Molecular mechanism of basic calcium phosphate crystal-induced activation of human fibroblasts. Role of nuclear factor kappab, activator protein 1, and protein kinase c.

Synovial fluid basic calcium phosphate (BCP) crystals are markers of severe joint degeneration in osteoarthritis. BCP crystals cause mitogenesis of articular cells and stimulate matrix metalloprotease production, thus promoting degradation of articular tissues. Previous work suggested that BCP crystal-induced cell activation required intracellular crystal dissolution, induction of proto-oncogene expression, and activation of signal transduction pathways involving protein kinase C and mitogen-activated protein kinases. Here we further elucidate the mechanisms of BCP crystal-induced cell activation as BCP crystals activate transcription factors nuclear factor kappaB and activator protein 1 in human fibroblasts. We confirm the role of protein kinase C in BCP crystal-induced mitogenesis in human fibroblasts. In contrast, we demonstrate that BCP crystals do not activate signal transduction pathways involving protein tyrosine kinases or phosphatidylinositol 3-kinase. These data further define the mechanism of cell activation by BCP crystals and confirm its selectivity, an observation that may have therapeutic implications.

Synovial fluid basic calcium phosphate (BCP) 1 (hydroxyapatite, octacalcium phosphate, and tricalcium phosphate) crystals are common in osteoarthritis (OA) and are associated with severe degenerative arthropathies (1). The prevalence of BCP crystals in synovial fluid from patients with knee OA is between 30 and 60%, and their presence correlates strongly with radiographic evidence of cartilage degeneration (2). Larger joint effusions are seen in affected joints when compared with joint fluid from OA knees without BCP crystals (3).
Clinical and pathological studies have demonstrated that synovial lining proliferation of varying degrees is associated with BCP crystals in osteoarthritis (4). BCP crystals themselves are at least partly responsible for such proliferation since they stimulate cell replication in vitro (5). Increased cellularity in the synovial lining enhances the capacity for secretion of cytokines, which may promote chondrolysis. Noninflammatory destruction of matrix-rich articular structures including cartilage, ligament, and tendon is also characteristic of BCP crystal deposition (1). BCP crystals promote tissue damage by induction of matrix metalloprotease (MMP) synthesis and secretion. Since there are no available drugs to inhibit deposition or affect reabsorption of these crystals, prevention of the biological consequences of the destructive processes initiated by BCP crystals is an attractive therapeutic strategy.
The in vitro effects of BCP crystals emphasize their pathogenic potential. BCP crystals induce mitogenesis in cultured fibroblasts (5,6). However, mechanisms by which BCP crystals induce mitogenesis have been incompletely studied. Endocytosis and intracellular dissolution of the crystals, producing an elevated cytoplasmic calcium concentration, are important (7). BCP crystals activate a protein kinase signal transduction pathway involving p42 and p44 mitogen-activated protein (MAP) kinases (8). Current data suggest that BCP crystals enhance phospholipase C activity in synovial fibroblasts (9). If BCP crystals increase phospholipase C activity, resultant diacylglycerol accumulation followed by increased protein kinase C (PKC) activity should ensue. This postulate is supported in a study by Mitchell et al. (6) who found that BCP crystal-induced mitogenesis in Balb/c/3T3 fibroblasts was inhibited when PKC was down-regulated using the tumor-promoting phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate (TPA), an analogue of diacylglycerol. Another index of PKC activation is translocation of the enzyme from the cytosolic compartment to the membrane compartment of the cell, and we have shown increased membrane-associated PKC activity in porcine chondrocytes treated with BCP crystals (10). These data suggest that the mitogenic response to BCP crystals involves activation of PKC. However, BCP crystal-induced activation of PKC in human cultures has not been reported.
A potentially important pathway of crystal-induced cell activation involves transcription factor, nuclear factor B (NF-B). Present in the cytoplasm of most cell types, NF-B is critical to many signal transduction pathways, including those leading to cell replication and oncogenesis, and is induced during the G 0 to G 1 transition of mitogenesis in murine fibroblasts (11). The best characterized form of NF-B consists of a heterodimer composed of a 50-kDa (p50) and a 65-kDa (p65) (also called RelA) subunit (12). In unstimulated cells, NF-B is found in the cytoplasm bound to IB (inhibitor B), a member of a family of inhibitor proteins that prevents it from entering the nucleus. When cells are exposed to inducers of NF-B, two serine residues of IB are phosphorylated by specific IB kinases causing its rapid degradation by the 26 S proteasome and loss of NF-B inhibitory activity (13). NF-B is thus released to translocate to the nucleus and activate transcription of target genes. BCP crystals induce mitogenesis, but their ability to activate NF-B has not previously been studied.
