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J. Biol. Chem., Vol. 281, Issue 34, 24124-24137, August 25, 2006

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Protein Kinase C Is Up-regulated in Osteoarthritic Cartilage and Is Required for Activation of NF-B by Tumor Necrosis Factor and Interleukin-1 in Articular Chondrocytes^*

Edward R. LaVallie¹, Priya S. Chockalingam^¶, Lisa A. Collins-Racie, Bethany A. Freeman, Cristin C. Keohan^¶, Michael Leitges^||, Andrew J. Dorner, Elisabeth A. Morris^¶, Manas K. Majumdar^¶, and Maya Arai

From the Departments of Biological Technologies and ^¶Women's Health and Musculoskeletal Biology, Wyeth Research, Cambridge, Massachusetts 02140-2325, ^||Hannover Medical School, Hannover 30625, Germany, and the Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118-2526

Received for publication, February 28, 2006 , and in revised form, June 8, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Protein kinase C (PKC) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor (TNF-), in some cell types. A study of gene expression in articular chondrocytes from osteoarthritis (OA) patients revealed that PKC is transcriptionally up-regulated in human OA articular cartilage clinical samples. This finding led to the hypothesis that PKC may be an important signaling component of cytokine-mediated cartilage matrix destruction in articular chondrocytes, believed to be an underlying factor in the pathophysiology of OA. IL-1 treatment of chondrocytes in culture resulted in rapidly increased phosphorylation of PKC, implicating PKC activation in the signaling pathway. Chondrocyte cell-based assays were used to evaluate the contribution of PKC activity in NF-B activation and extracellular matrix degradation mediated by IL-1, TNF, or sphingomyelinase. In primary chondrocytes, IL-1 and TNF- caused an increase in NF-B activity resulting in induction of aggrecanase-1 and aggrecanase-2 expression, with consequent increased proteoglycan degradation. This effect was blocked by the pan-specific PKC inhibitors RO 31-8220 and bisindolylmaleimide I, partially blocked by Gö 6976, and was unaffected by the PKC-sparing inhibitor calphostin C. A cell-permeable PKC pseudosubstrate peptide inhibitor was capable of blocking TNFand IL-1-mediated NF-B activation and proteoglycan degradation in chondrocyte pellet cultures. In addition, overexpression of a dominant negative PKC protein effectively prevented cytokine-mediated NF-B activation in primary chondrocytes. These data implicate PKC as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that result in catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Osteoarthritis (OA),² a progressive and ultimately debilitating orthopedic disorder, is the most common degenerative joint disease in man (1). More than 20 million individuals in the United States alone have symptomatic OA, and it has been estimated that many more, greater than 50% of people over 65 years of age and 80% of those over age 75, have radiographic evidence of this disease (2). Yet, despite the widespread incidence of the disease in the human population, the etiology of OA is still largely unknown. OA is characterized by a slow focal destruction of articular cartilage, causing a roughening and thinning of the weight-bearing regions of the articular surface resulting in progressive immobility and pain. Articular cartilage is the tissue that provides shock-absorptive resiliency as well as low friction articulation to joints. It contains only a single cell type, the chondrocyte, which is responsible for the homeostasis of the tissue by synthesizing extracellular matrix that surrounds the cells and provides the important biophysical characteristics of the tissue. However, chondrocytes are also capable of producing catabolic factors capable of destroying the matrix components. Thus, extracellular matrix synthesis and degradation are dynamic processes that must be balanced by the chondrocytes for proper homeostasis of the tissue. In osteoarthritic cartilage, this balance appears to be shifted toward degradation, resulting in progressive loss of matrix due to up-regulation of proteolytic activities such as matrix metalloproteinases and aggrecanases. There is some debate whether osteoarthritis is a noninflammatory arthrosis or an inflammatory arthritis; however, synovial inflammation has been documented in OA (3) and inflammatory cytokines, especially interleukin-1 (IL-1) and tumor necrosis factor (TNF), have been implicated as important mediators of the disease (4–8). These proinflammatory cytokines are known to be major regulators of chondrocytic expression of downstream proteases (such as aggrecanases and collagenases) that are ultimately involved in matrix breakdown resulting in the formation of osteoarthritic lesions (6, 8). Therefore, these cytokines themselves, or components of their intracellular signaling pathways, constitute possible therapeutic intervention points that could mitigate the destruction of articular cartilage in OA.

One group of signaling proteins that are shared by both the IL-1 and TNF signaling pathways are the members of the protein kinase C (PKC) family of intracellular serine/threonine kinases. In the course of their convergence on the activation of the nuclear factor B (NF-B) transcription factor, both the IL-1 and TNF pathways reportedly signal through PKC family members in various cell types (9–13). The PKC family is made up of several isoforms that are divided into three basic classes ("conventional," "novel," and "atypical"), by virtue of the structure of their regulatory domains and (consequently) their methods of activation (14). The conventional or cPKC isoforms contain two characteristic membrane targeting domains called C1 and C2 that are capable of binding diacylglycerol (or the synthetic analog phorbol ester) or calcium, respectively, ultimately resulting in activation of the kinase. The novel or nPKC family members also contain these domains, but they are reversed in their orientation, and the C2 is modified such that it is unresponsive to calcium. The human atypical or aPKC group consists of only two members, and (called in mouse), both of which lack a C2 domain and possess only a modified C1 domain. Atypical PKC isoforms are insensitive to both diacylglycerol and calcium but are activated by phosphatidylserine.

