Molecular Mechanism of the Induction of ...

JBC Papers in Press. Published on February 8, 2002 as Manuscript M200278200

Molecular Mechanism of the Induction of Metalloproteinases 1 and 3 in Human Fibroblasts by Basic Calcium Phosphate Crystals* ROLE OF CALCIUM-DEPENDENT PROTEIN KINASE C- α

Paul M. Reuben§, Michele A. Brogley¶, Yubo Sun§ and Herman S. Cheung§‡** From the § Department of Medicine, University of Miami School of Medicine, Miami, Florida 33101, the ¶ Department of Human Genetics, University of Michigan Medical School, Ann Arbor,

Veterans Administration Medical Center, Miami, Florida 33125 and the Department of Biomedical Engineering, University of Miami, Coral Gables, Florida 33146.

Corresponding Author: Herman S. Cheung, Ph. D Tel: (305) 324-4455 x3646 Fax: (305) 324-3365 E-mail: [email protected]

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Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Michigan, 48109-0618., the ‡ Research Service & Geriatric Research, Education and Clinical Center,

Running title: BCP crystal induction of matrix metalloproteinases


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SUMMARY - 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 crystalelicited signal transduction pathways have not been completely studied. Since 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

(RT/PCR), 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-α 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.

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expressions in human fibroblasts. Using reverse transcription/polymerase chain reaction

INTRODUCTION

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 (PDGF), epidermal growth

and chondrocyte mitogenesis in vitro (3), stimulate the production of prostaglandin (PGE2) via the phospholipase A2/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 (ERK 1 and ERK 2) 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 crystal-induced upregulation of MMP-1 and MMP-3 mRNA and protein expressions (16). Moreover,

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factor (EGF) and serum. It has been demonstrated that BCP crystals stimulate fibroblast, synoviocyte

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 crystal-induced 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 minutes after stimulation, due to

crystal induction 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 3’,5’-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 (PIP2) and production of the intracellular messengers, inositol triphosphate (IP3) 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) while diacyglycerol 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 the alpha (α), beta I (βI), beta

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crystal dissolution (18). Removal of calcium from the cell culture medium attenuates the BCP

II (βII) and gamma (γ) and their activities are calcium and phospholipid dependent. The novel isozymes comprise the delta (δ), epsilon (ε), eta (η) and theta (θ) whose activities are calciumindependent but phospholipid-dependent. The atypical isozymes are made up of zeta (ζ), iota (ι) and mu (µ) 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

activity associated with the membrane fraction is seen following BCP crystal stimulation of chondrocytes (26). Downregulation of PKC activity by chronic treatment with the phorbol ester, 12o-tetradecanoyl-phorbol 13-acetate (TPA), an analog of DAG, blocks crystal-induced c-fos and c-myc expressions and mitogenesis in Balb/c 3T3 cells (7) while 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 crystal-induced 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. Since 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.

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PKC inhibitors, Staurosporine and Bisindolylmaleimide I (25). Furthermore, an increase in PKC

EXPERIMENTAL PROCEDURES

Materials – Dulbecco’s modified Eagle’s medium (DMEM), Hank’s balanced salt solution (HBSS), Phosphate buffered saline (PBS), Fetal bovine serum (FBS), penincillin, streptomycin, fungizone, ThermoScript RT-PCR System, PCR primers, Trizol Reagent were obtained from Life Technologies, Inc., Gaithersburg, MD. MMP-1 specific probe (a 2.02 Kb HindIII/Sma I

plasmid) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) specific probe (a mouse 0.8 Kb HindIII insert from the pBS-GAPDH plasmid) were obtained from American Type Culture Collection, Rockville, MD. positive

control

proteins

was

Concentrated medium containing MMP-1 and MMP-3 from

Chemicon

International

Inc.,

Temecula,

CA.

