Molecular Mechanism of the Induction of Metalloproteinases 1 and 3 in Human Fibroblasts by Basic Calcium Phosphate Crystals

Synovial fluid basic calcium phosphate (BCP) crystals are common in osteoarthritis and are often associated with destructive arthropathies involving cartilage degeneration. These crystals are mitogenic and induce oncogene expression and matrix metalloproteinase (MMP) synthesis and secretion in human fibroblasts. To date, BCP crystal-elicited signal transduction pathways have not been completely studied. Because protein kinase C (PKC) is known to play an important role in signal transduction, we investigated the participation of this pathway in the BCP crystal induction of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts. Using reverse transcription/polymerase chain reaction (RT-PCR) and Northern and Western blotting techniques, we show here that BCP crystal stimulation of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts is dependent upon the calcium-dependent PKC signal transduction pathway and that the PKCα isozyme is specifically involved in the pathway. We have previously shown that BCP crystal induction of MMP-1 and MMP-3 is also dependent on the p44/42 mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We now show that these two pathways operate independently and seem to complement each other. This leads to our hypothesis that the two pathways initially function independently, ultimately leading to an increase in mitogenesis and MMP synthesis, and may converge downstream of PKC and p44/42 MAPK to mediate BCP crystal-induced cellular responses.


PKC) is known to play an important role in signal transduction, we investigated the participation of this pathway in the BCP crystal induction of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts. Using reverse transcription/polymerase chain reaction (RT-PCR) and Northern and Western blotting techniques, we show here that BCP crystal stimulation of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts is dependent upon the calcium-dependent PKC signal transduction pathway and that the PKC␣ isozyme is specifically involved in the pathway.
We have previously shown that BCP crystal induction of MMP-1 and MMP-3 is also dependent on the p44/42 mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway. We now show that these two pathways operate independently and seem to complement each other. This leads to our hypothesis that the two pathways initially function independently, ultimately leading to an increase in mitogenesis and MMP synthesis, and may converge downstream of PKC and p44/42 MAPK to mediate BCP crystal-induced cellular responses.
Calcium-containing crystals such as basic calcium phosphate (BCP) 1 and calcium pyrophosphate dihydrate (CPPD) are two of the most common forms of pathologic articular materials that are associated with destructive arthropathies involving cartilage degeneration (1,2). At concentrations found in pathologic human joint fluids, these crystals exert biological effects on cultured cells in a manner similar to growth factors like platelet-derived growth factor, epidermal growth factor, and serum. It has been demonstrated that BCP crystals stimulate fibroblast, synoviocyte, and chondrocyte mitogenesis in vitro (3); stimulate the production of prostaglandin via the phospholipase A 2 /cyclo-oxygenase pathway (4); activate phospholipase C and inositol phospholipid hydrolysis (5); induce the expression of the proto-oncogenes, c-fos and c-myc (6,7); and induce the synthesis and secretion of metalloproteinases (MMPs) 1, 3, 8, and 13 (8 -12).
In contrast to other mitogenic and growth factors, BCP crystal-elicited signal transduction pathways have not been completely studied. However, we have identified some of the component molecules involved in calcium-containing crystal signal transduction mechanisms. One pathway activated upon crystal stimulation of human fibroblasts (HF) is the p44 and p42 mitogen-activated protein kinase (p44/42 MAPK) pathway, also known as extracellular signal-related mitogen protein kinases 1 and 2 (ERK1 and ERK2), respectively. The MAPK cascade can be blocked by the selective inhibitors, PD98059 (13) and U0126 (14), which hinder the activation and phosphorylation of MEK (MAPK/ERK kinase). Co-treatment of HF with BCP crystals and PD98059 blocks crystal-induced p44/42 MAPK activation and mitogenesis (15) in addition to crystalinduced up-regulation of MMP-1 and MMP-3 mRNA and protein expressions (16). Moreover, phosphocitrate (PC), a specific inhibitor of the biological effects of BCP and CPPD crystals (17), also blocks crystal-induced activation of p44/42 MAPK, further supporting the role of this signal pathway in crystalinduced responses in HF (15).
