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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Excess accumulation of extracellular inorganic pyrophosphate (ePPi) in aged human cartilage is crucial in calcium pyrophosphate dihydrate (CPPD) crystal formation in cartilage matrix. Two sources of ePPi are ePPi-generating ectoenzymes (NTPPPH) and extracellular transport of intracellular PPi by ANK. This study was undertaken to evaluate the role of NTPPPH and ANK in ePPi elaboration, by investigating expression of NTPPPH enzymes (cartilage intermediate-layer protein [CILP] and plasma cell membrane glycoprotein 1 [PC-1]) and ANK in human chondrocytes from osteoarthritic (OA) articular cartilage containing CPPD crystals and without crystals.

Methods

Chondrocytes were harvested from knee cartilage at the time of arthroplasty (OA with CPPD crystals [CPPD], n = 8; OA without crystals [OA], n = 10). Normal adult human chondrocytes (n = 1) were used as a control. Chondrocytes were cultured with transforming growth factor β1 (TGFβ1), which stimulates ePPi elaboration, and/or insulin-like growth factor 1 (IGF-1), which inhibits ePPi elaboration. NTPPPH and ePPi were measured in the media at 48 hours. Media CILP, PC-1, and ANK were determined by dot-immunoblot analysis. Chondrocyte messenger RNA (mRNA) was extracted for reverse transcriptase–polymerase chain reaction to study expression of mRNA for CILP, PC-1, and ANK. NTPPPH and ANK mRNA and protein were also studied in fresh frozen cartilage.

Results

Basal ePPi elaboration and NTPPPH activity in conditioned media from CPPD chondrocytes were elevated compared with normal chondrocytes, and tended to be higher compared with OA chondrocytes. Basal expression of mRNA for CILP (chondrocytes) and ANK (cartilage) was higher in both CPPD chondrocytes and CPPD cartilage extract than in OA or normal samples. PC-1 mRNA was less abundant in CPPD chondrocytes and cartilage extract than in OA chondrocytes and extract, although the difference was not significant. CILP, PC-1, and ANK protein levels were similar in CPPD, OA, and normal chondrocytes or cartilage extracts. Both CILP and ANK mRNA expression and ePPi elaboration were stimulated by TGFβ1 and inhibited by IGF-1 in chondrocytes from all sources.

Conclusion

CILP and ANK mRNA expression correlates with chondrocyte ePPi accumulation around CPPD and OA chondrocytes, and all respond similarly to growth factor stimulation. These findings suggest that up-regulated CILP and ANK expression contributes to higher ePPi accumulation from CPPD crystal–forming cartilage.

Calcium pyrophosphate dihydrate (CPPD) crystal deposition disease is caused by CPPD crystals formed in the extracellular matrix of articular cartilage (1). These crystals may cause acute attacks of pseudogout, but more importantly, CPPD deposits are intimately associated with and may cause osteoarthritis (OA) of weight-bearing joints (2). CPPD crystals initiate or amplify cartilage destruction by stimulating mitogenesis of synovial lining cells as well as synthesis and secretion of proteases, prostanoids, and cytokines that have been implicated in cartilage matrix degeneration (3). Prevention or reversal of CPPD deposition would be expected to have a salutary effect on the degree of degenerative joint disease associated with CPPD. However, current understanding of the mechanisms involved in CPPD crystal formation is insufficient to devise effective therapeutic or prophylactic interventions for the associated degenerative arthritis.

Local accumulation of excess extracellular inorganic pyrophosphate (ePPi), the anionic component of CPPD crystals, promotes CPPD crystal formation. Previous studies indicate that synovial fluid PPi concentrations are consistently elevated in patients with CPPD deposition (4–10). Articular cartilage chondrocytes uniquely elaborate large amounts of ePPi (11, 12). Although intracellular PPi (iPPi) is a by-product of many synthetic intracellular reactions (13), it does not diffuse across healthy biomembranes (14). To explain ePPi elaboration by chondrocytes, one must invoke de novo ePPi formation by ectoenzymes or a transport mechanism for iPPi to reach the matrix where CPPD crystals form.

