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• W2053646056 abstract "Although basic calcium phosphate (BCP) crystals are common in osteoarthritis, the crystal-induced signal transduction pathways in human fibroblasts have not been fully comprehended. We have previously demonstrated that the induction of matrix metalloproteinases (MMP) 1 and 3 by BCP crystals follows both the calcium-dependent protein kinase C (PKC) pathway and the calcium-independent p44/42 mitogen-activated protein kinase (p44/42 MAPK) pathway. Although we showed that the calcium-dependent PKC pathway was characterized by calcium-dependent PKCα, here we show that the calcium-independent p44/42 MAPK pathway is mediated by calcium-independent PKCμ. Inhibition of PKCμ synthesis and activity by antisense oligodeoxynucleotides and H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide), respectively, results in the inhibition of p44/42 MAPK activation, thus demonstrating that p44/42 MAPK activity is dependent upon PKCμ. Reverse transcription-polymerase chain reaction and Western blotting also show that inhibition of PKCμ results in the inhibition of MMP-1 and MMP-3 mRNA and protein expression as a result of p44/42 MAPK inhibition. These results now lead us to the conclusion that BCP crystal activation of human fibroblasts follows two pathways: 1) the calcium-dependent PKC pathway characterized by PKCα and 2) the calcium-independent p44/42 MAPK pathway mediated by PKCμ, which operate independently leading to an increase in mitogenesis and MMP synthesis and ultimately complementing each other for the efficient regulation of cellular responses to BCP crystal stimulation of human fibroblasts. Although basic calcium phosphate (BCP) crystals are common in osteoarthritis, the crystal-induced signal transduction pathways in human fibroblasts have not been fully comprehended. We have previously demonstrated that the induction of matrix metalloproteinases (MMP) 1 and 3 by BCP crystals follows both the calcium-dependent protein kinase C (PKC) pathway and the calcium-independent p44/42 mitogen-activated protein kinase (p44/42 MAPK) pathway. Although we showed that the calcium-dependent PKC pathway was characterized by calcium-dependent PKCα, here we show that the calcium-independent p44/42 MAPK pathway is mediated by calcium-independent PKCμ. Inhibition of PKCμ synthesis and activity by antisense oligodeoxynucleotides and H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide), respectively, results in the inhibition of p44/42 MAPK activation, thus demonstrating that p44/42 MAPK activity is dependent upon PKCμ. Reverse transcription-polymerase chain reaction and Western blotting also show that inhibition of PKCμ results in the inhibition of MMP-1 and MMP-3 mRNA and protein expression as a result of p44/42 MAPK inhibition. These results now lead us to the conclusion that BCP crystal activation of human fibroblasts follows two pathways: 1) the calcium-dependent PKC pathway characterized by PKCα and 2) the calcium-independent p44/42 MAPK pathway mediated by PKCμ, which operate independently leading to an increase in mitogenesis and MMP synthesis and ultimately complementing each other for the efficient regulation of cellular responses to BCP crystal stimulation of human fibroblasts. Basic calcium phosphate (BCP) 1The abbreviations used are: BCP, basic calcium phosphate; AP-1, activating protein-1; DAG, 1,2-dioctanoyl-sn-glycerol; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-related protein kinase; FBS, fetal bovine serum; H-89, N-(2-[p-bromocinnamylamino]ethyl)-isoquinlinessulfonamide; HF, human fibroblasts; MAPK; mitogen-activated protein kinase; MEK, MAPK-ERK kinase; MMP, matrix metalloproteinase; PC, phosphocitrate; PDBu, phorbol 12,13-dibutyrate; PKC, protein kinase C; RT, reverse transcription; ODN, oligodeoxynucleotide; TPA, 12-O-tetradecanoylphorbol-13-acetate; PH, pleckstrin homology. crystals are among the most common forms of pathologic articular minerals. They frequently occur in the joints of osteoarthritis patients and can be phlogistic (1Ryan L.M. Cheung