Alternatively, crystal-induced mitogenesis may derive from other pathways. The cellular effects of many growth-promoting agents are mediated by high affinity receptor protein tyrosine kinases (PTK) (14). In mammalian cells, the binding of ligand to the extracellular domain induces tyrosine phosphorylation of the cytoplasmic domain. This leads to recruitment and downstream activation of a number of common signaling molecules including PTK and phosphatidylinositol 3-kinase (PI3 kinase) (15). Phosphoinositides play a key role in signal transduction, and the 3-phosphoinositide pathway has been implicated in growth factor-dependent mitogenesis (16). Production of 3-phosphoinositides requires PTK-mediated recruitment and activation of PI3 kinase (15). Calcium pyrophosphate dihydrate (CPPD) and monosodium urate crystals induce protein tyrosine phosphorylation in human neutrophils (17). PI3 kinase is also involved in CPPD and monosodium urate crystal-induced neutrophil activation (18), but the role of either in BCP crystalinduced activation of neutrophils, fibroblasts, or chondrocytes has not been reported.
Transduction pathways resulting in protease synthesis and secretion are likely active in BCP crystal-induced arthritis. Probable effectors of tissue damage from BCP crystals are MMPs, including collagenase-1 (MMP-1), stromelysin (MMP-3), 92-kDa gelatinase (gelatinase B/MMP-9), and collagenase-3 (MMP-13) (19 -21). The collagenase promotor contains a cis element termed the TPA response element (TRE), which is involved in increased transcription in response to agonists including tumor necrosis factor ␣ and phorbol esters (22,23). Increased transcription through the TRE depends on the transcription factor activator protein-1 (AP-1), a heterodimer composed of the protein products of c-fos and c-jun, both primary response genes, which interact to stimulate transcription of AP-1-responsive genes. BCP crystals induce c-fos and c-jun mRNA accumulation in human fibroblasts (HF) and inhibitors of protein synthesis such as cycloheximide block BCP crystal induction of MMP mRNA (19,24). This suggests that synthesis of a transactivating complex containing Fos and Jun proteins, such as AP-1, is necessary for BCP crystal induction of MMPs.
In the present study, we demonstrate that the mitogenic response to BCP crystals is associated with PKC activation in HF and NF-B induction in Balb/c/3T3 and HF. We confirm that BCP crystals induce the transcription factor AP-1, and we show that the PKC inhibitor, staurosporine, inhibits BCP crystal induction of c-fos, but not c-jun, mRNA in HF. In contrast, we show that BCP crystals do not activate PI3 kinase or tyrosine kinases confirming that cell activation by BCP crystals is selective in mechanism.

Cell Culture
A model system of human foreskin fibroblast (HF) cultures was used for these experiments since HF responses to BCP crystals have been shown to be similar to those of synovial fibroblasts (5). HF cultures were established from explants and transferred as described previously (28). They were grown and maintained in DMEM supplemented with 10% FBS containing 1% penicillin, streptomycin, and Fungizone (PSF). All experiments were performed on confluent cell monolayers that had been rendered quiescent by removing the media, washing with DMEM alone, and subsequently incubating in DMEM containing 0.5% FBS and 1% PSF for 24 h. All cultures used were 3rd or 4th passage cells.
Balb/c/3T3 (American Type Culture Collection, Rockville, MD) cultures were grown and maintained in DMEM with 10% Colorado calf serum and 1% penicillin/streptomycin. When approximately 70% confluent, cultures were rendered quiescent by incubating in DMEM containing 0.5% Colorado calf serum for 24 h prior to experiments. NIH 3T3 cells (American Type Culture Collection) were cultured in DMEM supplemented with 10% bovine calf serum and 1% penicillin/ streptomycin.

Determination of Mitogenesis by Uptake of [ 3 H]Thymidine
Cells were grown to confluence in 24-well plates and rendered quiescent by incubation in 0.5% FBS for 24 h. [ 3 H]Thymidine (1 Ci/ml) was added to the wells 23 h after the addition of 10% FBS, TPA (200 nM), or BCP crystals (18 g/cm 2 ) in the presence or absence of varying concentrations of staurosporine and pulse-labeled for 1 h. Control cultures were in 0.5% FBS with or without varying concentrations of staurosporine. The cells were then washed 3 times with phosphatebuffered saline (PBS), and macromolecules were precipitated with 5% trichloroacetic acid solution. The precipitate was washed again with PBS and dissolved in 1 ml 0.1 N NaOH, 1% SDS. Levels of trichloroacetic acid-precipitable 3 H were determined in triplicate, using a liquid scintillation counter (Packard Instruments, Downers Grove, IL).