Bisindolylmaleimide, [8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy benzoate,HCl] (TMB-8), Staurosporine,

1,2-bis(o-amino-phenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl

ester (BAPTA/AM), PD98059, U0126, Gö6976, monoclonal MMP-1 antibody, monoclonal MMP-3 antibody, monoclonal PKC antibody were from Calbiochem, La Jolla, CA. Monoclonal Phospho P44/42 MAPK antibody, polyclonal P44/42 MAPK antibody were from New England Biolabs, Inc., Beverly, MA. Polyclonal antibodies against the PKC α, βI, βII, and γ isozymes were from Panvera, Madison, WI. Anti-mouse IgG HRP conjugate was from Promega, Madison, WI. Ethylenediamine-N,N,N’,N’-tetraacetic acid (EDTA), [ethyleneglycol-bis(ßaminoethyl)-N,N,N’,N’-tetraacetic acid] (EGTA), 3,3’-Diaminobenzidine (DAB), leupeptin

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insert from the pCllase 1 clone), MMP-3 specific probe (a 1.7 Kb EcoR1 insert from the pTR1

and aprotinin were from Sigma, St. Louis, MO. 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 hours (MMPs and PKC) or for 48 hours

free DMEM was added to the cells. For the inhibition experiments, the cells were pretreated with the appropriate concentrations of the inhibitors for 30 minutes 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 carbonate-substituted hydroxyapatite mixed with octacalcium phosphate as indicated by Fourier transform infrared spectroscopy.The crystals were crushed and sieved to yield 10-20 µm aggregates, which were sterilized and rendered pyrogen-free by heating at 200°C for at least 90 minutes. PC was prepared as previously described (29). RT and PCR -

Total RNA was isolated using the Trizol Reagent according to the

manufacturer’s instructions. Then 1µg of each sample was reverse transcribed at 50°C for 60 minutes, followed by enzyme inactivation at 85°C for 5 minutes using the ThermoScript RTPCR System. The resulting cDNA samples were amplified by the PCR method. PCR primers for MMP-1 were:

sense, 5’-GATCATCGGGACAACTCTCCT-3’, corresponding to positions

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(P44/42 MAPK). Then this medium was removed, the cells were washed with PBS and serum-

567-587 and antisense, 5’-TCCGGGTAGAAGGGATTTGTG-3’, 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, 5’GAAAGTCTGGGAAGAGGTGACTCCAC-3’, corresponding to positions 414-440 and antisense, 5’-CAGTGTTGGCTGAGTGAAAGAGACCC-3’, corresponding to positions 671697 of the nucleotide and amino acid sequence for human MMP-3 and giving a PCR product of 284 bp (31). As an internal control, a 353-bp of the constitutively expressed housekeeping gene,

reaction. All primers were synthesized by Life Technologies, Inc. (Gaithersburg, MD). Amplifications were carried out for 30 cycles by denaturing at 95°C for 30 seconds, annealing at 55°C for 30 seconds and extending at 72°C for 45 seconds, with the final extension at 72°C for 10 minutes. The PCR products were analyzed by electrophoresis on 2% agarose gel containing ethidium bromide. Northern Blotting - Total RNA samples ( 10µg each) were denatured and electrophoresed through a 1.2% agarose gel containing 2.2M formaldehyde followed by transfer and crosslinking 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 hours and then hybridized at 42°C overnight to MMP-1 and MMP-3 specific cDNA probes that were radiolabeled with α-32P dATP (6000 Ci/mmol, Amersham Pharmacia Biotech, 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 –80°C.

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ß-actin, was also synthesized and used to normalize the amount of mRNA in each RT/PCR

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) SDS-polyacrylamide gel and then transferred onto

Immobilon-P PVDF

membranes (Millipore, Bedford, MA). After transfer, the membranes were incubated for 4 hours at room temperature in the blocking buffer, TBST (20mM Tris, 136mM NaCl, 0.1% Tween-20) containing 5% non-fat dry milk to eliminate non-specific binding. The membranes were washed several times and then incubated in TBST containing 5% BSA at 4°C overnight with the

antibody against PKC , a phospho-specific monoclonal MAPK antibody recognizing P44/42 MAPK phosphorylated at Tyr204 and Thr202 or a polyclonal P44/42 MAPK antibody or a polyclonal antibody against each of the PKC isozymes, α, βI, βII and γ. The membranes were again washed several times with TBST and incubated with the appropriate anti-mouse or antirabbit HRP-conjugated secondary antibody in TBST with 5% BSA for 1 hour 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 (25mg/100ml) and hydrogen peroxide in 0.05M 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 translocation buffer (20mM Tris-HCl, pH 7.5, 2mM EDTA, 0.5mM EGTA, 0.2 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 0.33M sucrose). The cells were sonicated on ice for 15 seconds and then centrifuged at 100,000 x g for 45 minutes. 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