H.S. Rheum. Dis. Clin. N. Am. 1999; 25: 257-267Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 2Halverson P.B. Koopman W.J. Arthritis and Allied Conditions. Lippincott Williams & Wilkins, 2001: 2371Google Scholar). There is compelling evidence that these crystals engender multiple biological effects that promote joint degeneration. The presence of BCP crystals correlates strongly with radiographic evidence of cartilaginous degeneration and synovial thickening and is associated with larger joint effusions when compared with joint fluid from osteoarthritis knees where BCP crystals are absent (3Halverson P.B. McCarty D.J. Ann. Rheum. Dis. 1986; 45: 603-605Crossref PubMed Scopus (96) Google Scholar, 4Schumacher H.R. Arthritis Rheum. 1968; 11: 426-435Crossref PubMed Scopus (29) Google Scholar). Conversely, osteoarthritis is both more common and more severe in patients with calcium-containing crystals. We have demonstrated that BCP crystals stimulate the proliferation and synthesis of matrix metalloproteinases (MMPs) in cultured human foreskin and synovial fibroblasts (5McCarty G.M. Mitchell P.G. Cheung H.S. Arthritis Rheum. 1991; 34: 1021-1030Crossref PubMed Scopus (43) Google Scholar, 6McCarty G.M. Mitchell P.G. Struve J.A. Cheung H.S. J. Cell. Physiol. 1992; 153: 140-146Crossref PubMed Scopus (47) Google Scholar, 7Bai G. Howell D.S. Roos B.A. Howard G.A. Cheung H.S. Osteoarthritis Cartilage. 2001; 9: 416-422Abstract Full Text PDF PubMed Scopus (33) Google Scholar, 8McCarty G.M. Westfall P.R. Masuda I. Christopherson P.A. Cheung H.S. Mitchell P.G. Ann. Rheum. Dis. 2001; 60: 399-406Crossref PubMed Scopus (61) Google Scholar, 9Reuben P.M. Wenger L. Cruz M. Cheung H.S. Connect. Tissue Res. 2001; 42: 1-12Crossref PubMed Scopus (42) Google Scholar). The addition of BCP crystals to the growth medium yielded an immediate 10-fold rise in the intracellular calcium level and a second rise starting at 60 min and lasting for 3 h. This second rise in the intracellular calcium level is probably because of the intracellular dissolution of phagocytosed crystals, which may activate a variety of calcium-dependent signals and induce sustained cell proliferation and MMP synthesis (10Halverson P.B. Greene A. Cheung H.S. Osteoarthritis Cartilage. 1998; 6: 324-329Abstract Full Text PDF PubMed Scopus (40) Google Scholar). To date, BCP crystal-induced signal transduction has not been fully comprehended. We have previously demonstrated that the induction of human MMP-I and MMP-3 expression by BCP crystals, in part, follows the p44/42 mitogen-activated protein kinase (p44/42 MAPK) signal transduction pathway (11Sun Y. Wenger L. Brinkerhoff C.E. Misra R.R. Herman H.S. J. Biol. Chem. 2002; 277: 1544-1552Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 12Brogley M.A. Cruz M. Cheung H.S. J. Cell. Physiol. 1999; 180: 215-224Crossref PubMed Scopus (48) Google Scholar). We have also shown that BCP crystal stimulation of MMP-1 and MMP-3 mRNA and protein expression in human fibroblasts are mediated, in part, through the calcium-dependent protein kinase C (PKC) signal transduction pathway and that the PKCα isozyme is specifically involved in the pathway (13Reuben P.M. Brogley M.A. Sun Y. Cheung H.S. J. Biol. Chem. 2001; 277: 15190-15198Abstract Full Text Full Text PDF Scopus (58) Google Scholar). We found that these two pathways operate independently since the p44/42 MAPK inhibitors, PD098058 and U0126, had no effect on the BCP crystal stimulation of PKC and the PKC inhibitors, staurosporine, bisindolylmaleimide I, and Gö6979, had no effect on the BCP crystal activation of p44/42 MAPK (13Reuben P.M. Brogley M.A. Sun Y. Cheung H.S. J. Biol. Chem. 2001; 277: 15190-15198Abstract Full Text Full Text PDF Scopus (58) Google Scholar). However, the possibility that a PKC isozyme that was not sensitive to the PKC inhibitors might be required for the BCP crystal activation of the p44/42 MAPK signal transduction could not be ruled out. Therefore, we undertook the current study to identify all of the PKC isozymes involved in the BCP crystal activation of human fibroblasts and to further examine the interrelationship between the PKC and the p44/42 MAPK signal transduction pathways involved in the process. Activation of p44/42 MAPK in response to various agonists can occur via mechanisms that may be PKC-dependent (14Todico A. Takeuchi Y. Urumov A. Yamado J. Stepan V.M. Yamada T. Am. J. Physiol. (Lond.). 