Crystal Synthesis and Preparation
BCP crystals were synthesized by a modification of published methods (29). Mineral prepared by this method has a calcium/phosphate molar ratio of 1.59 and contains partially carbonate-substituted hydroxyapatite with admixed octacalcium phosphate shown by Fourier transform infrared spectroscopy. The crystals were crushed and sieved to yield 10 -20-m aggregates, which were sterilized and rendered pyrogen-free by heating at 200°C for 90 min. Heating to 200°C did not alter the crystal character or the relationship of hydroxyapatite and octacalcium phosphate, 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 crystal suspension was sonicated again just prior to use. The amounts of crystals used were determined based on our previous studies of dose-response relationships between BCP crystals and the mitogenic response (5). In those studies, maximal mitogenic responses were achieved with BCP levels of 50 -100 g/ml. These levels are equivalent to concentrations of BCP crystals found in pathologic joint fluids.

Extract Preparation
Cells were washed in PBS and lysed in E buffer (0.3% Nonidet P-40, 10 mM Tris, pH 8.0, 60 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PMSF)) by 5 min of incubation on ice. Nuclei were pelleted by spinning for 5 min at 2,500 rpm at 4°C in an Eppendorf microcentrifuge, and the supernatant was saved as the cytoplasmic extract prior to SDS-PAGE through 10% polyacrylamide gels (30). Glycerol was added to cytoplasmic extracts to a final concentration of 20%, after which the extracts were stored at Ϫ80°C until use. Nuclear extracts were washed in buffer E without Nonidet P-40 and resuspended in 40 l of C buffer (20 mM HEPES, pH 7.9, 0.75 mM spermidine, 0.15 mM spermine, 0.2 mM EDTA, 2 mM EGTA, 2 mM dithiothreitol, 20% glycerol, 1 mM PMSF). NaCl was added to a concentration of 0.4 M, and the nuclei were gently shaken for 10 min at 4°C. The nuclei were then pelleted by spinning for 10 min at 14,000 rpm at 4°C in an Eppendorf microcentrifuge, and the nuclear extract supernatant was stored at Ϫ70°C until use. The protein content of cell extracts was determined using the method of Bradford (Bio-Rad) (31).

DNA Binding Reactions and Electrophoretic Mobility Shift Analysis (EMSA)
EMSA was performed using a radiolabeled probe, UV 21, designed for cross-linking studies. This probe is derived from a template oligonucleotide UV1 (CAGGGCTGGGGATTCCCCATCTCCACAGTTTCAC-TTC) corresponding to bases Ϫ178 to Ϫ141 of the region 1 enhancer of the H-2K b gene which contains a strong B motif. UV1 was annealed to primer UV2 (GAAGTGAAAC-TGTGG) and extended with Klenow fragment in the presence of [␣-32 P]dCTP as described previously (32). The AP-1 probe is described under "Materials." Binding reactions were performed by first incubating 2 g of nuclear extract with 1 g of poly(dI-dC) in binding buffer (10 mM Tris, pH 7.7, 50 mM NaCl, 20% glycerol, 1 mM dithiothreitol, 0.5 mM EDTA) for 10 min at room temperature. Approximately 10,000 cpm of probe was then added and allowed to bind for approximately 30 min. Some nuclear extracts were treated with IB (recombinant IB␣ expressed in Escherichia coli and purified using histidine tags) prior to addition of the UV 21 probe. The reaction mix was then loaded onto native 5% acrylamide gels, prepared with 0.25ϫ TBE (22.3 mM Tris, 22.3 mM borate, 0.5 mM EDTA). Supershift experiments were performed by incubating binding reaction mixes with 1 l of antiserum for 15 min at room temperature. The polyclonal antibody against p50 (Ab1141) is generated in rabbits and is raised against a peptide fragment made from amino acids 2-15 of human p105. The anti-p65 antibody (Ab5192) is polyclonal antisera against the C terminus of human p65. Gels were analyzed either by autoradiography or with a PhosphorImager (Molecular Dynamics).