Another messenger with an apparent role in crystal-activated signal transduction is calcium. We have previously shown that treatment of HF with BCP crystals induces a rapid transient rise of intracellular calcium levels in seconds due to calcium influx from outside the cell, followed by a slow and sustained increase of intracellular calcium within 60 min after stimulation, due to crystal dissolution (18). Removal of calcium from the cell culture medium attenuates the BCP crystal in-duction of c-fos mRNA (18), suggesting that an influx of extracellular calcium is required for maximal induction of c-fos expression. Perhaps related to the rise of intracellular calcium is the crystal activation of adenosine 3Ј,5Ј-cyclic monophosphate (cAMP) response element (CRE)-binding protein (CREB) (15), a key transcriptional regulator of the c-fos gene that has been shown to be important for mediating c-fos activation in response to elevated levels of intracellular calcium (19).
Treatment of cells with BCP crystals also results in the activation of phospholipase C, leading to the hydrolysis of phosphatidylinositol 4,5-biphosphate and production of the intracellular messengers, inositol triphosphate and diacylglycerol (DAG) (5,20). Inositol triphosphate modulates the activities of calcium-dependent enzymes such as protein kinases by releasing calcium from the endoplasmic reticulum (21) whereas diacylglycerol is a potent activator of protein kinase C (PKC) (22). In humans, the PKC family consists of at least 11 structurally related serine/threonine protein kinases. These isozymes are further divided into three subfamilies: the conventional, the atypical, and the novel isozymes. The conventional isozymes include alpha (␣), beta I (␤I), beta II (␤II), and gamma (␥), and their activities are calcium-and phospholipid-dependent. The novel isozymes comprise delta (␦), epsilon (⑀), eta (), and theta (), whose activities are calcium-independent but phospholipiddependent. The atypical isozymes are made up of zeta (), iota (), and mu (), and their activities are neither calcium-nor phospholipid-dependent (23,24).
We have previously shown that crystal treatment of HF results in the translocation of the PKC enzyme from the cytosolic to the membrane fraction of the cell, an indicator of PKC activation. The BCP crystal-induced PKC activity is blocked by co-treatment of crystal-stimulated cells with the PKC inhibitors, staurosporine and bisindolylmaleimide I (25). Furthermore, an increase in PKC activity associated with the membrane fraction is seen following BCP crystal stimulation of chondrocytes (26). Down-regulation of PKC activity by chronic treatment with the phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate, an analog of DAG, blocks crystal-induced c-fos and c-myc expressions and mitogenesis in Balb/c 3T3 cells (7) whereas co-treatment with the PKC inhibitor, staurosporine, blocks BCP-induced c-fos expression and mitogenesis in HF (25), indicating that PKC activity is essential for these crystalinduced effects to occur.
In this study, we investigated the participation of the PKC signal transduction pathway in the BCP crystal induction of MMP-1 and MMP-3 mRNA and proteins in HF. Because the PKC family comprises several isozymes, we further sought to identify the specific isozyme of the PKC family that is translocated to the putative membrane fraction as an indication of PKC activity in the human fibroblasts. We also examined the requirement of calcium signaling in the crystal activation of PKC in HF as well as the relationship between the BCP crystal-induced PKC and p44/42 MAPK signal transduction pathways.
Cell Culture-HF were established from explants and transferred as previously described (27). They were grown and maintained in DMEM supplemented with 10% heat-inactivated FBS containing 1% penicillin, streptomycin, and fungizone. All cultures were third or fourth passage cells. All experiments were performed on confluent monolayers that had been rendered quiescent by removing the medium, washing the cells with DMEM alone and subsequently incubating the cells in the same medium containing 0.5% FBS for 24 h (MMPs and PKC) or for 48 h (p44/42 MAPK). Then this medium was removed, the cells were washed with PBS, and serum-free DMEM was added to the cells. For the inhibition experiments, the cells were pretreated with the appropriate concentrations of the inhibitors for 30 min before being stimulated with BCP crystals or PMA for the indicated length of time.
BCP Crystals and PC Preparations-BCP crystals were synthesized by modification of previously published methods (28). These crystals have a calcium/phosphate ratio of 1.59 and contain partially carbonatesubstituted hydroxyapatite mixed with octacalcium phosphate as indicated by Fourier transform infrared spectroscopy. The crystals were crushed and sieved to yield 10-to 20-m aggregates, which were sterilized and rendered pyrogen-free by heating at 200°C for at least 90 min. PC was prepared as previously described (29).