1997; 273: G891-G898PubMed Google Scholar, 15Kometiani P. Li J. Gnudi L. Kahn B.B. Askari A. Xie Z. J. Biol. Chem. 1998; 273: 15249-15256Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) or PKC-independent (16Crespo P. Xu N. Daniotti J.L. Troppmair J. Rapp U.R. Gutkind J.S. J. Biol. Chem. 1994; 269: 21103-21109Abstract Full Text PDF PubMed Google Scholar, 17Eguchi S. Matsumoto T. Motley E.D. Utsunomiya H. Inagami T. J. Biol. Chem. 1996; 271: 14169-14175Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 18Berts A. Zhong A. Minneman K.P. Mol. Pharmacol. 1999; 55: 296-303Crossref PubMed Scopus (38) Google Scholar). Even in the same cell type, p44/42 MAPK activation can be PKC-dependent or PKC-independent depending upon the stimulus presented and the corresponding cellular response (19Buscher D. Hipskind R.A. Krautwald S. Reimann T. Baccarini M. Mol. Cell. Biol. 1995; 15: 466-475Crossref PubMed Scopus (165) Google Scholar). On the other hand, the PKC family of isozymes consists of three subfamilies: the conventional, the novel, and the atypical isozymes. Whereas the conventional isozymes (α, βI, βII, and γ) require calcium, phospholipid, and 1,2-dioctanoyl-sn-glycerol (DAG) for their activities, the novel isozymes (δ, ϵ, η, and θ) require only DAG and phorbol ester and the atypical isozymes (ζ, ι, and λ) require neither calcium nor DAG for their maximal activities (20Cohen R.I. Molina-Holgado E. Almazan G. Brain Res. Mol. Brain Res. 1996; 43: 193-201Crossref PubMed Scopus (64) Google Scholar, 21Larocca J.N. Almazan G. J. Neurosci. Res. 1997; 50: 743-754Crossref PubMed Scopus (52) Google Scholar). Human PKCμ and its murine analog, protein kinase D (PKD), form a distinct class (22Johannes F.-J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar, 23Van Lint J. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 24Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (851) Google Scholar) and are activated by DAG in the absence of calcium (23Van Lint J. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 25Johannes F.-J. Prestle J. Dietrich S. Oberhagemann P. Link Pfizenmaier K. Eur. J. Biochem. 1995; 227: 303-307Crossref PubMed Scopus (95) Google Scholar). Recently, this class has been named the PKD family, which comprises three isoforms: PKCμ/PKD1; PKD2; and PKD3 (26Rykx A. Kimpe L.D. Mikhalap S. Vantus T. Seufferlein T. Vandenheede J.R. Lint J.V. FEB Lett. 2003; 546: 81-86Crossref PubMed Scopus (196) Google Scholar). In contrast to other PKC isozymes, PKCμ has a hydrophobic N terminus, a transmembrane domain, a regulatory C1 domain with an extended spacing of two cysteine clusters, and an additional 270 amino acid sequence that separates the protein kinase domain from the regulatory C1 domain, resulting in an unusually large isozyme with an apparent molecular mass of 115 kDa (22Johannes F.-J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar, 25Johannes F.-J. Prestle J. Dietrich S. Oberhagemann P. Link Pfizenmaier K. Eur. J. Biochem. 1995; 227: 303-307Crossref PubMed Scopus (95) Google Scholar). In addition, PKCμ contains a pleckstrin homology (PH) domain that may exert an inhibitory effect on its kinase activity and also lacks the C2 domain that is responsible for the calcium sensitivity of the conventional PKC subgroup, thereby making it calcium-unresponsive (22Johannes F.-J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar). Because of the lack of a pseudosubstrate sequence present in the other PKC family members, PKCμ also displays a distinct inhibitor and substrate specificity (27Gschwendt M. Dieterich S. Rennecke J. Kittstein W. Mueller M.-J. Johannes F.-J. FEB Lett. 1996; 392: 77-80Crossref PubMed Scopus (566) Google Scholar, 28Rennecke J. Johannes F.-J. Richter K.H. Kittstein W. Marks F. Gschwendt M. Eur. J. Biochem. 