Northern Blot Analysis
Northern blot analysis of total cellular RNA was used to study the expression of c-fos and c-jun mRNA in HF after stimulation with BCP crystals in the presence or absence of the PKC inhibitor staurosporine. Confluent, quiescent monolayer cultures of HF in 100-mm plates were washed twice with cold PBS 24 h after treatment. Total RNA was recovered by precipitation with 4 M LiCl as described by Cathala et al. (33). Five micrograms of total cellular RNA was fractionated on a 1.2% agarose-formaldehyde gel; ribosomal RNA was visualized with ethidium bromide, and the fractionated RNA was transferred to nitrocellulose filters (34). Hybridization of the filters with DNA probes was performed overnight at 42°C. Probes were labeled using the random primer method, and [␣-32 P]dATP, to a specific activity of Ͼ5 ϫ 10 8 cpm/g (35). The filters were washed at a maximal stringency of 0.25ϫ standard saline citrate (SSC, 1ϫ SSC ϭ 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0) at 60°C for 30 min. Autoradiography was performed with Kodak XAR-5 film (Eastman Kodak Co.), and signal intensity was quantified by scanning laser densitometry (LKB Instruments, Stockholm, Sweden).

PKC Translocation
Confluent cultures of HF grown in 100-mm plates were washed twice in cold PBS and then harvested in 1.5 ml of translocation buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 0.2 mM PMSF, 10 g/ml leupeptin, 10 g/ml aprotinin, 0.33 M sucrose) (36). Cells were sonicated on ice for 15 s and then centrifuged at 100,000 ϫ g for 45 min. The supernatant was used as the cytosolic fraction. The pellet was then dissolved in 0.5 ml of translocation buffer containing Triton X-100 (0.1% w/v), shaken at 4°C overnight, and then centrifuged at 100,000 ϫ g for 45 min. The supernatant was used as the membrane fraction. Samples (20 l) of cytosolic and membrane fractions were subjected to SDS-PAGE in 10% polyacrylamide gels.

PKC Activity Assay
Plates were washed 2 times in cold PBS and then scraped into 1 ml of buffer A (0.3 M sucrose, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM EGTA, 0.3% ␤-mercaptoethanol, 0.1 mM PMSF, 10 mg/ml pepstatin, 10 mg/ml soybean trypsin inhibitor) and sonicated for 30 s. The cell lysate was then centrifuged at 100,000 ϫ g for 45 min, and the supernatant was removed. The pellet was resuspended in 1 ml of buffer A containing 1% Triton X-100 and then applied to 200 ml of DEAE-Sepharose minicolumns equilibrated in buffer B (buffer A without sucrose). The columns were then washed with 1 ml of buffer B and eluted with 1 ml of buffer C (buffer B ϩ 0.15 M NaCl). Prior experiments determined that 0.15 M NaCl eluted the majority of the PKC activity. 25 ml samples were then assayed for PKC activity using a PKC assay kit (Amersham Pharmacia Biotech). This assay is a modification of the mixed micelle assay and utilizes a peptide that is specifically phosphorylated on threonine by PKC (37,38). Assay conditions and separation of the phosphorylated peptide were as described in the instructions supplied with the kit.

Western Blotting
After electrophoresis, proteins were electrophoretically transferred to nitrocellulose membranes overnight at 4°C. After transfer, the membranes were blocked in 2.5% non-fat dry milk in Tris-buffered saline (TBS) (20 mM Tris, 500 mM NaCl, pH 7.5). The membranes were washed twice with Tween 20 wash solution (20 mM Tris, 500 mM NaCl, 0.05% Tween 20) (TTBS). The membranes were then incubated for 1 h with antibody buffer (0.1% non-fat dry milk-TTBS) containing a 1:500 or 1:1000 dilution of primary antibodies. The blots were then washed with TTBS and incubated with a 1:1000 dilution of biotin-conjugated goat anti-mouse or anti-rabbit IgG for 1 h. The blots were washed with TTBS and incubated with a 1:1000 dilution of alkaline phosphatase/streptavidin for 1 h. Membranes were washed twice with TTBS and once with TBS. Immunoreactive bands were detected using enhanced chemiluminescence reagents (ECL) (Amersham Pharmacia Biotech).

Statistics
Statistical analysis was performed using the Wilcoxon Rank Sum test (40).

BCP Crystals
Cause PKC Translocation-PKC activation is associated with translocation of the enzyme from the cytosolic to the membrane fraction of the cell. The addition of BCP crystals (18 g/cm 2 ) or TPA (200 nM) to HF cultures in 0.5% FBS/DMEM caused translocation of an upper band, immunoreactive with the monoclonal anti-PKC antibody (clone MC5), from the cytosolic compartment to the membrane compartment of HF harvested 15 min after treatment (Fig. 1). Treatment of HF with IL-1 (50 ng/ml) showed no such effect. Translocation of the immunoreactive band was maximal when cells were harvested 10 min after treatment with BCP crystals and returned to control levels within 30 min after stimulation (Fig. 2).