In an effort to gain understanding of the pathological processes that underlie the development and progression of OA, we performed experiments to identify transcriptional alterations in articular chondrocytes that distinguish patients with end stage OA from normal subjects. This work revealed that PKC was the only member of the protein kinase C family with dysregulated expression in chondrocytes from human osteoarthritic cartilage. PKC has been implicated previously in the NF-B signaling pathway in some cell types, but its function in chondrocytes has not been well characterized. In this study, we utilized chondrocyte-based assay systems to investigate the role of PKC on TNFand IL-1-mediated activation of NF-B, and on the consequent proteoglycan degradation that results from the activation of the NF-B pathway. We find that NF-B activation by TNF and IL-1 in chondrocytes requires PKC activity and, most importantly, that inhibition of PKC blocks cytokine-mediated up-regulation of aggrecanase expression and the resulting destruction of articular cartilage extracellular matrix proteoglycans that is a hallmark of the OA disease process. Thus, PKC constitutes a pivotal signaling molecule in the catabolic pathways initiated by the proinflammatory cytokines IL-1 and TNF in articular chondrocytes and may represent an important therapeutic target.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Media, Chemicals, and Reagents—Sphingomyelinase, collagenase, Pronase, penicillin/streptomycin, and RO 31-8220 (a broad spectrum PKC inhibitor also capable of activating JNK-1 (15)) were from Sigma. Recombinant human IL-1, IL-1, and TNF- were purchased from R & D Systems. The following inhibitors were purchased from Calbiochem: bisindolylmaleimide I (a staurosporin analog that is a pan-PKC inhibitor (16)); calphostin C (a PKC inhibitor that does not inhibit atypical PKCs (17, 18)); and the NF-B cell-permeable peptide inhibitors SN50 and the mutated control peptide SN50M (19). SN50 contains the nuclear localization sequence (residues 360–369) of the transcription factor NF-B p50 subunit linked to the hydrophobic region (h-region) of the signal peptide of Kaposi fibroblast growth factor; SN50M is identical except this control peptide has two amino acid changes (Lys-363 to Asn and Arg-364 to Gly) that abolish its inhibition of NF-B nuclear translocation. Triptolide (an NF-B inhibitor compound (20)), Gö 6976 (a PKC inhibitor with higher potency for conventional PKC family members (21)), and pseudosubstrate peptides for PKC (myristoylated and nonmyristoylated N-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu) and PKC/ (N-Myr-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-NH₂) were purchased from Biomol (Plymouth Meeting, PA). Adenovirus expressing an NF-B-luciferase reporter gene construct (22) was obtained from Dr. Doug Harnish (Wyeth Research). T/C-28a2 cells (23) were a generous gift from Dr. Mary Goldring.

Primary Chondrocyte Isolation and Culture—Bovine cartilage was obtained from the metacarpophalangeal joint of calves (2–10 days old), and chondrocytes were isolated by serial enzymatic digestion using Pronase (1 mg/ml, 37 °C for 30 min) and collagenase (1 mg/ml, 37 °C for overnight) in Dulbecco's modified Eagle's medium (DMEM) with 10 mM HEPES, and 100 units/ml penicillin, 100 µg/ml streptomycin. The digest was filtered through a 70-µm nylon cell strainer (Falcon) and processed as described (24). Cells were resuspended in growth media (HL-1 media, Cambrex catalog number 77201) containing 2 µM L-glutamine, 50 µg/ml ascorbate, antibiotics, and 10% fetal bovine serum (FBS) and aliquoted into 15-ml Falcon centrifuge tubes at a concentration of 1 x 10⁶ cells per tube. The tubes were centrifuged at 200 x g at room temperature to allow formation of cell pellets and subsequently incubated at 37 °C in a humidified atmosphere with low oxygen tension (5% O₂) to retain their differentiated chondrocytic phenotype and to maximize the production of extracellular matrix (25). For inhibitor studies, chondrocyte pellets were weaned from the serum gradually by replacing the media every 3–4 days with decreasing concentrations of FBS (5, 2.5, and 0%). Chondrocytes were then cultured in pellet form for 3 weeks in the absence of serum to allow the accumulation of the proteoglycanand collagen-containing extracellular matrix. Chondrocyte cell pellets were preincubated for 2 h with inhibitors prior to cytokine stimulation for 18 h. The level of sulfated glycosaminoglycan in the culture media and cartilage/pellet extracts was determined by the dimethylmethylene blue (DMMB) assay (26). Shark and whale chondroitin sulfate (Fluka Biochemika, Switzerland) was used as a standard. Cytotoxicity testing was performed for each of the inhibitors at the concentrations used in the pellet culture assays by measuring lactate levels in culture media as an indicator of cellular metabolism and viability using a kit from Sigma.

Isolation of RNA from Primary Cartilage Tissue and from Chondrocytes in Culture—RNA was isolated from human osteoarthritic articular cartilage samples obtained from patients (n = 18, mean age = 66.2 years, range 49–84 years) undergoing total knee replacement surgery (New England Baptist Hospital) or from nonosteoarthritic cartilage obtained from above-knee amputations (n = 10, mean age = 71.6 years, range 43–100) (Clinomics). The OA cartilage samples were obtained as whole joints within2hof surgery, and the articular cartilage was shaved from the joint surfaces taking great care to avoid any pannus, fibrotic tissues, subchondral bone, and other noncartilaginous regions of the joint (27). Nonosteoarthritic cartilage samples were obtained from individuals without a clinical diagnosis nor symptoms of OA, and the specimens were evaluated histologically to confirm the classification prior to inclusion in this study. Cartilage pieces were flash-frozen in liquid nitrogen and stored at –80 °C until processed for RNA isolation. The frozen cartilage was pulverized using a Spex Certiprep freezer mill (model 6750) at 15 Hz two times for 1 min each under liquid nitrogen. The frozen powdered cartilage was resuspended in 4 M guanidinium isothiocyanate (Invitrogen) containing 8.9 mM 2-mercaptoethanol and homogenized on ice with a Polytron homogenizer at maximum speed setting twice for 1 min each time, with a 1 min "rest" between homogenizations. The homogenate was centrifuged at 1500 x g for 10 min, and the supernatant was saved. The gelatinous pellet was resuspended in guanidinium isothiocyanate/2-mercaptoethanol and homogenized a second time as described above. The pellet was then discarded, and the two resulting supernatant fractions were combined and incubated with Triton X-100 (2% final concentration) and sodium acetate (pH 5.5, 1.5 M final concentration) sequentially for 15 min each. The samples were extracted once with an equal volume of acid phenol chloroform, pH 4.5, and twice with acid phenol, pH 4.5, phenol, pH 7.5, chloroform mix (1:1:1). RNA was subsequently precipitated by the addition of isopropyl alcohol, and further purified using an RNeasy mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. RNA quantity and purity were measured by ultraviolet absorbance at A₂₆₀/A₂₈₀, and RNA quality was assessed by the RNA6000 assay using the Agilent BioAnalyzer 2100 (Palo Alto, CA). RNA yields averaged between 5 and 10 µg of total RNA per g of cartilage tissue.