1996; 242: 428-432Crossref PubMed Scopus (43) Google Scholar). In our previous work, only the PKC isozymes in the conventional subfamily were examined (13Reuben P.M. Brogley M.A. Sun Y. Cheung H.S. J. Biol. Chem. 2001; 277: 15190-15198Abstract Full Text Full Text PDF Scopus (58) Google Scholar). In this work, we extended our study to include other PKC isozymes in the novel, atypical, and PKD subfamilies to determine their expression and potential involvement in the BCP crystal-induced activation of the p44/42 MAPK signal transduction pathway in the human fibroblasts (HF). We also further examined the relationship between the BCP crystal-induced PKC and p44/42 MAPK signal transduction pathways. Materials—Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline, fetal bovine serum (FBS), penicillin, streptomycin, fungizone, protein A-agarose, ThermoScript RT-PCR system, and TRIzol reagent were obtained from Invitrogen. Phorbol 12,13-dibutyrate (PDBu), l-α-phosphatidyl-l-serine, DAG, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89), leupeptin, and aprotinin were obtained from Sigma. U0126 and monoclonal antibodies against MMP-1 and MMP-3 were from Calbiochem. Concentrated serum-free medium containing MMP-1 and MMP-3 control proteins was from Chemicon International Inc. (Temecula, CA). Monoclonal phospho-p44/42 MAPK antibody and polyclonal p44/42 MAPK antibody were from Cell Signaling Technology, Inc. (Beverly, MA). Polyclonal antibodies against PKC α, βI, βII, γ, δ, ϵ, η, and ζ were from Pan Vera (Madison, WI). Monoclonal antibody against PKCα, polyclonal antibodies against PKCθ, PKCι, and PKCμ, rat brain extract, rat thymus, NIH 3T3, and K-562 whole cell lysates were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-mouse IgG-horseradish peroxidase conjugate and Anti-rabbit IgG-horseradish peroxidase conjugate were from Promega (Madison, WI). SuperSignal WestPico chemiluminescent substrate was from Pierce. Cell Culture—HF were established from explants and transferred as described previously (29Cheung H.S. J. Tissue Cult. Methods. 1980; 6: 39-40Crossref Scopus (21) Google Scholar). They were grown and maintained in DMEM supplemented with 10% heat-inactivated FBS containing 1% penicillin, streptomycin, and fungizone. All of the cultures were third or fourth passage cells. All of the 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 0.2% FBS for 24 h. This medium then was removed, the cells were washed with phosphate-buffered saline, and serum-free DMEM was added to the cells and allowed to equilibrate before being used for the experiments. For the inhibition experiments, the cells were pretreated with the appropriate concentrations of the inhibitors for 30 min, unless otherwise stated, before being stimulated with the required concentrations of the agonists and for the indicated length of time. Synthesis of BCP Crystals—BCP crystals were synthesized by a modification of previously published methods (30Evans R.W. Cheung H.S. McCarty D.J. Calcif. Tissue Int. 1994; 36: 645-650Crossref Scopus (55) Google Scholar). 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 min. RT-PCR—Total RNA was isolated using the TRIzol reagent according to the manufacturer's instructions. 1 μg of each sample then was reverse-transcribed at 60 °C for 60 min followed by enzyme inactivation at 85 °C for 5 min using the ThermoScript RT-PCR system. PCR primers for MMP-1, MMP-3, and β-actin were designed and synthesized as described previously (13Reuben P.M. Brogley M.A. Sun Y. Cheung H.S. J. Biol. Chem. 2001; 277: 15190-15198Abstract Full Text Full Text PDF Scopus (58) Google Scholar). PCR primers for PKCμ were as follows: sense, 5′-GAGATATCTCTGTGAGTATTTCAG-3′, corresponding to positions 1908–1931, and antisense, 5′-ACACATTTTCTGGTTTGAGGTCAC-3′, corresponding to positions 2349–2372 of the nucleotide sequence of the human PKCμ and giving a PCR product of 465 bp (22Johannes F.