Inhibition of BCP Crystal-induced HF Mitogenesis by the PKC Inhibitor Staurosporine-By using [ 3 H]thymidine incorporation as an index of mitogenesis, we demonstrated a 5-fold increase in [ 3 H]thymidine incorporation in HF cultures treated with BCP crystals (25 g/cm 2 ) compared with that of control HF cultures incubated with 0.5% FBS alone after 24 h (Fig. 3). Staurosporine, which inhibits PKC by interacting with the catalytic domain, attenuated the mitogenic response of HF to BCP crystals in a concentration-dependent fashion. Similarly, the phorbol ester TPA (200 nM), a known activator of PKC, caused a 2 1 ⁄2-fold increase in [ 3 H]thymidine incorporation in HF cultures. Staurosporine inhibited the mitogenic response to TPA in a concentration-dependent manner. When HF were incubated with staurosporine alone, there was a modest increase in [ 3 H]thymidine incorporation at 0.5 and 0.1 nM concentrations.
BCP Crystals Cause Increased PKC Activity-The addition of BCP crystals (25 g/cm 2 ) to HF cultures in 0.5% FBS/DMEM produced an approximately 3-fold increase in PKC activity after 30 min (Table I). TPA (200 nM) caused an approximately 5-fold increase in PKC activity after 30 min. The increases in PKC activity induced by both BCP crystals and TPA were attenuated by the PKC inhibitors staurosporine, bisindolylmaleimide-1 (a competitive inhibitor for the ATP-binding site of PKC), and H7. To preclude inhibition of a cAMP-dependent kinase, we also investigated the effects of HA1004, a compound structurally similar to H7 but with a higher K i for PKC (K i PKC, 40 M; K i , cAMP-dependent kinase 2.3 M) (37). HA1004 had no inhibitory effect on BCP-or TPA-induced increases in PKC activity.
BCP Crystals Induce NF-B in Balb/c/3T3 Fibroblasts-By using the UV21 probe, BCP crystal-induced NF-B was demonstrated in Balb/c/3T3 fibroblasts by formation of an NF-B⅐DNA complex within 30 min of treatment which was maximal within 60 min of treatment and which had returned to base line by 24 h after treatment (Fig. 4). When nuclear extracts were treated with recombinant IB prior to addition of the UV21 probe, DNA binding of the protein complex was completely inhibited. Co-incubation of the nuclear extracts with an anti-p65 antibody caused a supershift of the NF-B⅐DNA complex, confirming the specificity of binding. The addition of an anti-p50 antibody did not result in a supershifted NF-B⅐DNA complex, likely due to inability of the human antibody to recognize murine protein.
BCP Crystals Cause NF-B Induction in HF-BCP crystalinduced NF-B was demonstrated in HF by formation of an NF-B⅐DNA complex within 4 h of treatment which continued through 24 h after treatment, the longest time point tested (Fig. 5). Co-incubation of the nuclear extracts with either an anti-p65 or anti-p50 antibody resulted in a supershifted complex. The induction of NF-B by BCP crystals was delayed relative to the induction of NF-B in response to 10% FBS as treatment of cultures with 10% FBS resulted in formation of an NF-B⅐DNA complex within 1 h which had returned to base line within 24 h after treatment. Control cultures were in 0.5% FBS.
When BCP crystal-treated HF cultures were co-incubated with the PKC inhibitor staurosporine, there was a concentration-dependent inhibition of BCP crystal-induced NF-B. Such inhibition was not specific as NF-B induction in response to 10% FBS was also inhibited (Fig. 6). Concurrent with BCP crystal induction of NF-B, there was transient disappearance of immunoreactive I-Ba noted both 1 and 4 h after treatment with BCP crystals. There was reappearance of I-B by 24 h after treatment (Fig. 7).