For isolation of RNA from chondrocyte pellet cultures and from chondrocytes in monolayer culture, no pulverization was required. Pellets were digested with collagenase (2.5 mg/ml, Sigma), and RNA was subsequently prepared using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. Primary chondrocytes and chondrocytic cell lines in monolayer culture were lysed by direct addition of TRIzol reagent followed by standard TRIzol RNA purification methodologies.

Microarray Analysis of Osteoarthritic and Normal Cartilage—Gene expression changes in RNA from lesional (n = 14) and adjacent nonlesional (n = 13) osteoarthritic cartilage compared with nonosteoarthritic cartilage (n = 10) were analyzed using the Human Genome U95Av2 (HG-U95Av2) GeneChip® Array (Affymetrix, Santa Clara, CA) for expression profiling. The HG-U95Av2 chip contains 25-mer oligonucleotide probes representing 12,000 primarily full-length sequences (16 probe pairs/sequence) derived from the human genome. For each probe that is designed to be perfectly complementary to a target sequence, a partner probe is generated that is identical except for a single base mismatch in its center. These probe pairs allow for signal quantitation and subtraction of nonspecific noise.

RNA was extracted from individual articular cartilage tissue samples, converted to biotinylated cRNA, and fragmented according to the Affymetrix protocol. The fragmented cRNAs were diluted in 1x MES buffer containing 100 µg/ml herring sperm DNA and 500 µg/ml acetylated bovine serum albumin and denatured for 5 min at 99 °C followed immediately by 5 min at 45 °C. Insoluble material was removed from the hybridization mixtures by a brief centrifugation, and the hybridization mixture was added to each array and incubated at 45 °C for 16 h with continuous rotation at 60 rpm. After incubation, the hybridization mixture was removed, and the chips were extensively washed and stained with streptavidin (R)-phycoerythrin (Molecular Probes, Eugene, OR) using the GeneChip® Fluidics Station 400 following the manufacturer's specifications. The raw fluorescent intensity value of each transcript was measured at a resolution of 6 µm with a Hewlett-Packard Gene Array Scanner. GeneChip® software 3.2 (Affymetrix), which uses an algorithm to determine whether a gene is "present" or "absent," as well as the specific hybridization intensity values or "average differences" of each gene on the array, was used to evaluate the fluorescent data. The average difference for each gene was normalized to frequency values by referral to the average differences of 11 control transcripts of known abundance that were spiked into each hybridization mixture according to the procedure of Hill et al. (28). First, the frequency of each gene was calculated and represents a value equal to the total number of individual gene transcripts per 10⁶ total transcripts. Transcripts which were called present by the GeneChip® software in at least one of the arrays for both arthritis and normal cartilage, were included in the analysis. Second, for comparison between arthritis and normal cartilage, a t test was applied to identify the subset of transcripts that had a significant (p < 0.05) increase or decrease in frequency values. Third, average fold changes in frequency values across the statistically significant subset of transcripts were required to be 2.0-fold or greater. These criteria were established based upon replicate experiments that estimated the intra-array reproducibility.

Quantitative RT-PCR (TaqMan®)—RNA for TaqMan® analysis (ABI PRISM 7700 sequence detection; PerkinElmer Life Sciences) was isolated from primary cartilage tissue as described above and then further purified with two more rounds of phenol/chloroform extraction followed by RNeasy (Qiagen) column binding and elution. To ensure the elimination of genomic DNA, RNA was treated with DNase (Qiagen) during RNeasy column purification (as recommended by the supplier), and following the RNA purification any residual genomic DNA was removed using DNA-free (Ambion, Austin, TX), following that manufacturer's instructions.

For comparison of PKC and PKC mRNA levels in human OA chondrocytes, human primary chondrocytes were isolated as described above for bovine primary chondrocytes. The primary chondrocytes were plated in a 24-well format (2 x 10⁶/well) in DMEM/F-12 + 10% FBS + 100 units/ml penicillin, 100 µg/ml streptomycin for 2 days. The cells were lysed, and RNA was isolated using the RNeasy mini kit (Qiagen). 200 ng of RNA was used for each TaqMan assay in the TaqMan one-step RT-PCR method (Applied Biosystems). The human PKC and PKC probe/primer sets were "Assays on Demand" from Applied Biosystems, assay identification Hs00177051_m1 and Hs00702254_s1, respectively. Assay mixtures and cycling conditions were per the manufacturer's recommendation.