RT and PCR-Total RNA was isolated using the reagent TRIzol according to the manufacturer's instructions. Then 1 g of each sample was reverse-transcribed at 50°C for 60 min, followed by enzyme inactivation at 85°C for 5 min using the ThermoScript RT-PCR system. The resulting cDNA samples were amplified by the PCR method. PCR primers for MMP-1 were: sense, 5Ј-GATCATCGGGACAACTCTCCT-3Ј, corresponding to positions 567-587, and antisense, 5Ј-TCCGGGTA-GAAGGGATTTGTG-3Ј, corresponding to positions 980 -1000 of the published nucleotide sequence of the human skin collagenase cDNA and giving a PCR product of 434 bp (30). The primers for MMP-3 were: sense, 5Ј-GAAAGTCTGGGAAGAGGTGACTCCAC-3Ј, corresponding to positions 414 -440, and antisense, 5Ј-CAGTGTTGGCTGAGTGAAA-GAGACCC-3Ј, corresponding to positions 671-697 of the nucleotide and amino acid sequence for human MMP-3 and giving a PCR product of 284 bp (31). As an internal control, 353 bp of the constitutively expressed housekeeping gene, ␤-actin, was also synthesized and used to normalize the amount of mRNA in each RT-PCR reaction. All primers were synthesized by Invitrogen (Gaithersburg, MD). Amplifications were carried out for 30 cycles by denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and extending at 72°C for 45 s, with a final extension at 72°C for 10 min. The PCR products were analyzed by electrophoresis on 2% agarose gel containing ethidium bromide.
Northern Blotting-Total RNA samples (10 g each) were denatured and electrophoresed through a 1.2% agarose gel containing 2.2 M formaldehyde followed by transfer and cross-linking with a UV Stratalinker 1800 (Stratagene, La Jolla, CA) to Nytran Supercharge nylon membranes (Schleicher & Schuell, Inc., Keene, NH). The membranes were prehybridized at 42°C for 4 h and then hybridized at 42°C overnight to MMP-1-and MMP-3-specific cDNA probes that were radiolabeled with [␣-32 P]dATP (6000 Ci/mmol, Amersham Biosciences, Inc., Piscataway, NJ). The blots were subsequently stripped and reprobed with GAPDH cDNA as a control. After washing, the hybridized membranes were exposed to Kodak X-OMAT-AR films with intensifying screen at Ϫ80°C.
Western Blotting-Aliquots of conditioned media (MMPs), cell lysates (p44/42 MAPK), and membrane fractions (PKC) were electrophoresed through a 10% (MMPs) or 7.5% (PKC) or 12% (p44/42 MAPK) SDSpolyacrylamide gel and then transferred onto Immobilon-P PVDF membranes (Millipore, Bedford, MA). After transfer, the membranes were incubated for 4 h at room temperature in the blocking buffer, TBST (20 mM Tris, 136 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk to eliminate nonspecific binding. The membranes were washed several times and then incubated in TBST containing 5% bovine serum albumin at 4°C overnight with the following antibodies: a monoclonal antibody against MMP-1 or MMP-3, a monoclonal antibody against PKC, a phospho-specific monoclonal MAPK antibody recognizing p44/42 MAPK phosphorylated at Tyr-204 and Thr-202 or a polyclonal p44/42 MAPK antibody or a polyclonal antibody against each of the PKC isozymes, ␣, ␤I, ␤II, and ␥. The membranes were again washed several times with TBST and incubated with the appropriate antimouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody in TBST with 5% bovine serum albumin for 1 h at room temperature. Finally, the membranes were washed in TBST and TBS, and the protein bands were visualized colorimetrically with a solution containing 3,3-diaminobenzidine (25 mg/100 ml) and hydrogen peroxide in 0.05 M Tris-HCl, pH 7.5.
PKC Translocation-After treatment, the cells were washed twice with cold PBS. The cells were then harvested on ice in 1.5 ml of translocation buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.5 mM EGTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, and 0.33 M sucrose). The cells were sonicated on ice for 15 s and then centrifuged at 100,000 ϫ g for 45 min. The supernatant was collected as the cytosolic fraction. The pellet was then dissolved in 0.5 ml of translocation buffer containing 0.1% Triton X-100, shaken at 4°C overnight, and then centrifuged again at 100,000 ϫ g for 45 min. The supernatant was used as the membrane fraction. Samples (25 l each) of the fractions were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and Western blotting p44/42 MAPK Activation-Following experimental treatments, the cells were washed twice with ice-cold PBS. The cell lysates were then harvested on ice in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue). The cell lysates were scraped into microcentrifuge tubes and boiled for 5 min, and aliquots (25 l) were subjected to 12% SDSpolyacrylamide gel electrophoresis and Western blotting with a Phospho p44/42 MAPK monoclonal antibody or p44/42 polyclonal antibody.