-J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar). Amplifications were carried out as previously described (13Reuben P.M. Brogley M.A. Sun Y. Cheung H.S. J. Biol. Chem. 2001; 277: 15190-15198Abstract Full Text Full Text PDF Scopus (58) Google Scholar) with the exception that MMP-1 was done at 25 cycles while PKCμ and MMP-3 were done at 30 cycles. The PCR products were analyzed by electrophoresis on 2% agarose gel containing ethidium bromide. Cell Lysis—After the experiment, the cells were washed with cold phosphate-buffered saline and the plates were drained. 1 ml of cold lysis buffer (50 mm Tris, pH 7.6, 2 mm EDTA, 2 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 100 μg/ml leupeptin, 10 μg/ml aprotinin, and 1% Triton X-100) then was added to each 100-mm plate and placed on ice with occasional shaking. The cells were then scraped into 1.5-ml Microfuge tubes and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatants were transferred to fresh tubes, and the protein concentrations were determined with the Bio-Rad protein assay reagent (Hercules, CA) according to the manufacturer's instructions. The lysates were either used immediately or stored at –70 °C. Immunoprecipitation—Following an experiment, the cells were lysed and aliquots of the lysates were incubated with the appropriate antibodies on ice for 4 h to allow formation of the immune complexes. Protein A-agarose gel was added to each mixture and gently rotated overnight at 4 °C to allow the immune complexes to bind by adsorption of the antibody to protein A. The unbound proteins were removed by washing the solid phase several times with the lysis buffer by centrifugation, leaving the immune complexes bound to the agarose gel. The purified immune complexes were ready for further analysis by gel electrophoresis and immunoblotting after adding an equal volume of 2× SDS sample buffer and boiling for 5 min, or the immune complexes were used for enzyme activity studies. Expression of PKC Isozymes in HF—HF cells were grown on 100-mm plates to confluency in DMEM containing 10% FBS and then washed with phenol red-free DMEM containing 0.2% FBS and starved in the same medium for 24 h. This medium then was replaced with serum-free and phenol red-free DMEM and allowed to equilibrate in that medium before the cells were treated with or without BCP crystals for 15 min. After removing the medium and washing with phosphate-buffered saline, the cells were lysed and total proteins were extracted using the mammalian protein extraction reagent or M-PER obtained from Pierce according to the manufacturer's instructions. Protein concentrations in the extracts were determined as described before. Aliquots of the extracts with equal protein concentrations were electrophoresed on a 10% SDS-polyacrylamide gel and subjected to Western blotting. Western Blotting—Aliquots of cell lysates were electrophoresed through a 10 (PKC) or 12% (p44/42 MAPK) SDS-polyacrylamide gel and then transferred onto Immobilon-P polyvinylidene fluoride membranes (Millipore, Bedford, MA). After the transfer, the membranes were incubated for 4 h at room temperature in the blocking buffer TBST (20 mm Tris, 136 mm NaCl, 0.1% and 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 phosphospecific 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 (α, βI, βII, γ, δ, ϵ, η, ζ, θ, ι, and μ). The membranes were washed again several times with TBST and incubated with the appropriate anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody in TBST with 5% bovine serum albumin for 30 min at room temperature. Finally, the membranes were washed in TBST and Tris-buffered saline, and the protein bands were detected with the SuperSignal West Pico Chemiluminescent substrate according to the manufacturer's instructions and exposed to Kodak X-Omat AR films. Down-regulation of PKC with TPA—Cultured HF cells in 100-mm plates were starved and pretreated with or without TPA (1 μm) for 24 h to down-regulate PKC. They were treated with or without BCP crystals (50 mg/ml) for 15 min. The cells were also treated with PDBu (200 nm) and DAG (1 mm), both of which were used as positive controls for the BCP crystal stimulation of PKC. After cell lysis, aliquots of the lysates with equal protein concentrations were electrophoresed through a 10 (for PKC) or 12% (for phospho-p44/42) SDS-polyacrylamide gel, subjected to Western blotting, and probed with antibodies against PKCα, PKCμ and phospho-p44/42. Treatment of HF Cells with Sense and Antisense Oligodeoxynucleotides—An 18-base-long phosphorothioated antisense oligodeoxynucleotide (ODN) against PKCμ mRNA and its corresponding sense ODN were synthesized by Sigma. The antisense sequence for PKCμ was 5′-GACCGGAGGGGCGCTCAT-3′ based on the start codons (ATG) plus the 15 additional downstream bases in the human PKCμ sequence (22Johannes F.