Oligonucleotide primers and fluorescent-labeled TaqMan probes for bovine ADAMTS-4, ADAMTS-5, and GAPDH cDNA sequences were designed using Primer Express 1.0 software (Applied Biosystems, Warrington, UK). Sequences for primers and probes were as follows: bADAMTS-4 forward, 5'-TGTGTGGTGGGGATGGTT-3', reverse, 5'-GCACCAGGATGTGGGTG-3', and probe, 5'-FAM-CTGGCTCCTTCAAAAAATTCAGGTACGGA-TAMRA-3'; bADAMTS-5 forward, 5'ATTTCGGCTCCACGGAAGAT-3', reverse, 5'-TTCTGTGATGGTGGCTGAGG-3', and probe, 5'-FAM-ATTGACGCATCCAAACCCTGGTCCA-TAMRA-3'; bGAPDH forward, 5'-AAAGTGGACATCGTCGCCAT-3', reverse, 5'-GACTGTGCCGTTGAACTTGC-3', and probe, 5'-FAM-TTCACTACATGGTCTACATGTTCCAGTATGATTCCA-TAMRA-3'. TaqMan PCR analysis was performed using the Applied Biosystems ABI Prism 7700 sequence detection system (TaqMan). PCRs for all samples were performed in duplicate (20 ng of DNase-treated purified total RNA) using TaqMan one-step PCR master mix reagent kit (Applied Biosystems) following the manufacturer's protocol. Thermocycler conditions comprised an initial reverse transcription step with incubation at 48 °C for 30 min, followed by AmpliTaq Gold enzyme activation at 95 °C for 10 min, and finally PCR amplification performed at 95 °C for 15 s and 60 °C for 1 min for 40 cycles. Threshold cycle (CT) values were obtained for ADAMTS-4 and ADAMTS-5, and the values were divided by CT values for GAPDH to obtain the relative expression level of aggrecanases normalized to GAPDH expression.

Phosphoprotein Analysis of PKC—For the chondrocyte cell line, T/C-28a2 cells (23) (kindly provided by Mary Goldring) were plated in a 24-well format in DMEM/F-12 + 10% FBS + 1% antimycotic antibiotic solution and grown to confluence. After the cells became confluent, they were changed to serum-free medium, allowed to adapt to serum-free conditions overnight, and were then stimulated with 20 ng/ml recombinant human IL-1 (R & D Systems) for varying lengths of time (0–30 min). The cells were immediately lysed at the conclusion of each time point with 1x Cell Lysis buffer (Cell Signaling Technologies). PKC was immunoprecipitated from the cell lysates with a monoclonal antibody to p62 Lck ligand (BD Transduction Laboratories). The immunoprecipitated proteins were then run on 10% SDS-PAGE under reducing conditions, and Western analysis was performed using either a monoclonal antibody that specifically recognizes PKC phosphorylated at position Thr⁴¹⁰ (Cell Signaling Technologies) or with a monoclonal antibody raised to the carboxyl-terminal 20 amino acids of PKC (C-20, Santa Cruz Biotechnology). The C-20 antibody recognizes total PKC regardless of its phosphorylation state. For bovine primary chondrocytes, primary bovine chondrocytes were isolated from fresh bovine metacarpophalangeal joints as described above. The primary chondrocytes were plated in a 24-well format (2 x 10⁶ cells/well) in DMEM/F-12 + 10% FBS + 1% antimycotic antibiotic solution for 2 days and then switched to serum-free media overnight prior to cytokine induction. The cells were treated with IL-1 (20 ng/ml) for various time points; the cells were lysed, and the lysate was analyzed as described above for the T/C-28a2 cells.

Western blots were visualized using a goat anti-mouse horseradish peroxidase conjugate followed by detection using the ECL Western blotting detection kit (Amersham Biosciences). Band intensities were determined by scanning the developed Western blots with a Gel Doc 2000 PC using the Quantity One quantitation software (Bio-Rad). The degree of phosphorylation for each sample was determined by calculating the ratio of phospho-PKC to total PKC.

Immunodetection of Aggrecan Cleavage Product—Detection of aggrecan cleavage sites in pellet culture conditioned media was performed using neoepitope monoclonal antibody Agg-C1 (anti-NITGE³⁷³, detects aggrecanase cleavage at aggrecan interglobular domain site (24)). Equal volumes of conditioned media from pellet cultures following incubation with or without cytokines and/or inhibitors were deglycosylated with chondroitinase ABC and keratanase (Calbiochem) and separated by 4–12% NOVEX Tris-glycine gels (Invitrogen). Subsequently, the samples were electrophoretically transferred to Hybond membrane (Amersham Biosciences) and incubated with Agg-C1 antibody overnight at 4 °C in TSA (50 mM Tris-Cl, pH 7.4; 0.2 M NaCl; 0.02% sodium azide). Unbound antibody was removed by washing the membrane in 1x TSA buffer three times for 5 min, followed by incubation with goat anti-mouse IgG-alkaline phosphate conjugate secondary antibody (1:5000, Novagen) for 1 h at room temperature in 1x TSA buffer. Following a second set of three 5-min washes in 1x TSA, immunoreactive products were detected as described previously (28) by developing the Western blot with bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Promega), and the image was digitized using a Hewlett-Packard flatbed scanner.

Quantitation of Atypical PKC Protein Levels in Human Chondrocytes—Specific rabbit polyclonal antibodies against PKC / and PKC were generated by immunizing rabbits with peptides spanning either residues 184–234 (PKC /; GenBank^TM accession number NM_008857 [GenBank] .2) or 185–244 (PKC; GenBank^TM accession number NM_008860 [GenBank] .2). Primary human chondrocytes were isolated from live cartilage using Pronase and collagenase digestion as described above for bovine cartilage. Whole cell lysates from these primary human OA chondrocytes (150 µg per sample) were size-fractionated by 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Hybond ECL; Amersham Biosciences). The primary antibody reaction was performed overnight at 4 °C. Unbound antibodies were removed by washing the nitrocellulose membrane three times for 15 min in washing buffer (PBS, pH 7.4, 0.1% Tween 20) at room temperature. Subsequently the membrane was incubated with secondary antibody (goat anti-rabbit horseradish peroxidase, 1:5,000; Dianova) for 2 h at room temperature followed by washing as described above. Antibodies were detected by chemiluminescence using ECL Western blotting detection reagents (Amersham Biosciences).