Statistics-Statistical analysis was performed by the Student's t test in SigmaPlot Scientific Graphing software, and p Ͻ 0.05 was considered significant. Data were expressed as the means Ϯ S.E.

Participation of a Protein Kinase Pathway in the BCP Crystal-induced MMP-1 and MMP-3
Expression-Treatment of cultured human fibroblasts with calcium-containing crystals gives rise to increased expression levels of MMP-1 and MMP-3 (9,17). To determine whether a protein kinase signaling is necessary for the BCP crystal-induced expression of these MMPs, we examined the effect of staurosporine, a potent, cell-permeable and broad spectrum inhibitor of protein kinases on the BCP crystal-induced MMP mRNA and protein levels by reverse transcription (RT)-PCR and Northern and Western blots. The RT-PCR with MMP-1-and MMP-3-specific primers show that, after 24 h of stimulation of HF with BCP crystals, levels of MMP-1 and MMP-3 mRNA increased approximately 4-fold over the control levels as shown in Fig. 1, A and B, respectively. The inhibition of the MMP-1 and MMP-3 mRNA by staurosporine is concentration-dependent, with the greatest inhibition at 100 nM, which is similar to the inhibition by 1 mM of PC, a well known inhibitor of the biological effects of BCP crystals (17) and suggests the participation of a protein kinase signaling pathway. The corresponding expression of the housekeeping gene, using ␤-actin primers, did not show any change in Fig.  1C. The densitometric scan of the relative intensities (means Ϯ S.E.) of three such independent experiments showed a significant inhibition of the BCP crystal-induced MMP-1 and MMP-3 by staurosporine at 100 nM (p Ͻ 0.05) (data not shown).
Northern blotting of the RNA samples in Fig. 2 shows no degradation of the RNA at all concentrations of staurosporine in Fig. 2A. Fig. 2, B and C, shows that, at 100 nM staurosporine, the complete inhibition of MMP-1 and MMP-3 mRNA, respectively, is similar to the inhibition by 1 mM of PC whereas panel D shows no change in the housekeeping gene, GAPDH.
These results were confirmed with Western blotting of the culture medium in Fig. 3. Here, there is also a concentrationdependent inhibition of the BCP crystal-induced MMP-1-and MMP-3-secreted proteins in Fig. 3, A and B, respectively, with the greatest inhibition again at 100 nM staurosporine and with the molecular mass of the proteins corresponding to MMP-1 control standard at 53-55 kDa and MMP-3 control standard at 57-59 kDa. All the results suggest the participation of a protein kinase pathway.
Identification of Protein Kinase C as the Signaling Pathway-Our aim was to determine whether PKC was involved in the BCP crystal activation of MMP-1 and MMP-3 transcription in human fibroblasts. Using bisindolylmaleimide I (Bis I), a highly selective, cell-permeable PKC inhibitor that is structurally similar to staurosporine (33), we have shown, by Northern blotting, that there is a concentration-dependent inhibition of MMP-1 and MMP-3 mRNA by Bis I in Fig. 4, A and B, respectively, and that, at 10 M, the inhibition is similar to the inhibition by 1 mM PC. To determine the specificity of the inhibition, we also used bisindolylmaleimide V (Bis V), which is a structural analog of Bis I and a negative control inhibitor for PKC (34) and which shows no inhibition even at the same 10 M concentration as Bis I. We also used PMA as a positive control for PKC stimulation. The samples were normalized with GAPDH as the housekeeping gene (Fig. 4C). These results were confirmed by Western blotting for the MMP-1 and MMP-3 protein expressions in Fig. 5, A and B, respectively. The results identify the PKC signaling pathway as a participant in the BCP crystal induction of MMP-1 and MMP-3 in HF.
Requirement for Calcium in the PKC Signaling Pathway-To identify the particular subfamily of PKC that participates in the signaling pathway upon the BCP crystal induction of MMP-1 and MMP-3 mRNA, we used the indolocarbazole Gö6976, which is a specific inhibitor of the calcium-dependent PKC (35). Simultaneous treatment of the cells with BCP crystals and Gö6976 led to a concentration-dependent inhibition of MMP-1 and MMP-3 mRNA expression in the Northern blotting results in Fig. 6, A and B, respectively and with the maximum inhibition at 25 nM Gö6976 similar to the inhibition by PC at 1 mM. PMA was used as a positive control for the PKC activity, and the samples were again normalized with the housekeeping gene, GAPDH (Fig. 6C). These results were also confirmed by Western blotting for the MMP-1 and MMP-3 protein expressions in Fig. 7, A and B. The results show convincingly that the calcium-dependent PKC subfamily is required for the BCP crystal induction of MMP-1 and MMP-3 mRNA and protein expressions in human fibroblasts.