The ecto-NTPPPHs hydrolyze extracellular nucleoside triphosphates into their monophosphate esters and ePPi (15, 16). These enzymes are enriched in plasma membranes (17). Several studies indicate that the bulk of ePPi involved in CPPD crystal formation in joints is derived from the hydrolysis of extracellular ATP by ecto-NTPPPH (9, 10, 15, 18, 19). Excess NTPPPH in synovial fluid correlates with the elevated synovial fluid ePPi levels reported in CPPD crystal deposition disease (8, 9). In cultured chondrocytes and articular cartilage, reduction of substrate (ATP) or ectoenzyme activity (by trypsinization) markedly reduces ePPi formation (19, 20).

Two chondrocyte cell membrane proteins may exhibit NTPPPH activity. One is cartilage intermediate-layer protein (CILP), which has recently been described as a human homolog of the putative porcine 127-kd NTPPPH (21, 22). CILP distribution is restricted to articular tissues including hyaline cartilage, fibrocartilage, tendon, ligament (23), and synovial membrane (24), which are also the only tissues that spontaneously elaborate ePPi in organ culture (11). Although CILP has not been proven to have intrinsic NTPPPH activity to date and there is a possibility that CILP may not be the enzyme itself, the original isolation of CILP was from NTPPPH-enriched fractions (25), and its expression correlates with ePPi generation by porcine chondrocytes in response to growth factors and with aging (26). CILP protein content in human OA articular cartilage increases with age (27). Increased CILP expression with aging was confirmed by immunohistochemistry and in situ hybridization studies (28), paralleling the increased prevalence of CPPD deposition disease with aging.

A definitive NTPPPH is plasma cell membrane glycoprotein 1 (PC-1), a member of the phosphodiesterase nucleotide pyrophosphatase family that includes PC-1, autotaxin, and B10 (29). PC-1 is expressed broadly, including on skin fibroblasts, osteoblasts, and chondrocytes (30). Overexpression of PC-1 increases iPPi, ePPi, and matrix vesicle PPi in several cell types (31, 32). Both PC-1 and CILP may be directly involved in chondrocyte ePPi formation or have a regulatory role in ePPi elaboration.

ANK was recently proposed to be an important factor in transport of iPPi across the cell membrane (33). The ANK protein is a multipass transmembrane protein that serves either as an anion channel or as a regulator of such a channel. ANK may control egress of iPPi across the cell membrane, or possibly egress of intracellular substrates for NTPPPH. Ho et al have reported that the ank gene mutation in mouse fibroblasts increases iPPi concentration and reduces ePPi concentration (33). Overexpression of wild-type ANK in mutant ank/ank mouse fibroblasts reversed the alterations in ePPi and iPPi levels, indicating an important role for ANK in regulating PPi trafficking. The effect of ANK was blocked by probenecid, a general inhibitor of organic anion transport previously reported to decrease ePPi elaboration by articular chondrocytes (34).

Chondrocyte ePPi elaboration is a bioregulable process, responsive to growth factors and some cytokines. Transforming growth factor β1 (TGFβ1) is the major growth factor that elevates ePPi production by normal chondrocytes (35). Aged chondrocytes are much more responsive to TGFβ1 than are chondrocytes from young animals (26, 36). Insulin-like growth factor 1 (IGF-1) is a negative modulator of ePPi elaboration by articular chondrocytes (37).

The purpose of this study was to evaluate perturbations of chondrocyte NTPPPH and ANK expression and of ePPi accumulation in chondrocytes from diseased human cartilage. First we measured ePPi elaboration into conditioned media from CPPD, OA, and normal chondrocytes. Subsequently, we studied CILP, PC-1, and ANK expression in each group, and investigated the response to growth factors in these diseased chondrocytes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Materials.

Recombinant human TGFβ1 and IGF-1 were obtained from Austral Biologicals (San Ramon, CA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), bovine serum albumin, and penicillin/streptomycin/Fungizone (PSF) were from Life Technologies (Grand Island, NY). Chemical reagents, unless otherwise specified, were from Sigma (St. Louis, MO).