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following antibodies: a monoclonal antibody against MMP-1 or MMP-3 , a monoclonal

centrifuged again at 100,000 x g for 45 minutes. The supernatant was used as the membrane fraction. Samples (25µl 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 (62.5mM Tris-HCl, pH6.8, 2% w/v SDS, 10% glycerol, 50mM DTT and 0.1% bromophenol blue). The cell lysates were scraped into microfuge tubes, boiled for 5 minutes and aliquots (25µl) were

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 < 0.05 was considered significant. Data were expressed as the means ± SE.

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subjected to 12% SDS-polyacrylamide gel electrophoresis and western blotting with a Phospho

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 (17,32). 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/polymerase chain reaction (RT/PCR), northern and western blottings. The RT/PCR with MMP-1 and MMP-3 specific primers show that after 24 hours of stimulation of HF with BCP crystals, levels of MMP-1 and MMP-3 mRNA increased approximately four-fold over the control levels as shown in Fig.1, panels A and B respectively. The inhibition of the MMP-1 and MMP-3 mRNA by staurosporine is concentration- dependent, with the greatest inhibition at 100nM , which is similar to the inhibition by 1mM 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 ß-actin 12


RESULTS

primers, did not show any change in panel C. The densitometric scan of the relative intensities (mean ± S.E.) of three such independent experiments showed a significant inhibition of the BCP crystal-induced MMP-1 and MMP-3 by staurosporine at 100nM (p < 0.05) (figure not shown). Northern blotting of the RNA samples in Fig. 2 shows no degradation of the RNA at all concentrations of staurosporine in panel A. Panels B and C show that, at 100nM of staurosporine, the complete inhibition of MMP-1 and MMP-3 mRNA respectively is similar to the inhibition by 1mM of PC while panel D shows no change in the housekeeping gene,

These results were confirmed with western blotting of the culture medium in Fig. 3. Here, there is also a concentration-dependent inhibition of the BCP crystal-induced MMP-1 and MMP-3 secreted proteins in panels A and B respectively, with the greatest inhibition again at 100nM of staurosporine and with the molecular weight of the proteins corresponding to MMP-1 control standard at 53-55KDa and MMP-3 control standard at 57-59KDa. 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, panels A and B respectively and that, at 10µM, the inhibition is similar to the inhibition by 1mM of 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

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GAPDH.

which shows no inhibition even at the same 10µM 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 in panel C. These results were confirmed by western blotting for the MMP-1 and MMP-3 protein expressions in Fig. 5, panels 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

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, panels A and B respectively and with the maximum inhibition at 25nM of Gö6976 similar to the inhibition by PC at 1mM. PMA was used as a positive control for the PKC activity and the samples were again normalized with the housekeeping gene, GAPDH, in panel C. These results were also confirmed by western blotting for the MMP-1 and MMP-3 protein expressions in Fig. 7, panels 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. Since 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

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PKC that participates in the signaling pathway upon the BCP crystal induction of MMP-1 and

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 specific 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. 8, panel A. 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, and is totally inhibited by 2µM of

25nM that completely inhibited the MMPs in Figures 6 and 7. Our dose-dependent study (data not shown) found 2µM to be the concentration of Gö6976 that would inhibit PKC in HF. This is in agreement with a previous study which found that 2µM of 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 calcium-dependent subfamily of PKC is the conventional subfamily which is a pool of isozymes consisting of alpha (α), beta I (βI), beta II (βII) and gamma (γ) (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-α isozyme in panel B whereas there was no induction at all of the βI, βII and γ isozymes in panels C, D and E respectively. The specificity of the PKC-α isozyme was confirmed with the use of the Gö6976