Further evidence for the involvement of a calcium-dependent PKC signaling pathway is provided by determining PKC activity in the absence and presence of calcium. Because PKC is known to be physiologically active only in the membrane-associated state and translocation of the PKC enzyme from the cytosol to the membrane of the cell is used to monitor its intracellular activation (25,26), we determined PKC activity in the membrane fractions of the cells in calcium-and magnesium-free HBSS and compared it with the activity in HBSS containing calcium and magnesium. Using Gö6976 as the spe-cific inhibitor of the calcium-dependent PKC and PMA as the positive control for the PKC activity, we have shown that there was no PKC activity in the absence of calcium and magnesium as seen in Fig. 8A. On the other hand, BCP crystal induction resulted in increased PKC activity in the presence of calcium and magnesium, similar to that of PMA as the positive control,

FIG. 7. Western blotting analysis of the inhibition of BCP crystal-induced MMP-1 and MMP-3 protein expression by the Ca 2؉dependent PKC inhibitor, Gö 6976.
The culture medium of the experiment in Fig. 6 was concentrated 10-fold and electrophoresed on a 10% SDS-polyacrylamide gel, transferred to PVDF membranes, and subsequently blotted with monoclonal antibodies against MMP-1 (A) and MMP-3 (B). Concentrated serum-free medium containing Human MMP-1 and MMP-3 was used as the positive control standards. Blots shown are representatives of three independent experiments. and is totally inhibited by 2 M Gö6976 (Fig. 8B). However, this concentration is different from the concentration of 25 nM that completely inhibited the MMPs in Figs. 6 and 7. Our dose-dependent study (data not shown) found 2 M to be the concentration of Gö6976 that would inhibit PKC in HF. This is in agreement with a previous study, which found that 2 M Gö6976 was not toxic to NALM-6 cells (36). Taken together, all these results confirm the necessity for the calcium-dependent PKC in the signaling pathway for the BCP crystal induction of MMP-1 and MMP-3.
Identification of the Specific PKC Isozyme-The only calciumdependent subfamily of PKC is the conventional subfamily, which is a pool of isozymes consisting of alpha (␣), beta I (␤I), beta II (␤II), and gamma (␥) (23,24). To evaluate the extent and specificity of the PKC activation induced by BCP crystals, we also sought to identify the specific isozyme/isozymes involved in the induction. As seen in Fig. 9, blotting of the membrane fractions with the total pool PKC antibody and with the antibodies to the individual isozymes showed an induction of PKC in the total pool by BCP crystals in panel A and a more specific induction of PKC␣ isozyme in panel B, whereas there was no induction at all of the ␤I, ␤II, and ␥ isozymes in panels C, D, and E, respectively. The specificity of the PKC␣ isozyme was confirmed with the use of the Gö6976, which is a specific inhibitor of the calcium-dependent PKC ␣ and ␤I isozymes (35). Complete inhibition was seen in the PKC in the total pool as well as in the PKC␣, thus unequivocally identifying the ␣ isozyme of the calcium-dependent PKC as being activated by BCP crystals.
Cooperativity of PKC with PKC-independent MAPK-We have previously shown that treatment of human fibroblasts with calcium-containing crystals activate the p44/42 MAPK signal transduction pathway (15) and recently reported that this pathway is required for maximal induction of MMP-1 and MMP-3 mRNA and proteins by BCP crystals (16). Here, we were interested in determining whether p44/42 MARK induction by BCP crystals is PKC-dependent and whether the two pathways are coupled in HF. Treatment of the cells with BCP crystals resulted in an increased level of Phospho p44/42 MAPK activation shown in Fig. 10. When the same concentrations of the protein kinase inhibitor, staurosporine, which in-hibited BCP crystal-induced MMP-1 and MMP-3 mRNA and proteins shown in Figs. 2 and 3, respectively, were used with the BCP crystal-treated cells, there were no changes in the BCP crystal-induced Phospho p44/42 levels in Fig. 10A. To show that BCP crystal-induced Phospho p44/42 could be inhibited, 1 mM PC was used as a control inhibitor, which resulted in a marked inhibition of the BCP crystal-induced Phospho p44/ 42. To the contrary, the constitutively expressed or nonactivated p44/42 was seen with no changes in all the samples in Fig. 10B. These results demonstrate that the BCP crystal activation of the p44/42 signal transduction pathway is independent of the PKC pathway.