Preparation of human cartilage.

Human cartilage specimens were harvested from spent tissues removed from knees at the time of arthroplasty for arthritis. Joint fluids obtained at the time of surgery were examined under polarizing light, and the results were used to divide individual specimens into 2 groups: OA with CPPD crystal deposition disease (CPPD group) (mean ± SD age 68.4 ± 12.5 years; n = 8) and OA without CPPD crystals (OA group) (age 65.9 ± 10.7 years; n = 10). One portion of articular cartilage was used for chondrocyte isolation and culture. The remainder was frozen for later isolation of messenger RNA (mRNA) and protein. Control specimens of frozen cartilage from healthy subjects were obtained from the Musculoskeletal Transplant Foundation (age 25.6 ± 7.7 years; n = 8).

Isolation and culture of chondrocytes.

Chondrocytes were released from the harvested cartilage by a sequential enzyme digestion method described previously (38). Normal adult human chondrocytes (from 1 50-year-old individual) were purchased from Clonetics (Walkersville, MD). Cells were plated at high-density monolayer and used only in second-passage culture to preserve chondrocyte phenotype (39). The expression of type II collagen and aggrecan in these chondrocytes was confirmed by reverse transcriptase–polymerase chain reaction (RT-PCR) using gene-specific primer pairs (40, 41). Cells were cultured to confluence in DMEM supplemented with 10% FBS and 1% PSF. The day before initiation of experiments, growth medium was removed and replaced with serum-depleted medium (DMEM only).

Addition of growth factors.

Culture medium was changed to DMEM, 25 mM HEPES (pH 7.4), 0.5% heat-inactivated FBS, and 1% PSF. TGFβ1 (10 ng/ml) and/or IGF-1 (20 ng/ml) were added to some cultures. Growth factors at these concentrations have maximal effects on ePPi elaboration (37). Aliquots of ambient media and cells were harvested at 48 hours.

Biochemistry assay.

The PPi assay was performed on conditioned media by the 14C-labeled uridine diphosphoglucose method as previously described (42). NTPPPH enzyme activity was determined using a colorimetric method as previously described (9). PPi and NTPPPH values were normalized to protein content of cell lysate, as determined by the Lowry method (43).

RT-PCR.

Total RNA from cultured chondrocytes was isolated, after a 24-hour incubation with or without growth factors, by the acidified guanidinium isothiocyanate method, using TRIzol reagent (Life Technologies). To isolate total RNA from native cartilage, frozen cartilage was ground into powder with liquid nitrogen, homogenized mechanically, and then RNA extracted with TRIzol. Samples of total RNA (0.5 μg for chondrocytes and 0.2 μg for cartilage extract) were reverse transcribed into complementary DNA (cDNA) in the presence of 50 pM oligo(dT)12-18 primer (Life Technologies), 10 units of ribonuclease inhibitor (Amersham, Piscataway, NJ), 10× RT buffer, dNTP mix (5 mM each dNTP), and reverse transcriptase (Omniscript RT kit, Qiagen, Valencia, CA), for 60 minutes at 37°C.

Amplification of generated cDNA was performed in a GeneAmp PCR System 2400 thermocycler (Perkin-Elmer Biosystems, Foster City, CA) using Platinum PCR Supermix (Life Technologies). The gene-specific primer pairs were as follows: CILP (21, 22) sense 5′-GGAATCCGAGATGTGAGGAG-3′, antisense 5′-AATCAGTAGCATGGGACAAAG-3′; PC-1 (44) sense 5′-CACAAGAAACCCCAGAGATAAC-3′, antisense 5′-CCACTGACGACATTGACAC-3′; ANK (45) sense 5′-TGGGATGTGCCTCAATCTCA-3′, antisense 5′-CACAGAGTTCTGCAAAGGCAA-3′; GAPDH (46) sense 5′-GGTGAAGGTCGGAGTCAACG-3′, antisense 5′-CAAAGTTGTCATGGATGACC-3′. Cycling parameters were as follows: for CILP, 32 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds; for PC-1, 28 cycles of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds; for ANK, 32 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds; for GAPDH, 25 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. An additional elongation step at 72°C for 7 minutes was included. The number of cycles was selected in the exponential phase of the amplification curve (data not shown).