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Gö6976 as seen in panel B. However, this concentration is different from the concentration of

which is a specific inhibitor of the calcium-dependent PKC α and βI isozymes (35). Complete inhibition was seen in the PKC in the total pool as well as in the PKC-α, thus unequivocally identifying the α isozyme of the calcium-dependent PKC as being activated by BCP crystals. Cooperativity of PKC with PKC-independent mitogen activated protein kinase—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

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 inhibited BCP crystal induced MMP-1 and MMP-3 mRNA and proteins shown in Figures 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 panel A. To show that BCP crystal induced Phospho P44/42 could be inhibited, 1mM of 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 panel B. 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

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(16). Here, we were interested in determining whether P44/42 MARK induction by BCP crystals

whereas treatment with the Phospho P44/42 inhibitors, PD98059 and U0126, did not inhibit PKC at all as shown in panel A. Conversely, the PKC inhibitors did not inhibit Phospho P44/42 which was only inhibited by its own inhibitors, PD98059 and U0126 as shown in panel B, 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 panel C. BCP crystal activated P44/42 MAPK pathway is calcium-independent—We have shown in Fig.

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 (IP3) and diacylglycerol (DAG) (20) which are involved in intracellular calcium mobilization (21) and PKC activation (22) respectively. While extracellular calcium chelation by EGTA and intracellular calcium chelation by BAPTA-AM and TMB blocked the BCP crystal induced PKC in Fig.12, panel A, they had no effect on Phospho P44/42 in panel B. 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.

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8 that BCP crystal induction of MMP-1 and MMP-3 requires the calcium-dependent PKC

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, while mitogenesis leads to an increase in synoviocytes that generate more MMPs (37). Of interest are the signal transduction mechanisms by which crystal-induced upregulation of MMP synthesis and secretion and increased mitogenesis are mediated. Here, we demonstrate for the first time that the calciumdependent PKC signal transduction pathway is required for maximal BCP crystal induction of

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DISCUSSION

MMP-1 and MMP-3 mRNA and protein expressions in HF and also identify PKC-α as the specific isozyme that is activated upon BCP crystal stimulation. We also show that the calciumdependent PKC signal transduction pathway works in cooperation with the distinct calciumindependent 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

However, this inhibition is not specific for PKC since staurosporine is a broad spectrum indolocarbazole which not only inhibits the calcium-dependent PKC but also the cAMPdependent 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 Figures 1-3 was 100nM. 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 which inhibit mitogenesis (25). A similar phenomenon was observed with Gö6976 which inhibited MMP-1 and MMP-3 mRNA and protein expressions at one concentration (25nM) in Figures 6 and 7 and PKC activation at a different concentration (2µM) in Figures 8, 9 and 11. However, the concentration of BCP crystals (50µg/ml) used in these studies is consistent with our previously established optimal range of 50-100µg/ml in vitro, depending on the cell type and is also consistent with the in vivo concentration in articular joint fluids isolated from osteoarthritic patients which ranges from 10-120µg/ml depending on the severity of the disease (38). Since PKC has previously been

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and MMP-3 in Figures 1, 2 and 3, only suggesting the involvement of a protein kinase pathway.

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 Figures 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 stimulation of HF results in a

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. Since there are several PKC subfamilies, each with a number of different isozymes which 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-

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rapid transient increase in intracellular calcium levels due to an influx of extracellular calcium

dependent PKC α and β isozymes (35). In our results in Fig. 9, we have identified PKC-α 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 to 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 crystal induced mitogenesis (15) and MMP induction (16). Activation of P44/42 MAPK in response to various agonists can occur via mechanisms which may be PKC-dependent (39,40) or PKC-independent (41-43). Even in the same cell type, P44/42 MAPK activation can be PKCdependent or –independent, depending upon the stimulus presented and the corresponding cellular response (44). Since 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 crystal-activated PKC with staurosporine did not block the activation of P44/42 (Fig.12), 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.