NF-B-Luciferase Cell Line Construction—To construct a chondrocytic cell line that stably expresses an NF-B-luciferase reporter gene, the T/C-28a2 cell line (23) was transfected with vectors pIRESpuro3 (catalog number 6986, Clontech) and pNF-B-Luc (catalog number 6053, Clontech), and cells were selected for resistance to puromycin. Cells that survived the selection were screened by a luciferase reporter assay (Promega) after IL-1 (10 ng/ml) induction. Puromycin-resistant T/C-28a2/NF-B-luciferase cell lines were first selected for response to IL-1 induction with a minimum signal/background ratio of 5 in the luciferase reporter assay (Promega). The clones that passed this primary screen were further tested using TNF- (5 and 20 ng/ml)/IL-1 (5 and 20 ng/ml). In this secondary screening, a T/C-28a2/NF-B-luciferase clone was selected that showed the highest signal:background ratio and dose-dependent response to both TNF and IL-1 compared with other clones at the same cell density. Optimal conditions for using this cell line to test NF-B response to cytokines ± inhibitors were determined empirically. The conditions found to be optimal were plating density of 30,000 cells/well in a 96-well format, 10 ng/ml IL-1 concentration, 25 ng/ml TNF- concentration, 1 h of preincubation with inhibitors prior to cytokine induction, and a 3-h incubation with cytokines before luciferase assay. Cytotoxicity testing was performed for each of the inhibitors at the concentrations used in the NF-B-luciferase assays either by measuring lactate levels in culture media as a measure of cellular metabolism and viability using a kit from Sigma, or by directly measuring cellular proliferation using the WST-1 assay (Roche Applied Science).

Adenovirus Constructs and Infection Conditions—The vectors used to produce adenovirus were replication-defective human adenovirus type 5 with complete deletion of the E1a and E1b regions and partial deletion of the E3 regions of the viral genome. Separate adenoviral expression constructs were created that contained cDNAs encoding full-length active human PKC (FL-PKC; GenBank^TM accession number Q05513 [GenBank] ); a mutant human PKC (DN–PKC) in which the alteration of a key residue in the ATP-binding site (K281W) results in a dominant negative kinase (29, 30); an NF-B-luciferase reporter gene construct; and a green fluorescent protein virus as a transfection normalization standard (Ad-GFP). In experiments utilizing infection of primary bovine chondrocytes with the NF-B-luciferase reporter virus, cells were infected 4 days prior to the experiment with equal m.o.i. (typically 100 m.o.i.) that resulted in 100% infectivity as determined by titrated Ad-GFP infections. These m.o.i. levels were tested and confirmed to be noncytotoxic using the lactate assay. All constructs (except the NFB-luciferase reporter gene virus) expressed cDNAs under the control of the cytomegalovirus (CMV) promoter. These vectors were used to propagate recombinant viruses in human embryonic kidney cells (HEK293) (ATCC, Manassas, VA), which were then purified by two rounds of cesium chloride centrifugation (31). The purified virus was dialyzed in phosphate-buffered saline (PBS) and stored at –80 °C in 10% glycerol in PBS at a concentration of 10⁹ plaque-forming units/µl. Adenovirus were generated, purified, and titered by ViraQuest Inc. (North Liberty, IA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Protein Kinase C Is Transcriptionally Up-regulated in Human Osteoarthritic Cartilage—Gene expression changes in articular chondrocytes from lesional (n = 14) and adjacent nonlesional (n = 13) osteoarthritic cartilages compared with nonosteoarthritic cartilages (n = 10) were analyzed using Affymetrix GeneChip® U95Av.2 arrays. Analysis of these data to identify the global transcriptional changes in articular chondrocytes that were associated with osteoarthritis is beyond the scope of this paper, and will be published separately.³ However, a focused analysis of the data was performed to specifically identify OA-associated expression changes in protein kinase C family members. Probe sets for nine PKC isoforms were represented on the U95Av.2 array as follows:, , , , , , , , and . Only four of these PKC isoforms, , , , and , were judged to be present by virtue of their hybridization intensity levels and the consistent hybridization performance of their probes on the arrays. Of these four PKC isoforms, only PKC and PKC exceeded an empirically determined signal intensity threshold of 50 signal units allowing reliable quantitation of their transcript levels on the gene chips, and only PKC appeared to be transcriptionally altered in OA articular cartilage compared with normal articular cartilage (Fig. 1A and data not shown). Transcript levels for PKC, the only other human atypical PKC isoform, were weakly detected on the gene chips and did not appear to show disease-related expression changes, but it was expressed at levels too low for accurate quantitation by this methodology. When the raw gene chip signal intensity for PKC hybridization was converted to normalized mRNA quantities (in parts per million) using an intrinsic standard curve on the chips (28), the PKC transcript levels were found to be 2.5–3.5-fold higher in the nonlesional and lesional OA cartilage samples compared with normal cartilage (Fig. 1B). The up-regulation of PKC mRNA in the lesional OA samples reached statistical significance (p < 0.004), but variability in the PKC values in the nonlesional samples limited the significance of the up-regulation in those samples on the gene chips. To confirm the gene chip results and to increase the sensitivity of the transcriptional analysis, PKC transcript levels were measured in RNA from these same human samples by TaqMan quantitative RT-PCR (Fig. 1C). These TaqMan data supported the gene chip results, confirming that PKC mRNA was up-regulated 3.5-fold in nonlesional OA articular cartilage (p < 0.002 versus normal cartilage) and 2.0-fold in lesional OA articular cartilage (p < 0.02 versus normal cartilage).