Further evidence for the two independent pathways is provided by treatment of the BCP crystal-stimulated cells with inhibitors of the two different pathways in Fig. 11. Treatment of the BCP crystal stimulated cells with the PKC inhibitors, Bis I and Gö6976, inhibited only PKC whereas treatment with the Phospho p44/42 inhibitors, PD98059 and U0126, did not inhibit PKC at all as shown in Fig. 11A. Conversely, the PKC inhibitors did not inhibit Phospho p44/42, which was only inhibited by its own inhibitors, PD98059 and U0126, as shown in Fig.  11B, thus indicating that the two pathways are independent of each other. On the other hand, the constitutively expressed or nonactivated p44/42 was not affected by any of the inhibitors in Fig. 11C.
BCP Crystal-activated p44/42 MAPK Pathway Is Calciumindependent-We have shown in Fig. 8 that BCP crystal induction of MMP-1 and MMP-3 requires the calcium-dependent PKC signaling pathway. Similarly, we wanted to know if the p44/42 MAPK pathway was also calcium-dependent. Cellular calcium requirement can be met by either an influx of extracellular calcium from the culture medium into the cells (18) or by the stimulation of a phosphatidylinositol-specific phospholipase C (PI-PLC), leading to the generation of inositol triphosphate and diacylglycerol (DAG) (20), which are involved in intracellular calcium mobilization (21) and PKC activation (22), respectively. Although extracellular calcium chelation by EGTA and intracellular calcium chelation by BAPTA-AM and TMB blocked the BCP crystal-induced PKC in Fig. 12A, they had no effect on Phospho p44/42 (Fig. 12B). These results show that neither calcium influx nor calcium release is necessary for the BCP crystal-mediated activation of the p44/42 MAPK pathway, thus establishing that this pathway is distinct from the calcium-dependent PKC pathway through which BCP crystals activate MMP-1 and MMP-3 in the human fibroblasts.

DISCUSSION
The ultimate biological effects of calcium-containing crystals on cells in vitro are an increase in MMP synthesis and secretion and increased mitogenesis. These effects are hypothesized to be correlated with calcium deposition disease in vivo. The increased production of matrix-degrading MMPs by synoviocytes results in articular damage and degeneration and the release of additional crystals from the surrounding tissue, whereas mitogenesis leads to an increase in synoviocytes that generate more MMPs (37). Of interest are the signal transduction mechanisms by which crystal-induced up-regulation of MMP synthesis and secretion and increased mitogenesis are mediated. Here, we demonstrate for the first time that the calcium-de-pendent PKC signal transduction pathway is required for maximal BCP crystal induction of MMP-1 and MMP-3 mRNA and protein expressions in HF and also identify PKC␣ as the specific isozyme that is activated upon BCP crystal stimulation. We also show that the calcium-dependent PKC signal transduction pathway works in cooperation with the distinct calcium-independent p44/42 MAPK pathway, which is also elicited by BCP crystals in HF.