-J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar, 31Fleming I. MacKenzie S.J. Vernon R.G. Anderson N.G. Houslay M.D. Kilgour E. Biochem. L. 1998; 333: 719-727Crossref PubMed Scopus (52) Google Scholar). The corresponding sense sequence, 5′-ATGGCTGACGTTTACCCG-3′, was used as the control. As previously reported (31Fleming I. MacKenzie S.J. Vernon R.G. Anderson N.G. Houslay M.D. Kilgour E. Biochem. L. 1998; 333: 719-727Crossref PubMed Scopus (52) Google Scholar), semi-confluent HF cells in 100-mm plates were treated with or without 20 μm ODN, 20 μg/ml Lipofectin for 6 h at 37 °C. The cells then were washed, and the medium was replaced with fresh medium containing 2 mm glutamine and 10% heat-inactivated FBS and in the presence or absence of 20 μm ODN and without Lipofectin at 37 °C for 72 h. After these steps, the cells were treated with or without BCP crystals (50 μg/ml) for 15 min (for PKCμ and phospho-p44/42) and 24 h (for MMP-1 and MMP-3). The lysates then were assessed for immunoreactive PKCμ, phospho-p44/42, MMP-1, and MMP-3 protein levels by Western blotting and for PKCμ, MMP-1, and MMP-3 mRNA levels by RT-PCR. Inhibition of BCP Crystal-induced PKCμ and Phospho-p44/42 Activation—HF cells were cultured, starved, and preincubated with or without PKCμ inhibitor H-89 (100 μm) and phospho-p44/42 inhibitor U0126 (10 μm) for 30 min before the addition of BCP crystals (50 mg/ml) and PDBu (200 nm), which was used as a positive control for PKCμ activation. Cell lysates were immunoprecipitated with anti PKCμ, and the immune complex was used to determine PKCμ activity by autophosphorylation assay. Autophosphorylation of PKCμ—After cell lysis and immunoprecipitation with anti-PKCμ, the immune complexes were washed several times with the lysis buffer and then with the phosphorylation buffer (20 mm Tris, pH 7.4, 5 mm magnesium acetate, 2 mm sodium orthovanadate, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). Autophosphorylation was started by adding 30 μl of the phosphorylation buffer containing 0.3 μl of [γ-32P]ATP (5000 Ci/mmol and 10 mCi/ml) and l-α-phosphatidyl-l-serine (100 μg/ml) to the immunocomplexes and incubating at 37 °C for 20 min (23Van Lint J. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 25Johannes F.-J. Prestle J. Dietrich S. Oberhagemann P. Link Pfizenmaier K. Eur. J. Biochem. 1995; 227: 303-307Crossref PubMed Scopus (95) Google Scholar, 32Dieterich S. Herget T. Link G. Böttinger H. Pfizenmaier K. Johannes F.-J. FEBS Lett. 1996; 381: 183-187Crossref PubMed Scopus (76) Google Scholar). The reactions were terminated by adding an equal volume of 2× SDS sample buffer to each one. The proteins were released by boiling for 5 min, resolved by electrophoresis on 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The 32P-labeled proteins were visualized after a 4-hour exposure to autoradiography. p44/42 MAPK Activation—Following experimental treatments, cell lysis, and immunoprecipitations, aliquots of the cell lysates or immunoprecipitates were subjected to 12% SDS-polyacrylamide gel electrophoresis and Western blotting with a monoclonal antibody specific for the activated and phosphorylated p44/42 MAPK (phospho-p44/42) and a polyclonal antibody specific for the constitutive and nonactivated p44/42. Statistics—Statistical analysis was performed with the Student's t test in Sigma Plot scientific graphing software, and p < 0.05 was considered significant. Data were expressed as the means ± S.E. Expression of PKC Isozymes in HF—Because