PKC Is the Predominant Atypical PKC Isoform in Articular Cartilage—Because PKC shares a high degree of sequence similarity with PKC (72% amino acid identity overall and even greater within the catalytic domain (32, 33)), differentiating between PKC and PKC using most available biochemical reagents is difficult or impossible. Therefore, we were interested in determining how PKC transcript levels compared with PKC in an effort to judge the potential extent or proportion of the functional contribution of PKC in subsequent experiments. Because PKC mRNA levels were too low for accurate quantitation in the gene chip transcriptional profiling, more sensitive TaqMan assays were performed with probe/primer sets designed to distinguish PKC from PKC. These assays were first calibrated with known input amounts of PKC and PKC cDNA to allow direct comparison of transcript abundance between genes (data not shown). The correction factor based upon assay efficiencies in these calibration assays was 0.988 (PKC = PKC/0.988). RNA was isolated from articular chondrocytes from three separate donors with end stage OA, and PKC and PKC transcript levels were measured in each donor sample by TaqMan Q-PCR. The threshold cycle (Ct) value for PKC averaged 9 cycles lower than for PKC (Fig. 2A). In Fig. 2B, transcript abundance for each gene was converted to TaqMan units (raw Ct value normalized by comparison to GAPDH in the same samples), and the normalized value was corrected for assay efficiency by calculation of absolute transcript levels using the cDNA calibration curves. The result showed that the abundance of PKC mRNA in human OA articular cartilage was more than 800 times greater than PKC, which was detectable but present at consistently low levels in all three of the OA articular cartilage samples (Fig. 2B).

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Transcriptional regulation of protein kinase C isoforms in human osteoarthritic articular cartilage. A, transcript abundance of human PKC isoforms that were detected at measurable levels by Affymetrix GeneChips in human clinical samples of normal cartilage (n = 10) and in lesional (severely affected, n = 14) and nonlesional (adjacent mildly affected, n = 13) osteoarthritic articular cartilage. RNA levels are expressed in raw signal intensity units. B, measurement of average mRNA levels of PKC in RNA from normal human articular cartilage and from nonlesional and lesional human osteoarthritic cartilage using GeneChips. Transcript levels are reported as normalized mRNA abundance, expressed as parts per million. C, quantitative RT-PCR measurement of PKC mRNA levels in a subset of the same samples shown in A, using TaqMan®. Transcript levels are reported as TaqMan® units, normalized to an internal standard (GAPDH). All values are reported as the mean ± S.D. of all of the samples from each cohort subset (n = 6 for each group), and significance was determined by Student's t test.

PKC Is Activated by IL-1 Signaling in Chondrocytes—The role of IL-1 in the initiation of extracellular matrix destruction in chondrocytes has been well established (24, 34–37). IL-1 has been shown to trigger the phosphorylation of PKC in some cell types (38, 39). To investigate whether PKC may be downstream of IL-1 signaling in chondrocytes, a human immortalized chondrocyte cell line T/C-28a2 (23) and primary bovine articular chondrocytes were treated separately in culture with 20 ng/ml IL-1 or IL-1, respectively. We have shown previously that at equivalent doses the recombinant human IL-1 is more potent than recombinant human IL-1 on bovine chondrocytes in terms of activation of NF-B and degradation of matrix proteoglycan (24), whereas humanand porcine-derived chondrocytes show greater response to IL-1.⁴ Because IL-1 and IL- signal through the same cell-surface receptor and elicit the same downstream responses (40), we assumed that the differences in potency reflected differences in cross-species binding affinities for the two isoforms of IL-1 to the cognate receptors and therefore chose the most active cytokine for each system. At various time points up to 10 min after addition of IL-1, cultures were lysed and the cell lysates were immunoprecipitated using an antibody to p62 (41). p62 is a scaffolding protein known to associate with PKC when it is activated by upstream protein kinases (42, 43). Thus, immunoprecipitation of p62 provides enrichment for PKC that has been phosphorylated by upstream kinases. p62 protein, along with proteins from the cell lysates that were bound to it, was recovered and run on an SDS-polyacrylamide gel, and a Western blot was performed either with an antibody that specifically binds PKC when it is phosphorylated at threonine 410 (Fig. 3, top panels of A and C) or with an antibody raised to a peptide representing the carboxyl-terminal 20 amino acids of PKC that binds to PKC regardless of its phosphorylation state (Fig. 3, bottom panels of A and C). Phosphorylation of PKC at Thr⁴¹⁰ is known to be due to the activity of PDK-1, and this phosphorylation event initiates the activation of PKC enzymatic activity (44). In the T/C-28a2 cell line, basal levels of phospho-Thr⁴¹⁰ PKC were low (Fig. 3, A and B). Upon addition of IL-1, phosphorylation of PKC at the Thr⁴¹⁰ position was significantly increased after 1 min (p < 0.001), and peaked at 3 min of IL-1 exposure. This increase in PKC phosphorylation remained at roughly the same significantly elevated level throughout 10 min of IL-1 exposure (Fig. 3, A and B). The timing and persistence of PKC phosphorylation in the IL-1-treated primary bovine chondrocytes were very similar to T/C-28a2 cells that were stimulated with IL-1 (Fig. 3, C and D). The increase in PKC phosphorylation elicited by IL-1 treatment of the primary bovine chondrocytes was also detectably increased after 1 min but did not reach statistical significance (p < 0.05) until 3 min of IL-1 exposure; this significant increase in bovine chondrocyte phospho-PKC levels then persisted throughout the remaining 10-min time course of the experiment. The greater variability of response of the primary bovine chondrocytes to IL-1 treatment compared with the human chondrocytic cell line probably arose from variability between donors, because the bovine chondrocytes were harvested from articular cartilage of different individual calves for each of the replicate experiments.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Comparison of PKC and PKC mRNA and protein levels in human articular chondrocytes. Quantitative RT-PCR assays (TaqMan®) were used to evaluate the relative abundance of mRNA for PKC and PKC in human osteoarthritic cartilage samples. A, threshold cycle values (mean ± S.D.) for PKC and PKC in articular cartilage RNA from three different human donors with end stage OA. B, "TaqMan units" were calculated for each transcript by comparing Ct values for each sample (assayed in triplicate) to a standard curve consisting of known quantities of cDNA for each gene. The extrapolated transcript abundance from the standard curve was then normalized to an internal standard (GAPDH) for each assay. The calculated values are shown within the bar graph because the PKC levels were too low for graphing on this scale. C, immunoblot analysis of primary chondrocyte cell lysates from two different ("H3" and "H4") human donors with end stage OA. Identical blots were prepared from 10% SDS-polyacrylamide gels loaded with 150 µg of cell lysate protein per lane, and the immobilized proteins were individually subjected to Western blotting with either a rabbit polyclonal antibody that recognizes both PKC and PKC ("aPKC C-20", left panel), or with rabbit polyclonal antibodies that specifically recognize PKC (middle panel) or PKC (right panel). Data shown for these two donors typify the results seen for additional samples that were tested.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Effect of IL-1 treatment of articular chondrocytes on PKC phosphorylation state. The human chondrocytic cell line T/C-28a2 (A) or primary bovine chondrocytes in culture (B) were treated with either 20 ng/ml IL-1 (A) or 20 ng/ml IL-1 (B) for various time points. At the indicated times, the cells were lysed, and PKC from the cell lysates was indirectly immunoprecipitated using a monoclonal antibody to p62 Lck ligand (BD Transduction Laboratories). Western analysis was performed on the immunoprecipitated proteins using antibodies against phospho-(Thr410) PKC (A and C, top panels) and total PKC (A and C, bottom panels). The Western blots are representative of at least three independent experiments with comparable results. The ratio of phosphorylated PKC to total PKC protein for the T/C-28a2 cells (B) and the primary bovine chondrocytes (D) was determined from densitometry scans of the replicate Western blots, and is shown as the mean ± S.D. of replicate experiments. *, p < 0.05; **, p 0.01; ***, p 0.001, as determined by Student's t test.