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BCP crystals in HF (15,16). Like the PKC pathway, the P44/42 MAPK pathway is required for

BCP crystal activation of HF likely involves an interplay or “crosstalking” 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. Since 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

(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 upregulation 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 upregulation 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

22


with BAPTA-AM and TMB, had no effect on BCP crystal activation of P44/42 MAPK

calcium, P44/42 MAPK and PKC, depending upon the cell type and the stimulus being assessed. Our laboratory has shown that BCP crystal upregulation of c-fos mRNA expression in HF occurs via a PKC-dependent mechanism as 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

The upregulation 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 MMP3 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

23


showing PKC and extracellular calcium requirements for c-fos induction (48,49).

calcium-dependent

PKC-α isozyme. Finally, we have shown that BCP crystal induced

activation of P44/42 MAPK is independent of PKC, as 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

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/AP1/MMP1 signaling pathway (55) and that BCP crystals activate c-fos expression through a Ras/ERK dependent signaling mechanism (56). These facts and our present observations, therefore, lead us to the proposed model 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.

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Rheumatism

FOOTNOTES

* This work was supported by a USPHS grant AR-38421-13 and a Veteran Administration Merit Review grant (to H.S.C.).

Medical Center, 1201 NW 16th Street, Miami, Florida 33125. Tel.: (305) 324-4455 x 3646; Fax: (305) 324-3365;

E-mail: [email protected].

1 The abbreviations used are: AP-1, activating protein-1; BCP, basic calcium phosphate; BSA, bovine serum albumin; BAPTA-AM, 1,2-bis(o-amino-phenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester; Bis, bisindolylmaleimide; CPPD, calcium pyrophosphate dihydrate; DAB, 3,3’-diaminobenzidine, DAG, diacylglycerol; DTT, dithiotreitol;; DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediamine-N,N,N’,N’-tetraacetic acid; EGTA, [ethyleneglycol-bis(ß-aminoethyl)-N,N,N’,N’-tetraacetic acid]; ERK, extra cellular signalrelated protein kinase, FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; HF, human fibroblasts; HRP, horse radish peroxidase; HBSS, Hank’s balanced salt solution; MAPK, mitogen activated protein kinase, MEK, MAPK-ERK kinase; MMP, matrix metalloproteinase; PBS, phosphate buffered saline; PC, phosphocitrate; PDGF, platelet-

29


** To whom correspondence should be addressed: Research Service, Veterans Administration

derived growth factor; PKC, protein kinase C; PMA, phorbol,12-myristate,13-acetate; PMSF, phenylmethylsulfonyl

fluoride;

PVDF,

polyvinylidene

flouride;

RT/PCR,

reverse

transcription/polymerase chain reaction; SDS, sodium dodecl sulfate; SE, standard error; TBS, tris-buffered saline; TMB-8, [8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy benzoate,HCl]; TPA,12-o-tetradecanoly-phorbol 13-acetate . FIGURE LOCATIONS

Put Figures 4 and 5 after the “Identification of Protein Kinase C as the signaling pathway” part in the Results Section.

Put Figures 6, 7 and 8 after the “Requirement for calcium in the PKC signaling pathway” part in the Results section.

Put Figure 9 after the “Identification of the specific PKC isozyme” part in the Results Section.

Put Figures 10 and 11 after the “Cooperativity of PKC with PKC-independent mitogen activated protein kinase” part in the Results Section Put Figures 12 and 13 after the “BCP crystal activated P44/42 pathway is calcium independent” part in the Results Section.

30


Put Figure 1 after the first paragraph and Figures 2 and 3 after the second paragraph in the Results section.

FIGURE LEGENDS

the protein kinase inhibitor, staurosporine. Confluent HF cells were starved with 0.5% heat inactivated FBS for 24 hours, followed by pre-treatment of the cells with or without different concentrations of the protein kinase inhibitor, staurosporine (ST) and 1mM of PC as the control inhibitor for 30 minutes before being stimulated with BCP crystals (50µg/ml) for 24 hours. Total RNA was isolated and RT/PCR was performed on 1µg of each RNA sample using primers for MMP-1 (A), MMP-3 (B) and ß-actin (C) as described under “Experimental Procedures”. Bands shown are representatives of three independent experiments.