BCP Crystals Induce c-fos and c-jun mRNA and AP-1 in HF-BCP crystals caused maximal expression of c-fos mRNA in HF 30 min after treatment which returned to base line within 2 h (Fig. 8, A and B). When cultures were treated with staurosporine prior to stimulation with BCP crystals, c-fos mRNA expression was greatly attenuated. BCP crystals induced maximal expression of c-jun mRNA within 2 h after treatment which continued through 3 h, the longest time point tested (Fig. 8, C and D). In contrast with c-fos, there was minimal inhibition of c-jun mRNA when cultures were treated with staurosporine. Complementing these findings, BCP crystals induced nuclear translocation of the transcription factor AP-1 which continued for at least 2 h after treatment, the longest time point tested (Fig. 9, A and B). Nuclear binding was specific as none resulted from incubation with a mutant AP-1 probe. Control HF cultures were in 0.5% FBS, 10% FBS, or treated with TPA (200 nM). Co-incubation of the nuclear extracts with an anti-Fos antibody resulted in a supershifted complex.
BCP Crystals Do Not Induce Protein Tyrosine Phosphorylation or Activation of PI3 Kinase-Base-line levels of four tyrosine-phosphorylated proteins were observed in lysates from control and, unstimulated cultures. Bands at 50 and 27 kDa correspond to heavy and light chain antibody, respectively, reacting with secondary HRP-conjugated goat anti-mouse IgG. When cultures were treated with PDGF (50 ng/ml), there was enhanced tyrosine phosphorylation of a number of proteins with molecular masses of 185, 170, 145, 116, 80, 72, 62, 60, 40,  EMSA of equal amounts of protein from nuclear extracts of untreated, BCP crystal-stimulated, or 10% FBS-treated HF was performed using the probe described under "Experimental Procedures." Cells were harvested at the indicated times in hours after stimulation. C, unstimulated control cultures; B, BCP (18 g/cm 2 ) crystal-stimulated cultures; F, 10% FBS-treated cultures; numbers refer to time in hours after treatment. p50, polyclonal anti-peptide antibody to p50 (antibody 1141); p65, polyclonal anti-peptide antibody against p65; pep, corresponding peptide. Unlabeled arrows show the position of the supershifted complexes. and 33 kDa. In contrast, cells treated with either BCP crystals or the non-mitogenic particulate controls, latex beads or diamond dust, resulted in no increase in tyrosine phosphorylation relative to unstimulated cultures (Fig. 10A). Similarly, when HF cultures were treated with PDGF and lysates immunoprecipitated with anti-phosphotyrosine antibody, as expected, PI3 kinase activity on the immune complexes was dramatically increased over unstimulated cells (Fig. 10B, lane 3 versus 4). However, PI3 kinase activity on anti-phosphotyrosine immune complexes from BCP-treated cells was no different from untreated cells (lane 3 versus 5). NIH 3T3 cells treated with PDGF and immunoprecipitated with either phosphotyrosine or anti-PI3 kinase antibodies served as positive controls (lanes 1 and  2). Untreated, PDGF-treated, or BCP-treated HF cells immunoprecipitated with PI3 kinase antibody also served as positive controls for PI3 kinase activity (lanes 6 -8). DISCUSSION The results of this investigation show that BCP crystalinduced cell activation is associated with NF-B induction in Balb/c/3T3 and human fibroblasts (HF) and PKC activation and AP-1 induction in HF. Moreover, we have demonstrated that inhibition of BCP crystal-induced mitogenesis by the PKC inhibitor staurosporine is accompanied by inhibition of BCP crystal induction of NF-B and c-fos, but not c-jun, mRNA. Although the cellular effects of many other growth-promoting agents are mediated by high affinity receptor protein tyrosine kinases (PTK) (14) with resultant recruitment and activation of PI3K (15), out data show that neither tyrosine phosphorylation nor PI3K activity appears to be required for BCP crystalinduced cell activation. Thus, these observations further define the biological effects of BCP crystals.
BCP crystals cause mitogenesis in Balb/c/3T3 murine fibroblasts, and NF-B is induced when quiescent Balb/c/3T3 fibroblasts are stimulated with serum growth factors (6,11). Therefore, we initiated our investigation by studying the ability of BCP crystals to induce NF-B in this cell type. We were unable to demonstrate the presence of the p50 subunit in the NF-B⅐DNA complex by supershift in Balb/c/3T3 cells treated with BCP crystals. However, the antibody was raised against a peptide fragment from human, and not murine, p105, the cytoplasmic precursor of p50 and therefore may not recognize murine protein.