To test this assumption, total RNA was extracted from the pellet cultures at the end of the culture period, and quantitative RT-PCR was performed using probe/primer sets designed to bovine ADAMTS-4 (Agg-1) and bovine ADAMTS-5 (Agg-2) mRNA sequences (24). Bovine GAPDH was used as a normalization control. Both TNF- and IL-1 treatment induced Agg-1 and Agg-2 mRNA levels in these chondrocyte cultures (Fig. 4, C and D). Agg-1 mRNA induction by TNF- and IL-1 was significantly suppressed by RO 31-8220 and SN50 (p < 0.01), and the PKC PS peptide also appeared to reduce Agg-1 mRNA levels, but the effect did not reach statistical significance (Fig. 4C). However, up-regulation of Agg-2 mRNA by TNF and IL-1 in the primary chondrocyte cultures was significantly suppressed by all three inhibitors (Fig. 4D), demonstrating that PKC inhibition can effectively block induction of aggrecanase expression in chondrocytes by these inflammatory cytokines. Thus, the proteoglycan degradation caused by exposure of primary chondrocytes to IL-1 or TNF involves PKC-dependent aggrecanase up-regulation that results in accumulation of aggrecan fragments in the conditioned media that are cleaved at the Glu³⁷³–Ala³⁷⁴ site in the interglobular domain of aggrecan. Because aggrecan is the predominant proteoglycan in cartilage matrix (52), these data support a model in which suppression of cytokine-mediated aggrecanase induction by inhibition of PKC activity is the mechanism by which proteoglycan is preserved in this system.

To further explore the effects of specific inhibition of PKC, primary bovine chondrocyte pellet cultures were pretreated with increasing concentrations of the myristoylated (cell-permeable) PKC PS peptide prior to addition of 10 ng/ml IL-1 and overnight (18 h) incubation. Doses of peptide as low as 10 µM significantly (p < 0.05) reduced induction of proteoglycan degradation by IL-1, and doses of 20 µM or more of the PKC PS peptide led to even more significant reduction (p < 0.01) of the IL-1 effect on proteoglycan release from the pellet cultures (Fig. 5A). The inhibition of cytokine-induced proteoglycan release by the PKC PS peptide was not attributable to decreased synthesis of proteoglycan due to cytotoxicity, because lactate assays on conditioned media from these same pellet cultures showed no decrease in cellular metabolism (Fig. 5B). In addition, evaluation of the proteoglycan content of the cell pellets following papain digestion at the end of the experiment showed that total proteoglycan synthesis was not impaired (data not shown). In this same cytokine-induced chondrocyte proteoglycan release assay using TNF-, comparison of the myristoylated PKC PS peptide to a nonmyristoylated PKC PS peptide with the identical sequence, and to a myristoylated peptide containing the pseudosubstrate sequence of PKC/, revealed that the inhibitory effect was specific to PKC and required cell permeability (Fig. 5C). An additional myristoylated control peptide containing the same amino acid content as the PKC PS peptide but with the order of the amino acids scrambled has also been repeatedly tested in this assay and showed no inhibitory effect on cytokine-induced matrix degradation (data not shown).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Up-regulation of aggrecanase expression by TNF- and Il-1 in primary articular chondrocytes is dependent upon PKC and NF-B. Primary bovine chondrocyte pellet cultures were pretreated 2 h prior to cytokine addition either with no inhibitors or with RO 31-8220 (RO, 10 µM), a myristoylated PKC pseudosubstrate peptide inhibitor (PKC PS, 40 µM), a cell-permeable peptide inhibitor of NF-B nuclear translocation (SN50, 100 µM), or a mutated inactive SN50 control peptide (SN50M, 100 µM). TNF or IL-1 was then added, and the cells were incubated prior to assay. All culture treatments were performed in triplicate. A, total proteoglycan released to the conditioned media from the pellet culture extracellular matrix after 18 h of cytokine treatment, as measured by DMMB assay. DMMB assays were performed in duplicate and averaged. Data are expressed as the mean ± S.D. of the replicate cultures (n = 3). B, aliquots of culture media from the experiment shown in A were subjected to Western blotting using the monoclonal neoepitope antibody Agg-C1 to allow detection of aggrecanase-cleaved aggrecan fragment as a measure of aggrecanase activity in the pellet cultures. The figure is a composite of two separate blots prepared at the same time, immunostained, and developed together for the same amount of time. C and D, RNA harvested 18 h after cytokine addition was assayed for ADAMTS-4 (Agg-1; C) and ADAMTS-5 (Agg-2; D) mRNA levels using TaqMan® probe/primer sets designed to the bovine sequences. Data are expressed as the mean ± S.D. of replicate experiments (n = 3), and significance was determined by Student's t test.