FIG. 2. Northern blotting analysis of BCP crystal induced MMP-1 and MMP-3 mRNA

31


FIG. 1. RT/PCR analysis of BCP crystal induced MMP-1 and MMP-3 mRNA inhibition by

expression by the protein kinase inhibitor, staurosporine. Total RNA (10µg), isolated in Fig.1, was electrophoresed to determine the intactness of the mRNA (A). These were then blotted and probed with α-32PdATP-labeled cDNA probes specific for MMP-1 (B) and MMP-3 (C) as described under “Experimental Procedures”. The blots were subsequently stripped and reprobed with GAPDH cDNA as a control. Blots shown are representatives of three separate experiments.

expressions by the protein kinase inhibitor, staurosporine. The culture medium of the experiment in Fig. 1 was concentrated ten-fold and electrophoresed on a 10% SDS-polyacrylamide gel. The protein bands were transferred to PVDF membranes and subsequently blotted with monoclonal antibodies against MMP-1 (A) and MMP-3 (B). Concentrated medium containing MMP-1 and MMP-3 was used as positive control MMP standards. Molecular weight markers in KDa are indicated on the right. Blots shown are representatives of three separate experiments.

FIG. 4.

Northern blotting analysis of the inhibition of BCP crystal induced MMP-1 and

MMP-3 mRNA expression by the PKC inhibitors, Bis I and Bis V.

32


FIG. 3. Western blotting analysis of BCP crystal induced MMP-1 and MMP-3 protein

HF cells were starved in DMEM with 0.5% heat inactivated FBS for 24 hours and then pretreated with or without the indicated concentrations of Bis I, Bis V and PC for 30 minutes before being stimulated for 24 hours with either BCP (50µg/ml) or PMA (200nM) as a positive control. Total RNA was isolated and mRNA levels for MMP-1 (A), MMP-3 (B) and GAPDH (C) were determined as described under “Experimental Procedures”. Blots shown are representatives of three independent experiments.

Western blotting analysis of the inhibition of BCP crystal induced MMP-1 and

MMP-3 protein expression by the PKC inhibitors, Bis I and Bis V. The culture medium of the experiment in Fig. 4 was concentrated ten-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.

FIG. 6.

Northern blotting analysis of the inhibition of BCP crystal induced MMP-1 and

MMP-3 mRNA expression by the Ca2+-dependent PKC inhibitor, G‘6976. HF cells were starved in DMEM with 0.5% heat inactivated FBS for 24 hours and then pretreated with or without the indicated concentrations of G‘6976 and PC for 30 minutes before

33


FIG. 5.

being stimulated for 24 hours with either BCP (50µg/ml) or PMA (200nM) as a positive control. Total RNA was isolated and mRNA levels for MMP-1 (A), MMP-3 (B) and GAPDH (C) were determined as described under “Experimental Procedures”. Blots shown are representatives of three independent experiments.

Western blotting analysis of the inhibition of BCP crystal induced MMP-1 and MMP-

3 protein expression by the Ca2+-dependent PKC- inhibitor, G‘6976. The culture medium of the experiment in Fig. 6 was concentrated ten-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.

FIG. 8.

Calcium requirement for BCP crystal induced PKC translocation. Two sets of HF cells (4 plates/set) were starved in DMEM with 0.5% heat inactivated

FBS for 24 hours. One of the sets was washed with BHSS containing calcium and magnesium and the other with HBSS without calcium and magnesium and then equilibrated in the respective

34


FIG. 7.

medium for 1 hour. Then the cells in each set were pretreated with or without G‘6976 (2µM) for 30 minutes before being stimulated for 15 minutes with either BCP crystals (50µg/ml) or PMA (200nM) as a positive control. The cytosolic and membrane fractions of the cells were isolated as described in “Experimental Procedures”. Aliquots of the membrane fractions were electrophoresed on a 7.5% SDS-polyacrylamide gel and subjected to western blotting to determine the translocation of PKC to the membrane as an indication of the PKC activity. PKC translocation is shown in the absence (A) or presence (B) of calcium and magnesium. Blots

FIG. 9.