Transcripts were analyzed by 1.2% agarose/Tris–acetate–EDTA gel electrophoresis and visualized with SYBR Green-I (Sigma) using image scanner Storm (Molecular Dynamics, Sunnyvale, CA). The expected transcript sizes were 760 bp for CILP, 540 bp for PC-1, 600 bp for ANK, and 496 bp for GAPDH. Each PCR product was isolated from the agarose gel and the sequence confirmed by dideoxy chain-termination sequencing (47) after subcloning of the PCR product into Topo TA cloning vector (Invitrogen, Carlsbad, CA). The relative intensities of bands of interest were analyzed with scan analysis software (Image Quant version 5.0; Molecular Dynamics). Results were expressed as the ratio of band intensities relative to that of the GAPDH mRNA band. Response to growth factors was expressed as the percentage change compared with control cultures grown in the absence of growth factors.

Dot-immunoblot analysis.

Equal amounts of conditioned media containing 1 μg cell proteins in Tris buffered saline (20 mM Tris HCl [pH 8.0], 150 mM NaCl) were absorbed onto nitrocellulose membrane using a dot-blot apparatus (Bio-Rad, Hercules, CA). Dot-blotting for immunodetection of CILP, PC-1, and ANK, using specific antibodies to each, was performed as previously described (24). To detect CILP, we used affinity-purified antisynthetic N-terminal peptide from purified soluble 61-kd NTPPPH, which contains the carboxyl domain of CILP (24). For detection of products of PC-1, we used a purified rabbit polyclonal antibody to the carboxyl domain of human PC-1 (32) (provided by Dr. R. Terkeltaub, Veterans Administration Medical Center, San Diego, CA). To detect ANK, we used a previously characterized rabbit antiserum (Ab3) to mouse ANK (33) (provided by Dr. D. Kingsley, Stanford University School of Medicine, Stanford, CA). The primary antibody was used at a 1:5,000 dilution (final concentration of IgG ∼1 μg/ml); secondary antibody (horse anti-rabbit IgG/horseradish peroxidase conjugate [Pierce, Rockford, IL]) was used at 1:40,000 (final concentration 0.25 ng/ml). An enhanced chemiluminescence kit (SuperSignal West Pico kit; Pierce) was used to visualize immunoreactive protein.

We also studied CILP, PC-1, and ANK expression in frozen human cartilage extracts. Cartilage protein was obtained after isolation of RNA according to the TRIzol protocol, and dot-immunoblot analysis was then performed. After immunodetection of each protein, nitrocellulose membranes were stained using India ink to normalize for the amount of loaded protein. Results were expressed as the ratio of immunoband intensities relative to that of protein stained with India ink.

Statistical analysis.

The nonparametric Kruskal-Wallis test with Fisher's protected least significance difference (PLSD) was applied to determine the differences between disease groups. Differences among cells from explants treated with various growth factors were evaluated by one-way analysis of variance followed by Fisher's PLSD for multiple comparisons. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Excess elaboration of ePPi and increased expression of NTPPPH activity by CPPD chondrocytes.

Basal ePPi elaboration and NTPPPH activity in conditioned media from CPPD chondrocytes were elevated compared with values in media from normal chondrocytes (P < 0.05 and P < 0.01, respectively) (Table 1 and Figures 1 and 2). OA chondrocytes also elaborated more ePPi and showed higher NTPPPH activity in the media than did normal chondrocytes (P = 0.2 [not significant] and P < 0.05, respectively). NTPPPH and ePPi values for OA chondrocytes were lower than those for CPPD chondrocytes, but the differences were not significant.