BCP crystals possess many of the characteristics typical of a growth factor. Most water-soluble hormones or growth factors bind to specific receptors on the cell surface which induce the intracellular activation of signaling cascades. The mediator of the earliest events induced by contact of insoluble BCP crystals with the cell surface, or with an as yet unknown surface "receptor," has not yet been identified. We have used PDGF as a control in our work because the PDGF receptor has been characterized, and the signal transduction pathways induced by the PDGF receptor have been analyzed in detail (41). In contrast to that of BCP crystals, PTK and PI3 kinase are required for PDGF-induced DNA synthesis (16). Furthermore, PDGF-induced DNA synthesis occurs through a protein kinase C (PKC)independent pathway, whereas BCP crystals require at least basal levels of PKC to induce mitogenesis (6).
PTK and PI3K are involved in CPPD crystal-induced neutrophil activation (17,18). Although both are calcium-containing, CPPD and BCP crystals differ in their clinical manifestations. CPPD is commonly associated with attacks of acute arthritis, "pseudogout," characterized by joint effusions packed with neutrophils. BCP crystals in synovial fluid, in contrast, are usually associated with few neutrophils. It is not known whether BCP crystals induce PTK or PI3K in neutrophils. BCP crystals did not induce PTK or PI3K in human fibroblasts, in contrast with PDGF (6). These results emphasize the specificity of cell activation by BCP crystals and suggest that BCP crystals, unlike PDGF, directly or indirectly activate G-proteinlinked surface receptor(s) (42).
Many stimuli other than BCP crystals activate NF-B. These include cytokines and activators of protein kinase C such as the phorbol ester TPA (43). Several signal transduction pathways may be involved, but all of these stimuli act by means of protein kinases that phosphorylate (and thus degrade) IB. It was originally proposed that IB might serve as a substrate for kinases such as PKC and protein kinase A, based on in vitro studies (44,45). Recently, however, a high molecular mass IB kinase complex containing at least two cytokine-responsive IkB kinases has been identified (46). These kinases specifically phosphorylate critical serine residues of IB. Activation of NF-B by tumor necrosis factor-␣ and IL-1 requires the successive action of NF-B-inducing kinase and IB kinase (47). The molecular mechanisms by which NF-B-inducing kinase becomes activated are not yet understood. Although BCP crystals activate both PKC and NF-B, it is not known to what extent these two events are linked. Since the critical modification for IkB kinases activation appears to be phosphorylation, it is possible that PKC plays a role in BCP crystal induction of NF-B by inducing phosphorylation and activation of IkB kinases (48). In support of this concept, the PKC inhibitor staurosporine inhibited both BCP crystal-induced mitogenesis and NF-B. Alternatively, it is possible that staurosporine inhibits IkB kinases since, although it is a potent inhibitor of PKC, it also inhibits PKA and other protein kinases (49).
BCP crystals induce MMP synthesis and secretion in fibroblasts and chondrocytes, but the mechanism by which this occurs remains poorly understood. We have shown that while intracellular dissolution of BCP crystals augments the mitogenic response, it does not play a role in BCP induction of MMPs (50). The mechanism of stimulation of the synthesis and secretion of MMPs in response to stimuli other than BCP crystals is partly understood. For example, PKC activation is required for MMP induction in fibroblasts treated with epidermal growth factor (51) or interleukin-1 (52). Although PKC activation occurs with BCP crystal-induced mitogenesis, its role in the associated MMP production has not been established. Staurosporine does not inhibit BCP crystal-induced collagenase mRNA accumulation in HF 2 at concentrations which inhibit mitogenesis. We have shown here that staurosporine inhibited BCP crystal-induced c-fos but not c-jun mRNA accumulation in HF. This suggests that BCP crystal-induction of c-fos requires PKC activity, whereas induction of c-jun does 2 G. M. McCarthy, unpublished observations. A and B) and c-jun (C and D) mRNA accumulation, effect of staurosporine. Confluent cultures of HF in 100-mm plates were incubated in DMEM and 0.5% FBS for 24 h before being treated with BCP crystals (18 g/cm 2 ). Some cultures were treated with staurosporine (1 nM) 30 min prior to stimulation with BCP crystals. Unstimulated control cultures were in 0.5% FBS. At varying time points, cultures were harvested, and total RNA was isolated. Northern blot analysis was performed using radiolabeled c-fos cDNA, followed by autoradiography. The blot was then stripped of the c-fos probe and re-probed with radiolabeled c-jun cDNA, and autoradiography repeated. The blot was stripped one more time and re-probed with radiolabeled glyceraldehyde-3-phosphate dehydrogenase (GAPD) cDNA as a control. Autoradiographs were scanned using a laser densitometer with results presented as bar graphs and expressed as the percent of maximal expression, using glyceraldehyde-3-phosphate dehydrogenase to correct for small variations in total RNA analyzed. B, BCP crystals; St, staurosporine. The positions of the 18 S and 28 S ribosomes are shown.

