates with BCP crystals and 7 control plates). Thus, there was almost a 10% increase in cell number among the BCP crystal-stimulated cells. The kinetics of BCP crystal stimulation and PDGF stimulation of thymidine uptake in porcine chondrocytes were compared. Peak DNA synthesis stimulated by BCP crystals was delayed by 12 6 hours (mean 2 SD) compared with that by PDGF (Figure 2). Maximal thymidine incorporation stimulated by PDGF occurred between 18 and 24 hours, whereas the BCP-stimulated peak did not occur until 30-36 hours. In addition, the PDGF-induced peak was relatively sharp, whereas BCP crystal-stimulated cultures were still incorporating thymidine at an elevated rate 52 hours after stimulation.

*

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--

3000

e

g 2500-

-

T

0

10

20

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30

H7 ("MI

10

20

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HA1004 ( P M )

B

A 6001

1 Figure 3. Effect of protein kinase inhibitors on BCP crystalstimulated DNA synthesis. Confluent cultures of chondrocytes, in 24-well plates, were incubated in DMEM containing 0.5% lactalbumin hydrolysate. Forty-eight hours later, fresh DMEM containing 1% platelet-poor plasma (ppp) (m)and I pCi/ml 'H-thymidine was added. Wells were pretreated for 30 minutes with either H7 (A), H A W 4 (B), or staurosporine (C), and then BCP crystals (50 pg/cm2) ( 10% HS ( ESd ), or 400 nM 12-o-tetradecanoylphorbol 13-acetate (TPA) ( tZ3 ) was added. After 48 hours, the plates were processed and 'H-thymidine incorporation was determined as described in Materials and Methods. Results are expressed as the percent 'H-thymidine incorporation relative to control (1% ppp). Values are the mean f 1 SD of triplicate determinations. See Figure 1 for other abbreviations.

m),

0

1

10

20

Staurospor i n e ( d

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PKC-dependent DNA synthesis and stimulation of membrane PKC activity. To investigate the role of PKC in the stimulation of DNA synthesis in adult chondrocytes, 2 different PKC inhibitors, H7 (25) and staurosporine (26), were used. As shown in Figure 3A, there was a dose-dependent decrease in BCP crystalstimulated thymidine incorporation in response to increasing concentrations of H7 (Ki PKC 6.0 p M ;Ki CAMP-dependent protein kinase 3.0 pM).With 20 pit4 H7, >70% of the BCP-stimulated response was inhibited, while only a small decrease in human serumstimulated DNA synthesis was seen. TPA, which directly stimulates PKC (33), also induced a >Sfold increase in thymidine incorporation, and this increase was similarly inhibited by H7 in a dose-responsive manner. In contrast, the structurally similar compound HA1004 (25) (Ki PKC 40 pM; Ki CAMP-dependent protein kinase 2.3 pM) had no inhibitory effects over the same range of concentrations (Figure 3B).

Since H7 has other, nonspecific effects on cellular processes, apart from inhibition of PKC (34), we also investigated the effects of the PKC inhibitor staurosporine (Ki P K C 0.0007 nM; Ki CAMPdependent kinase 0.007 nM) o n BCP crystalstimulated DNA synthesis (Figure 3C). Similarly to H7, staurosporine inhibited both BCP crystal and TPA stimulation of thymidine incorporation in a dosedependent manner. With both 10 nM staurosporine and 20 nM staurosporine, BCP crystal and TPA stimulation of DNA synthesis were completely inhibited, while there was no effect on human serum-stimulated DNA synthesis. An early response to many cytokines is a rapid increase in PKC activity associated with the membrane fraction (32,33). We investigated whether BCP crystals could produce an increase in membraneassociated PKC activity in chondrocytes. Figure 4 demonstrates that 30 minutes after stimulation with BCP

BCP-STIMULATED MITOGENESIS AND COLLAGENASE GENE TRANSCRIPTION 347 crystals, there was a > 10-fold increase in membraneassociated PKC activity. The fact that this was indeed PKC was shown by 2 criteria: 1) the activity was dependent on the presence of calcium and phosphatidylserine, and 2) the activity stimulated by calcium and phosphatidylserine was substantially inhibited by 10 pit4 of the PKC inhibitor H7. BCP crystal stimulation of collagenase message. We investigated whether BCP crystals had any effect on collagenase gene expression in chondrocytes. Under serum-free conditions, BCP crystals stimulated a dose-dependent increase in collagenase message expression at 16 hours post-stimulation (Figure 5). BCP crystals at 50 pg/cm2 stimulated an almost 5-fold increase in collagenase message above control. No further increases in message were observed with higher doses, and levels remained elevated until at least 48 hours post-stimulation (results not shown). Maximal message levels induced by BCP were consis-