Table 1. Biochemical analysis in cultures of CPPD, OA, and normal human chondrocytes*
 PPiNTPPPHCell protein
CPPDOANormalCPPDOANormalCPPDOANormal
  • *

    For inorganic pyrophosphate (PPi), gross data and cell protein–corrected data are μM and μmoles/mg protein, respectively. For NTPPPH, gross data and cell protein–corrected data are pmoles/hour and pmoles/hour/mg protein, respectively. For cell protein, data are mg/ml. Values are the mean ± SD. CPPD = calcium pyrophosphate dihydrate; TGFβ1 = transforming growth factor β1; IGF-1 = insulin-like growth factor 1.

  • P < 0.05 versus normal chondrocytes, by Kruskal-Wallis test with Fisher's protected least significance difference (PLSD).

  • P < 0.05 versus osteoarthritis (OA) chondrocytes, by Kruskal-Wallis test with Fisher's PLSD.

  • §

    P < 0.05 versus control group (with no growth factor), by one-way analysis of variance followed by Fisher's PLSD.

Gross data, addition         
 Control (none)52 ± 3637 ± 269 ± 1195 ± 89153 ± 5924 ± 394 ± 5492 ± 4729 ± 6
 TGFβ1115 ± 71§96 ± 71§13 ± 1§248 ± 118196 ± 66§34 ± 3§122 ± 67113 ± 5335 ± 7
 IGF-141 ± 3434 ± 257 ± 0219 ± 119172 ± 6324 ± 2115 ± 63103 ± 5034 ± 6
 TGF + IGF-196 ± 60§79 ± 61§12 ± 1§279 ± 140201 ± 78§39 ± 4§130 ± 75118 ± 6539 ± 8
Data corrected for cell protein, addition         
 Control (none)0.6 ± 0.40.5 ± 0.30.3 ± 0.12.5 ± 1.42.1 ± 1.20.8 ± 0.1   
 TGFβ11.0 ± 0.5§1.0 ± 0.5§0.4 ± 0.1§2.4 ± 1.12.3 ± 1.21.0 ± 0.2§   
 IGF-10.5 ± 0.40.4 ± 0.40.2 ± 0.0§2.3 ± 1.32.1 ± 1.20.7 ± 0.1   
 TGF + IGF-10.9 ± 0.40.8 ± 0.5§0.3 ± 0.02.7 ± 1.32.5 ± 1.50.9 ± 0.1   
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Figure 1. Basal levels of extracellular inorganic pyrophosphate (PPi) elaboration into media after 48 hours, in cultures of calcium pyrophosphate dihydrate (CPPD), osteoarthritis (OA), and normal human chondrocytes. PPi values were normalized to cell protein. Individual experiments were performed in triplicate on each human sample. Values are the mean and SEM. N.S. = not significant.

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Figure 2. Basal levels of NTPPPH enzyme activity in media after 48 hours, in cultures of CPPD, OA, and normal human chondrocytes. Individual experiments were performed in triplicate on each human sample. Values are the mean and SEM. See Figure 1 for definitions.

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Differential CILP and ANK expression in CPPD chondrocytes versus OA chondrocytes.

The basal level of CILP mRNA expression in CPPD chondrocyte monolayer cultures was significantly higher than in OA and normal chondrocyte cultures (P < 0.01) (Figure 3). OA chondrocytes expressed higher levels of CILP mRNA than did normal chondrocytes, although the difference was not significant. In contrast to CILP expression, PC-1 mRNA expression was similar among all 3 groups. ANK mRNA expression paralleled CILP expression, being highest in the CPPD group, but without significant differences. Similarly, in cartilage extracts, CILP mRNA expression and ANK mRNA expression were higher in CPPD cartilage than in OA or normal cartilage, with significant differences for ANK (Figure 4). PC-1 mRNA expression in CPPD and OA cartilage extracts tended to be higher than in normal cartilage. However, PC-1 mRNA expression was similar in CPPD and OA cartilage extracts.