the expression and interactions of the PKC isozymes are cell type-specific (33Jaken S. Curr. Opin. Cell Biol. 1996; 8: 168-173Crossref PubMed Scopus (407) Google Scholar), we sought to determine the specific PKC isozymes expressed in HF upon BCP crystal (50 μg/ml) stimulation. By Western blotting, we show in Fig. 1 that, of all the conventional isozymes (A–D), only the α isozyme (A) is expressed as previously reported (13Reuben P.M. Brogley M.A. Sun Y. Cheung H.S. J. Biol. Chem. 2001; 277: 15190-15198Abstract Full Text Full Text PDF Scopus (58) Google Scholar). None of the novel (E–H) or atypical (I–J) isozymes is expressed. However, of the PKD family, PKCμ is highly expressed (K). These results indicate that both PKCα and PKCμ may have specific and distinct functions in the HF. The concentration of BCP crystals (50 μg/ml) used in these studies is consistent with our previously established optimal range of 50–100 μg/ml in vitro and is consistent with the in vivo concentration in articular joint fluids isolated from osteoarthritic patients, which range from 10 to 20 μg/ml, depending on the severity of the disease (34Cheung H.S. Ryan L.M. Woessner J.F. Howell D.S. Joint Cartilage Degradation: Basic and Clinical Aspects. Marcel Dekker, Inc., New York1993: 209-223Google Scholar). Role of PKC in the BCP Crystal Stimulation of Phospho-p44/42—Down-regulation of PKC is often used to determine its role in MAPK activation. Pretreatment of αT3-1 cells with TPA had been shown to down-regulate PKCα and PKCϵ with no effects on ERK 1 (p44) (35Sundaresan S. Colin I.M. Pestell G. Jameson J.L. Endocrinology. 1996; 137: 304-311Crossref PubMed Scopus (143) Google Scholar). To determine the roles of PKCα and PKCμ in the BCP crystal activation of phospho-p44/42 in HF, the cells were pretreated with TPA (1 μm) for 24 h to down-regulate the PKCs before stimulation with BCP crystals (50 μg/ml). Fig. 2 shows that pretreatment with TPA (+) completely depleted PKCα (A) but affected neither the expression of PKCμ (B) nor the stimulation of phospho-p44/42 by BCP, PDBu, and DAG (C) in lanes 5–8 compared with their respective controls that were not pretreated with TPA (–) in lanes 1–4. These results were further proof of our previous work that the PKCα and the p44–42 MAPK signal transduction pathways in response to BCP crystal stimulation in HF are independent of each other (13Reuben P.M. Brogley M.A. Sun Y. Cheung H.S. J. Biol. Chem. 2001; 277: 15190-15198Abstract Full Text Full Text PDF Scopus (58) Google Scholar). However, the results do not exclude the fact that PKCμ and phospho-p44/42 may share a common pathway in which the stimulation of phospho-p44/42 may be dependent upon the expression and/or activation of PKCμ. BCP Crystal Stimulation of Phospho-p44/42 Is Dependent upon PKCμ—Since it was suggested in Fig. 2 that PKCμ and phospho-p44/42 might share a common signal transduction pathway, the ideas that the two pathways were indeed related and/or one pathway was dependent upon the other were tested by treating the BCP crystal and PDBu-stimulated cells with the inhibitors of the two pathways in Fig. 3. The PKCμ inhibitor used, H-89, is a competitive ATP antagonist previously regarded as a highly specific inhibitor of protein kinase A (36Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar). However, it had been reported that PKCμ had an ∼60-fold higher sensitivity to H-89 than the other PKCs and that it required 60 μm H-89 to inhibit PKCμ autophosphorylation in A549 carcinoma cell line (25Johannes F.-J. Prestle J. Dietrich S. Oberhagemann P. Link Pfizenmaier K. Eur. J. Biochem. 1995;" @default.
• W2053646056 created "2016-06-24" @default.
• W2053646056 creator A5013328356 @default.
• W2053646056 creator A5052881743 @default.
• W2053646056 creator A5070231359 @default.
• W2053646056 date "2004-08-01" @default.
• W2053646056 modified "2023-10-07" @default.
• W2053646056 title "Basic Calcium Phosphate Crystals Activate p44/42 MAPK Signal Transduction Pathway via Protein Kinase Cμ in Human Fibroblasts" @default.
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