Identification of the specific PKC isozyme. HF cells were starved in DMEM with 0.5% heat inactivated FBS for 24 hours and then

pretreated with or without G‘6976 (2µM) for 30 minutes before being stimulated with or without BCP crystals (50µg/ml) for 15 minutes. The membrane fractions were isolated as described in “Experimental Procedures”. Aliquots of the membrane fractions were electrophoresed on a 7.5% SDS-polycrylamide gel and subjected to western blotting with a monoclonal antibody against the total pool of the isozymes (A), and polyclonal antibodies against PKC-α (B), PKC-βI (C), PKC-βII (D) and PKC-γ (E). Blots shown are representatives of three independent experiments.

FIG. 10.

The PKC inhibitor, staurosporine, has no effect on BCP crystal induced P44/42

MAPK activation.

35


shown are representatives of three independent experiments.

HF cells were starved in DMEM with 0.5% heat inactivated FBS for 48 hours followed by pretreatment with or without the indicated concentrations of staurosporine (ST) and PC for 30 minutes before being stimulated for 15 minutes with or without BCP crystals (50µg/ml). Cell lysates were harvested as described in “Experimental Procedures”. Levels of Phospho P44/42 (A) and P44/42 (B) were determined by western blotting analysis. Molecular weight markers in KDa are indicated on the left. Blots shown are representatives of three independent experiments.

Independent Pathways for PKC and Phospho P44/42 MAPK.

HF cells were starved for 24 hours (PKC) or 48 hours (Phospho P44/42) in 0.5% heat inactivated FBS and then pretreated for 30 minutes with the PKC inhibitors, Bis I (10µM), G‘6976 (2µM) and the P44/42 MAPK inhibitors, PD98059 (100µM) and UO126 (10µM), before being stimulated for 15minutes with BCP crystals (50µg/ml) or PMA (200nM) as a positive control. Membrane fractions and cell lysates were obtained as described in “Experimental Procedures”. Aliquots of the membrane fractions were used to determine the levels of PKC translocation to the membrane (A) while the cell lysates were used to determine the levels of activated Phospho P44/42 (B) and the constitutively expressed P44/42 (C) by western blotting. Blots shown are representatives of three independent experiments.

36


FIG. 11.

FIG. 12.

BCP crystal activated P44/42 MAPK pathway is calcium independent.

HF cells were pretreated with the extracellular chelator, EGTA (5mM) for 5minutes, and the intracellular chelators, BAPTA-AM (50µM) and TMB-8 (100µM) for 30 minutes before being stimulated for 15 minutes with or without BCP crystals (50µg/ml). Membrane fractions and cell lysates were obtained and subjected to western blotting to determine the levels of BCP crystal induced PKC translocation to the membrane (A) and activated Phospho P44/42 (B). Results shown are representatives of three independent experiments.

Proposed model of BCP crystal induced signal transduction in human fibroblasts.

The P44/42 MAPK and PKC signal transduction pathways activated upon BCP crystal stimulation initially function independently, ultimately leading to an increase in mitogenesis and MMP synthesis. The pathways may converge downstream of P44/42 MAPK and PKC to mediate BCP crystal induced cellular responses. A question mark (?) indicates that this component of the signal transduction pathway is currently unknown.

37


FIG. 13.

Fig. 1

37

Fig.2

38

Fig. 3

39

Fig. 4

40

Fig. 5

41

Fig. 6

42

Fig. 7

43

Fig. 8

44

Fig. 9

45

Fig. 10

46

Fig. 11

47

Fig. 12

48

Fig. 13

49

Molecular mechanism of the induction of metalloproteinases 1 and 3 in human fibroblasts by basic calcium phosphate crystals role of calcium-dependent protein kinase C-a Paul M. Reuben, Michele A. Brogley, Yubo Sun and Herman S. Cheung J. Biol. Chem. published online February 8, 2002

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