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Figure 3. Basal expression of mRNA for cartilage intermediate-layer protein (CILP), plasma cell membrane glycoprotein 1 (PC-1), and ANK in calcium pyrophosphate dihydrate (CPPD), osteoarthritis (OA), and normal human chondrocytes. A, Isolated RNA (total 0.5 μg) from each sample after 24-hour incubation was prepared, reverse transcribed, and studied by reverse transcriptase–polymerase chain reaction (RT-PCR). The quality and quantity of reverse-transcribed cDNA were assessed using primer pairs for CILP, PC-1, ANK, and the housekeeping gene GAPDH. B, Densitometric analysis of CILP, PC-1, and ANK expression by RT-PCR, with data normalized to GAPDH expression. Values are the mean and SEM. N.S. = not significant.

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Figure 4. Basal expression of mRNA for CILP, PC-1, and ANK in CPPD, OA, and normal human cartilage extracts. A, Frozen cartilage was homogenized and then RNA extracted by the acidified guanidinium isothiocyanate method. Isolated RNA (total 0.2 μg) from each frozen sample was prepared, reverse transcribed, and studied by RT-PCR. B, Densitometric analysis of CILP, PC-1, and ANK expression by RT-PCR, with data normalized to GAPDH expression. Values are the mean and SEM. See Figure 3 for definitions.

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Immunoreactive CILP, PC-1, and ANK protein levels were similar among CPPD, OA, and normal chondrocyte cultures (Figure 5). Extracts from CPPD, OA, and normal cartilage did not differ significantly with respect to CILP, PC-1, or ANK protein content (Figure 6).

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Figure 5. Basal expression of CILP, PC-1, and ANK protein in conditioned media after 48 hours, in cultures of CPPD, OA, and normal human chondrocytes. A, Equal amounts of conditioned media (1 μg proteins) were absorbed onto nitrocellulose membrane, and dot-blotting for immunodetection of CILP, PC-1, and ANK was performed using specific antibodies. B, Densitometric analysis of CILP, PC-1, and ANK expression by dot-blot analysis, with data normalized to protein stained with India ink. Values are the mean and SEM. See Figure 3 for definitions.

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Figure 6. Basal expression of CILP, PC-1, and ANK protein in CPPD, OA, and normal human cartilage extracts. A, Protein (1 μg of each sample) was absorbed onto nitrocellulose membrane, and dot blotting for immunodetection of CILP, PC-1, and ANK was performed. B, Densitometric analysis of CILP, PC-1, and ANK expression by dot-blot analysis, with data normalized to protein stained with India ink. Values are the mean and SEM. See Figure 3 for definitions.

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Response to growth factors.

As previously shown in normal porcine chondrocytes (26), ePPi elaboration and NTPPPH activity in conditioned media from normal human chondrocytes were increased by TGFβ1 (P < 0.01) and inhibited by IGF-1 (for ePPi, P < 0.01) after 48 hours of treatment (Table 1 and Figures 7 and 8). IGF-1 inhibited the stimulating effect of TGFβ1 on ePPi elaboration into conditioned media in normal chondrocytes, but not in CPPD or OA chondrocytes. Elaboration of ePPi by CPPD and OA chondrocytes also was increased by TGFβ1 (P < 0.01) and tended to be decreased by IGF-1; however, NTPPPH enzyme activity varied little in response to these growth factors. With TGFβ1 treatment, media ePPi and NTPPPH activities were significantly higher in OA and CPPD chondrocyte cultures than in cultures of normal chondrocytes. There were no significant differences between CPPD and OA groups with respect to ePPi or NTPPPH activity with TGFβ1 treatment (Table 1).

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Figure 7. Effect of transforming growth factor β1 (TGFβ1) and/or insulin-like growth factor 1 (IGF-1) on extracellular PPi elaboration by CPPD, OA, and normal human chondrocytes. CPPD, OA, and normal chondrocytes were incubated for 48 hours with 10 ng/ml TGFβ1, 20 ng/ml IGF-1, or both TGFβ1 and IGF-1. Individual experiments were performed in triplicate on each human sample. Values are the mean ± SEM. ∗ = P < 0.05 versus control (without TGFβ1 or IGF-1 treatment). See Figure 3 for other definitions.