TNF or IL-1 Induction of NF-B and Resulting Proteoglycan Degradation in Chondrocytes Is Blocked by Pan-PKC Inhibitors but Not by an Atypical-sparing PKC Inhibitor—Additional PKC inhibitors with different specificities were used with the T/C-28a2 NF-B-luciferase cell line in an effort to further implicate the isoform as the responsible protein kinase C family member in the pathways by which TNF and IL-1 signal through NF-B. Bisindolylmaleimide I (BIS) inhibits all PKC isoforms with a potency rank order of cPKC > nPKC > aPKC (16, 57). IL-1 induction of the NF-B-luciferase reporter gene in the T/C-28a2 cell line was inhibited by almost 80% by 12.5 µM BIS, with less inhibition noted with decreasing concentrations of BIS (Fig. 6A). This level of inhibition is consistent with the reported IC₅₀ of 5.8 µM for BIS on PKC, which is almost 300-fold higher than the IC₅₀ for BIS on cPKCs (0.02 µM) and more than 30-fold higher than the IC₅₀ for BIS on nPKCs (58). Gö 6976 reportedly is also most potent against the Ca²⁺-requiring (conventional) PKC isoforms and is less potent against the nPKCs and aPKCs, with an IC₅₀ for PKC of >10 µM (21, 59). Gö 6976 showed some inhibition of both IL-1 and TNF induction of NF-B-luciferase activity, but it was no more potent in this assay than BIS, despite the fact that Gö 6976 has an IC₅₀ 4-fold lower than BIS on cPKCs (21). Calphostin C is a compound that competes for the diacylglycerol/phorbol ester-binding site in the regulatory domain of the conventional and novel PKCs and competitively inhibits their activity (17). Because the atypical PKCs ( and ) lack this domain, their activity is unaffected by calphostin C. Calphostin C was totally ineffective at blocking IL-1 or TNF induction of NF-B (Fig. 6A), even at a concentration 100 times higher than its IC₅₀ for cPKC and nPKC (17). Similar results were obtained in the bovine primary chondrocyte pellet culture assay (Fig. 6B). Increased degradation and release of extracellular matrix proteoglycan by addition of TNF- was totally blocked in this assay by 40 µM BIS, whereas calphostin C had no effect at concentrations up to 100 nM. Similar data were obtained in bovine and porcine chondrocytes treated with either IL-1 or TNF-, even at calphostin C concentrations well above the IC₅₀ for cPKC and nPKC (data not shown). Previous studies have shown that calphostin C is capable of inhibiting PKC activation in primary chondrocytes (18), and 100 nM calphostin C significantly inhibited proteoglycan synthesis induced by CCN2 in the chondrocytic cell line HCS-2/8 (60), proving that calphostin C is active on chondrocytes in culture. These data strongly implicate an atypical PKC as a necessary component of IL-1 and TNF signal transduction to NF-B in chondrocytes.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Proteoglycan degradation and NF-B activation mediated by either IL-1 or TNF is blocked by a PKC pseudosubstrate peptide inhibitor in a dose-dependent manner. A and B, primary bovine chondrocyte pellet cultures were pretreated with increasing concentrations of myristoylated PKC pseudosubstrate peptide inhibitor for 2 h prior to the addition of IL-1 or TNF to the culture. After 48 h, pellet culture conditioned media were harvested and assayed for proteoglycan release by DMMB assay (A) or for lactate levels (B). C, the myristoylated PKC pseudosubstrate peptide (Myr PKC PS, 40 µM) was compared with a nonmyristoylated version of the same peptide (Non-Myr PKC PS, 40 µM) and to a myristoylated peptide representing the pseudosubstrate sequence for PKC/ (Myr PKC/ PS, 40 µM) in the primary bovine chondrocyte pellet culture proteoglycan release assay. D, the T/C-28a2 cell line with NF-B-luciferase reporter stably integrated was pretreated with the same pseudosubstrate peptides as in C (50 µM each), compared with the NF-B inhibitor triptolide (50 nM). Luciferase levels were measured after 3 h of incubation following cytokine addition. Control cultures without inhibitor added were used to define 100% NF-B-luciferase activity. Bars represent the mean ± S.D. of replicate experiments and significant differences were determined by comparisons using the Student's t test.

View larger version (22K): [in this window] [in a new window]   FIGURE 6. TNF and IL-1 induction of NF-B and resulting proteoglycan degradation is blocked by pan-PKC inhibitors but not by an atypical sparing PKC inhibitor. A, the T/C-28a2 NF-B-luciferase reporter cell line was pretreated for 1 h with increasing concentrations of either Gö 6976, calphostin C (0.6 µM-5 µM); BIS (1.6 µM-12.5 µM); or triptolide (6.3 nM-50 nM) prior to addition of either IL-1 (10 ng/ml) or TNF- (25 ng/ml). All compounds were shown to by noncytotoxic on these cells in the concentration ranges used in these assays by WST-1 proliferation assays. Luciferase expression was measured by luciferase assays on cell lysates 3 h after addition of cytokines. Control cultures without inhibitor added were used to define 100% NF-B-luciferase activity. B, bovine pellet cultures received either no inhibitor (none) or were treated with BIS (40µM) or with increasing concentrations of calphostin C (25-100 nM) prior to addition of 100 ng/ml TNF-. Proteoglycan degradation was measured 18 h later using the DMMB assay on the culture conditioned media. Values represent the mean ± S.D. of three replicate experiments, and p values were derived from comparisons using the Student's t test.