60

Figure 5. Dose-responsive increase in basic calcium phosphate (BCP) crystal-stimulated collagenase messenger RNA. Confluent cultures of chondrocytes, in 60-mm plates, were incubated in serumless media for 24 hours before being stimulated with different amounts of BCP crystals. After 16 hours, the cultures were harvested and total RNA was isolated. Northern blot analysis was carried out, and the blot was probed with a collagenase (Colt.) probe. After exposure to film, the blot was stripped of the collagenase probe and reprobed with a-tubulin (tub.) complementary DNA. The positions of the 28s and 18s ribosomal bands are shown.

\

e

30

z

a.

20

0

!i

10

0 control

BCP

Figure 4. BCP crystal stimulation of membrane-associated protein kinase C (PKC) activity. Confluent cultures of chondrocytes, in 60-mm plates, were incubated in DMEM containing 0.5% lactalbumin hydrolysate. Cultures were stimulated with 50 &cm2 BCP crystals, and 30 minutes later 2 BCP crystal-stimulated plates and 2 control plates were harvested. Membrane-associated PKC activity was isolated by chromatography on DEAE cellulose mini-columns. Triplicate 50-pl samples were assayed for '*P incorporation into histone 111-S both in the presence and in the absence of 2 mM calcium, I00 Lcg/ml phosphatidylserine. and 10 pg/ml diolein. EDTA ( I mM) was added to the samples that were assayed in the absence of the above factors. Counts incorporated in the absence of phosphatidylserine and diolein were subtracted from counts obtained in the presence of these factors. Samples were also assayed in the absence (open bars) and the presence (shaded bars) of H7. Results are expressed as pmol of phosphate incorporated/minute/106cells. Values are the mean f 1 SD of triplicate determinations.

tently substantially less than those stimulated by tumor necrosis factor Q (TNFa). Stimulation of c-fos, but not c-jun, message accumulation by BCP crystals. We investigated whether BCP crystal induction of collagenase was preceded by increased accumulation of c-fos andor c-jun message. As shown in Figure 6, c-fos message levels began to increase at 0.5 hours, and peaked at 2 hours before declining again. The same blot was reprobed with c-jun and no BCP crystal stimulation of c-jun was evident, although after extended exposure, low levels of c-jun were evident in all lanes. In contrast, EGF stimulates significant accumulation of the c-jun message in porcine chondrocytes at both 0.5 hours and 1 hour (27). Additional experiments demonstrated that no stimulation of c-jun occurred at times up to 12 hours after stimulation by BCP crystals.

DISCUSSION BCP crystals are mitogenic in a number of fibroblast cell lines (13,14), and it has been suggested

348

Figure 6. Basic calcium phosphate (BCP) crystal-stimulated accumulation of c-fos messenger RNA. Confluent cultures of chondrocytes, in 60-mm plates, were incubated in serumless media for 24 hours before being stimulated with BCP crystals at 50 1Lg/cm2.At the times indicated, cultures were harvested and total RNA was isolated. Northern blot analysis was carried out, and the blot was probed with a c-fos probe. After exposure to film, the blot was stripped of the c-fos probe and reprobed with a-tubulin (tub.) complementary DNA. The positions of the 28s and 18s ribosomal bands are shown.

that these crystals may contribute to synovial hyperplasia (13,15). Crystals of calcium compounds are found within the cartilage matrix in some arthritic conditions (6-9), and it is possible that BCP crystals could stimulate chondrocyte proliferation in vivo and contribute to abnormal cartilage remodeling. In this study, we demonstrated that BCP crystals stimulated DNA synthesis and cell proliferation in a differentiated chondrocyte culture system. In a manner similar to the findings with fibroblast growth factors such as PDGF, BCP crystals required plasma factors in order to induce chondrocytes to proceed through DNA synthesis. In this respect, BCP crystals act as a “competence factor,” rendering cells able to respond mitogenically to “progression” factors, such as IGF-1, found in plasma (17). Both the maximum induction of c-fos and the peak uptake of ’H-thymidine stimulated by BCP crystals were delayed when compared with results observed using soluble growth factors as stimulators. It has been postulated that the BCP crystal-stimulated signaling process involves an interaction between crystals and the cell membrane (14). Because of the particulate nature of BCP crystals, it might be ex-