FIG. 8. BCP crystal induction of c-fos (
not. Members of the AP-1 family of transcription factors include Fos/Jun or Jun/Jun dimers that bind preferentially to the TRE of the collagenase promotor (53). It is possible that the lack of inhibition of BCP crystal-induced c-jun or collagenase mRNA accumulation in the presence of staurosporine reflects binding of Jun/Jun dimers to the TRE during BCP crystal induction of MMP1 and other MMPs. Alternatively, since MAP kinases also modulate AP-1 activity (53), BCP crystal-induced activation of MAP kinases could facilitate MMP production in the presence of staurosporine (8).
The AP-1 sequence or TRE is necessary but not sufficient for phorbol ester induction of collagenase in fibroblasts (54). Furthermore, in some cells MMP expression is dependent on activation of and DNA binding to NF-B but not AP-1 (55). Conversely, the functional and physical interplay of the NF-B and AP-1 families of transcription factors have recently been reported to result in enhanced DNA binding and biological function of each (56). Finally, the potential role of the transcription  10. PDGF-or BCP crystal-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in fibroblasts. A, confluent HF in 100-mm plates were incubated overnight in 0.5% FBS and either left untreated (C) or treated with latex beads (18 g/cm 2 ), diamond dust (18 g/cm 2 ), BCP crystals (18 g/cm 2 ), or PDGF (50 ng/ml) for the times (Ј, min) indicated. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody 4G10 as described under "Experimental Procedures." Immune complexes were solubilized in SDS sample buffer, separated by 8.75% SDS-PAGE, and immunoblotted with 4G10 antibody as described under "Experimental Procedures." Tyrosine-phosphorylated proteins were detected by chemiluminescence. Molecular mass markers in kDa are indicated at the left. B, NIH 3T3 or human fibroblasts were either untreated (C) or treated as in A with either BCP crystals (18 g/cm 2 ) or PDGF (50 ng/ml) for 15 min. Cell lysates were immunoprecipitated (PPT) with either antiphosphotyrosine antibody 4G10 or anti-PI3K antibody as described under "Experimental Procedures." The PI3K assay, as described under "Experimental Procedures," was used to assess PI3K activity on the washed immune complexes using PtdIns(4,5)P 2 as substrate. Lipids were separated by thin layer chromatography and radiolabeled PtdIns (3,4,5) factor PEA3 in the transcriptional regulation of BCP crystalinduced MMP expression needs to be considered as the collagenase promotor contains a PEA3-binding site that acts synergistically with AP-1 to achieve maximal levels of transcriptional activation in response to TPA (57). BCP crystals induce both NF-B and AP-1, and our data suggest that NF-B plays a role in BCP crystal-induced mitogenesis and that AP-1 is involved in BCP crystal induction of MMPs. Further study will be necessary to show whether NF-B and/or PEA3 activation is necessary for BCP crystal induction of MMPs.
Glucocorticoids are potent inhibitors of NF-B activation in mice and cultured cells. Such inhibition appears to be mediated by induction of IB␣ inhibitory protein (58). Glucocorticoids also inhibit MMP-1 induction by interfering with AP-1 (59 -61). Intra-articular glucocorticoids are effective for at least shortterm relief of pain in OA, but no clinical predictors of response have been found (62). The presence or absence of synovial fluid BCP crystals was not evaluated, however, and based on the current data, glucocorticoids likely inhibit the major biological effects of BCP crystals in vivo. In addition, aspirin and sodium salicylate, both of which have been used to treat the symptoms of joint degeneration, also inhibit the activation of NF-B so that the anti-inflammatory effects of these drugs may be partly attributable to the inhibition of NF-B (63). Finally, a number of PKC modulators are currently undergoing clinical evaluation as anticancer drugs, and tamoxifen, already in clinical use, is a moderately potent inhibitor of PKC (64). PKC inhibition could be an effective target for attenuation of BCP crystalinduced mitogenesis.
The association of BCP crystals with osteoarthritis and joint destruction is well established. Synovial hypertrophy is likely a response to the mitogenic effects of the crystals, and degradation of cartilage likely results from MMP activity induced by the crystals in synoviocytes and chondrocytes. However, no known drug prevents or treats the consequences of BCP crystal deposition. An improved understanding of the molecular mechanisms leading to BCP crystal-induced mitogenesis and MMP induction is essential to the development of a rational approach to ultimate prevention or reversal of the consequences of BCP crystal deposition.