One of the objectives in this study was to determine the role of the PKC signal transduction pathway in the BCP crystal induction of MMP-1 and MMP-3 in HF. Our studies show that the protein kinase inhibitor, staurosporine, inhibits BCP crystal induction of MMP-1 and MMP-3 in Figs. 1, 2, and 3, only suggesting the involvement of a protein kinase pathway. However, this inhibition is not specific for PKC, because staurosporine is a broad spectrum indolocarbazole that not only inhibits the calcium-dependent PKC but also the cAMP-dependent PKA and cGMP-dependent PKG, as well as phosphorylase kinase, S6 kinase, and src kinase with similar efficiency (35). Additionally, it was noted that the concentration of staurosporine that had a significant inhibition of the MMPs in Figs. 1-3 was 100 nM. This is in agreement with a previous observation that staurosporine does not inhibit BCP crystal-induced collagenase (MMP-1) mRNA accumulation in HF at concentrations that inhibit mitogenesis (25). A similar phenomenon was observed with Gö6976, which inhibited MMP-1 and MMP-3 mRNA and protein expressions at one concentration (25 nM) in Figs. 6 and 7 and PKC activation at a different concentration (2 M) in Figs. 8, 9, and 11. However, the concentration of BCP crystals (50 g/ml) used in these studies is consistent with our previously established optimal range of 50 -100 g/ml in vitro, depending on the cell type, and is consistent with the in vivo concentration in articular joint fluids isolated from osteoarthritic patients, which ranges from 10 to 120 g/ml, depending on the severity of the disease (38). Because PKC has previously been shown to participate in the BCP crystal activation of fibroblasts and chondrocytes (7,25,26), we therefore wanted to determine whether PKC was also involved in the BCP crystal activation of MMP-1 and MMP-3 in HF. We used Bis I, a highly selective PKC inhibitor, which is structurally similar to staurosporine, and found a dose-dependent inhibition of MMP-1 and MMP-3 mRNA and protein expressions in Figs. 4 and 5, respectively, thus proving that the PKC pathway is indeed involved in the BCP crystal activation of these MMPs in HF.
In our previous work, we have shown that BCP crystal stim- ulation of HF results in a rapid transient increase in intracellular calcium levels due to an influx of extracellular calcium from the culture medium into the cells (18). To determine whether BCP crystal activation of PKC in HF also requires an influx of extracellular calcium, we determined PKC activity in a culture medium with and without calcium. Our results show conclusively that, in the absence of any calcium influx from the culture medium into the cells, there is no PKC activity, contrary to the PKC activity in the medium containing calcium as seen in Fig. 8. Further proof of this phenomenon is provided in Fig. 12A in which the chelation of both extracellular calcium with EGTA and intracellular calcium with BAPTA-AM and TMB results in no BCP crystal activation of PKC.
Because there are several PKC subfamilies, each with a number of different isozymes that can be calcium-dependent or calcium-independent (23,24), we then sought to identify the specific PKC isozyme that is activated upon BCP crystal stimulation. To this end, we used Gö6976, a methyl-and cyanoalkyl-substituted nonglycosidic indolocarbazole, which selectively inhibits the calcium-dependent PKC isozymes but does not affect the kinase activity of the isozymes that have no calcium requirement (23,24). Specifically, Gö6976 inhibits the calcium-dependent PKC ␣ and ␤ isozymes (35). In our results in Fig. 9, we have identified PKC␣ as the only isozyme that is activated upon BCP crystal stimulation and inhibited by Gö6976 in HF. Such selective inhibition of an overactivated PKC isozyme may provide a potential target for the design of pharmacological drugs and thereby offer a unique therapeutic approach for the management of crystal-induced diseases such as arthritis.
Another objective of this study was to examine the interrelationship of the PKC pathway with the p44/42 MAPK signal transduction pathway, which has also been shown to be elicited by BCP crystals in HF (15,16). Like the PKC pathway, the p44/42 MAPK pathway is required for BCP crystal-induced mitogenesis (15) and MMP induction (16). Activation of p44/42 MAPK in response to various agonists can occur via mechanisms that may be PKC-dependent (39,40) or PKC-independ-ent (41)(42)(43). Even in the same cell type, p44/42 MAPK activation can be PKC-dependent or -independent, depending upon the stimulus presented and the corresponding cellular response (44). Because both the PKC and p44/42 MAPK pathways are required for maximal induction of mitogenesis and MMP synthesis, it could be reasoned that the crystal-induced activation of p44/42 MAPK is a PKC-dependent event, whereby PKC acts as a direct activator of c-Raf, resulting in the subsequent activation of p44/42 MAPK. Surprisingly, evidence presented here indicates otherwise. Inhibition of BCP crystalactivated PKC with staurosporine did not block the activation of p44/42 (Fig. 10), thereby indicating that the activation of p44/42 MAPK by BCP crystals occurs via a PKC-independent pathway. However, these results cannot rule out the possibility that a PKC isozyme that is not sensitive to staurosporine may be required for the BCP crystal activation of the p44/42 MAPK signal transduction pathway.