MITCHELL ET AL pected that this interaction would lead to a delayed and asynchronous response when compared with the response prompted by a soluble growth factor with a specific membrane receptor. Such a delay was not observed with BCP crystal stimulation of BALBk-3T3 fibroblasts (14). However, the delay observed with chondrocytes may be due to the more extensive extracellular matrix secreted by chondrocytes. If the induction of c-fos is part of the BCP-stimulated signaling pathway leading to DNA synthesis, then the observed delay in its maximum induction (1.5 hours) cannot totally explain the long lag (mean k SD 12 -+ 6 hours) in the observed peak uptake of ’H-thymidine. Crystal dissolution is necessary for BCP stimulation of DNA synthesis in fibroblasts (lo), and it may be that this dissolution is the rate-determining step in the process. In previous investigations performed in our laboratory, down-regulation of PKC by long-term treatment with TPA effectively blocked BCPstimulated DNA synthesis (14). In the present work we have demonstrated that chondrocyte stimulation with BCP crystals leads to an increase in membraneassociated PKC activity. In addition, concentrations of staurosporine and H7, which had little effect on serum-stimulated DNA synthesis, blocked both BCPand TPA-induced DNA synthesis. These results suggest that BCP crystals, like TPA, act through a PKCdependent mechanism to stimulate DNA synthesis. The observed inhibition by H7 is probably not due to inhibition of CAMP-dependent kinases since HA1004, a structurally related inhibitor with a similar Ki for CAMP-dependent kinase but a higher K,for PKC, had no effect on BCP-stimulated mitogenesis. BCP crystals induced accumulation of collagenase message in a dose-responsive manner. Serum factors were not required for this effect, since BCPinduced induction of the message occurred under serum-free conditions. Message levels continued to be elevated until at least 48 hours post-stimulation. We currently do not know whether this is due to a change in message stability, increased transcription, or a combination of the two. At a concentration of BCP crystals that produced maximal DNA synthesis (50 pg/cm2), the response was still substantially less than that produced by 10 ng/ml TNFa. The collagenase (35) and stromelysin (36) promoter regions both contain a cis element termed the TPA response element (TRE). The TRE will act as an enhancer for heterologous gene constructs and can be induced by TPA (35) and TNFa (32). Enhanced transcription occurs after binding of a heterodimer, com-

BCP-STIMULATED MITOGENESIS AND COLLAGENASE GENE TRANSCRIRION 349 prising the Fos and Jun proteins, to the TRE (37,38). Elevated levels of c-fos and c-jun are reported to precede cytokine stimulation of collagenase transcription (32). Recently, McDonnell et a1 (39) demonstrated that in rat fibroblasts, optimal induction of stromelysin required the expression of both c-fos and c-jun, as well as activation of PKC. In our study, BCP crystals stimulated both c-fos and PKC; however, the level of transcription of collagenase was substantially less than the maximum potential. The reduced response may be related to the absence of any BCP-induced c-jun accumulation in chondrocytes. The lack of BCP crystal-stimulated c-jun was surprising, since BCP crystals stimulated cjun in both NIH 3T3 cells and human foreskin fibroblasts (Mitchell PG, Cheung HS: unpublished observations). It has been shown that cjun can be stimulated in chondrocytes, since EGF stimulation induced substantial accumulation of the message at 0.5 hours and 1 hour (27). In conclusion, we have demonstrated that BCP crystals are mitogenic in adult porcine chondrocytes and that the mechanism involves stimulation of membrane-associated PKC activity. As in fibroblasts, BCP crystals appear to be acting as a competence factor in chondrocytes, although the substantial delay in the peak of thymidine incorporation suggests that BCP crystals also influence events later in the G, phase. Induction of c-fos and activation of PKC preceded a sustained induction of collagenase message accumulation, but no c-jun induction was found. BCP crystal stimulation of DNA synthesis and collagenase mRNA accumulation may explain some of the in vivo pathologic manifestations of crystal deposition diseases.

ACKNOWLEDGMENTS We thank Dr. D. J. McCarty for valuable discussions during this work and the Klement Sausage Co. for supplying _ .. the porcine bones used as a source of chondrocytes.

REFERENCES 1. Halverson PB, McCarty DJ: Identification of hydroxyapatite crystals in synovial fluid. Arthritis Rheum 22:

389-395, 1979 2. McCarty DJ, Halverson PB, Carrera GF, Brewer BJ, Kozin F: “Milwaukee shoulder”: association of microspheroids containing hydroxyapatite crystals, active collagenase, and neutral protease with rotator cuff defects. I. Clinical aspects. Arthritis Rheum 24:464473, 1981 3. Halverson PB, Cheung HS, McCarty DJ, Garancis JC,

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