BCP crystal activation of HF likely involves an interplay or "cross-talking" among several second messengers and signal transduction pathways. Our present results and previous work (18) indicate that calcium plays an important role in BCP crystal activation of HF. Because PKC does not appear to be required for the BCP crystal activation of p44/42 MAPK, the prospect arises that calcium may be a necessary factor in the activation. Results of our investigation into the role of calcium in BCP crystal activation of p44/42 MAPK argue against this prospect. As seen in Fig. 12B, chelation of extracellular calcium influx with EGTA and intracellular calcium release with BAPTA-AM and TMB, had no effect on BCP crystal activation of p44/42 MAPK (Phospho p44/42), showing that neither external calcium influx nor internal calcium release is required for the activation of this signal transduction pathway by BCP crystals in HF. Similar studies have previously shown p44/42 activation to be independent of PKC, extracellular calcium, and intracellular calcium (43,45). Other studies have also shown p44/42 MAPK activation to be independent of PKC and extracellular calcium but dependent upon intracellular calcium levels (42,46). We have hypothesized that BCP crystal induction of MMP synthesis involves the up-regulation of activating protein-1 (AP-1) DNA binding activity (16,17,25). AP-1, a dimeric transcription factor typically composed of the protein products of c-fos and c-jun, recognizes a consensus DNA binding sequence present in the promoters of various AP-1-responsive genes, including MMP-1 and MMP-3 (47). Indeed, we have previously demonstrated that BCP crystal stimulation of HF results in the up-regulation of both c-fos and c-jun mRNA and in the activation of nuclear AP-1 DNA binding activity (17,18,25). The signal transduction pathways involved in the transcriptional regulation of c-fos itself may have differential requirements for calcium, p44/42 MAPK, and PKC, depending upon the cell type and the stimulus being assessed. Our laboratory has shown that BCP crystal up-regulation of c-fos mRNA expression in HF occurs via a PKC-dependent mechanism, because co-treatment of BCP crystal-stimulated cells with staurosporine greatly attenuated c-fos mRNA expression (25). Our previous work has also demonstrated that removal of calcium from the cell culture medium results in the reduction of BCP crystal-induced c-fos mRNA expression, indicating that an influx of extracellular calcium is required for maximal c-fos induction (18). These results are similar to the work of others showing PKC and extracellular calcium requirements for c-fos induction (48,49).
The up-regulation of c-fos and c-jun mRNA expression induced by BCP crystals is also blocked by PC, a specific inhibitor of BCP crystal-mediated biological effects (17). PC may have an important protective role in preventing calcium phosphate precipitation in cells or cellular compartments maintaining high concentrations of calcium and phosphate. We have demonstrated that PC interferes with many biological effects of calcium-containing crystals. Crystal-induced MMP synthesis and mitogenesis (17) and p44/42 MAPK activation (15) in HF are specifically inhibited by PC, although it has no effect on similar processes induced by growth factors or serum. Additionally, PC prevents BCP crystal deposition and disease progression in murine progressive ankylosis, an animal model of BCP crystal deposition disease (50), and blocks calcium-containing crystal formation in matrix vesicles and intact cartilage in an in vitro model of chondrocalcinosis (51).
In conclusion, we have demonstrated that BCP crystal stimulation of MMP-1 and MMP-3 mRNA and protein expressions in HF is through a calcium-dependent PKC signal transduction pathway. We have also provided evidence that BCP crystal treatment of HF induces the calcium-dependent PKC␣ isozyme. Finally, we have shown that BCP crystal-induced activation of p44/42 MAPK is independent of PKC, because the PKC inhibitors, staurosporine, Bis I, and Gö6976, have no effects on BCP crystal activation of p44/42 MAPK. The converse is also shown that the p44/42 MAPK inhibitors, PD098058 and U0126, have no effects on BCP crystal activation of PKC. The p44/42 MAPK is a family of serine/threonine kinases known to be important intermediary factors in converting extracellular signals into intracellular responses (52,53). In their phosphorylated and activated forms, they migrate from the cytoplasm to the nucleus and transmit extracellular stimuli by phosphorylating several transcription factors (54). We have recently demonstrated that the induction of human MMP-1 expression by BCP crystals in canine fibroblast-like synoviocytes, in part, follows the Ras/MAPK/c-fos/AP-1/MMP1 signaling pathway (32) and that BCP crystals activate c-fos expression through a Ras/ERK-dependent signaling mechanism. 2 These facts and our present observations, therefore, lead us to the proposed model shown in Fig. 13 and to the hypothesis that the PKC and p44/42 MAPK signal transduction pathways, activated by BCP crystals in HF, initially function independently, ultimately leading to an increase in mitogenesis and MMP synthesis, and may converge downstream of PKC and p44/42 MAPK to mediate BCP crystal-induced cellular responses.