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Figure 8. Effect of transforming growth factor β1 (TGFβ1) and/or insulin-like growth factor 1 (IGF-1) on NTPPPH activity. CPPD, OA, and normal chondrocytes were incubated for 48 hours with 10 ng/ml TGFβ1, 20 ng/ml IGF-1, or both TGFβ1 and IGF-1. Individual experiments were performed in triplicate on each human sample. Values are the mean ± SEM. ∗ = P < 0.05 versus control (without TGFβ1 or IGF-1 treatment). See Figure 3 for other definitions.

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CILP mRNA expression, PC-1 mRNA expression, and ANK mRNA expression were all stimulated by TGFβ1 in CPPD, OA, and normal chondrocytes (Figure 9). ANK mRNA expression in response to growth factors was similar to CILP expression, but the ANK mRNA response to TGFβ1 was much more vigorous in OA chondrocytes than in normal or CPPD chondrocytes. In contrast, PC-1 mRNA expression in CPPD, OA, and normal chondrocytes responded little to growth factors, with the sole exception of down-regulated PC-1 mRNA expression in normal chondrocytes treated with IGF-1. Immunoreactive CILP, PC-1, and ANK in both conditioned media and cell lysates varied much less than did mRNA expression in response to growth factors in these short-term cultures (Figure 10).

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Figure 9. Effect of transforming growth factor β1 (TGFβ1) and/or insulin-like growth factor 1 (IGF-1) on expression of mRNA for CILP, PC-1, and ANK in CPPD, OA, and normal chondrocytes. A, Representative results with adult and young chondrocytes. Total RNA was extracted from chondrocytes incubated for 24 hours in the presence of media without additive (Control), with 10 ng/ml TGFβ1, with 20 ng/ml IGF-1, or with both TGFβ1 and IGF-1. Isolated RNA was prepared, reverse transcribed, and studied by RT-PCR. The quality and quantity of reverse-transcribed cDNA were assessed using primer pairs for CILP, PC-1, ANK, and the housekeeping gene GAPDH. B, Densitometric analysis of CILP, PC-1, and ANK expression by RT-PCR, with data normalized to GAPDH expression. Results shown are representative of separate experiments with chondrocytes from 8 subjects with CPPD, 10 subjects with OA, and 1 normal subject. Values are the mean ± SEM. ∗ = P < 0.05 versus control (without TGFβ1 or IGF-1 treatment). See Figure 3 for other definitions.

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Figure 10. Effect of transforming growth factor β1 (TGFβ1) and/or insulin-like growth factor 1 (IGF-1) on expression of CILP, PC-1, and ANK protein in CPPD, OA, and normal chondrocytes. A, CPPD, OA, and normal chondrocytes were incubated for 48 hours with 10 ng/ml TGFβ1, 20 ng/ml IGF-1, or both TGFβ1 and IGF-1. Equal amounts of conditioned media were absorbed onto nitrocellulose membrane, and dot-blotting for immunodetection of CILP, PC-1, and ANK was performed using specific antibodies. B, Densitometric analysis of CILP, PC-1, and ANK expression by dot-blot analysis, with data normalized to protein stained with India ink. Values are the mean ± SEM. See Figure 3 for other definitions.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Chondrocytes from CPPD-containing cartilage elaborate more ePPi than do chondrocytes from OA cartilage without crystals, or normal chondrocytes. This is the first reported study comparing ePPi production by human chondrocytes from degenerative hyaline cartilage with and without CPPD crystals. Since CPPD crystals form extracellularly and since excess ePPi accumulates locally in synovial fluid in CPPD crystal deposition disease (4, 6), we measured ePPi levels in relation to CPPD crystal deposition. Previous studies of iPPi levels in chondrocytes (48) and skin fibroblasts (48, 49) and ePPi levels in synovial fluids (6, 50, 51) have shown higher values in samples from patients with CPPD crystal deposition than from normal controls. In addition, studies comparing CPPD and OA groups indicate that levels of iPPi in chondrocytes (48) and skin fibroblasts (48, 49) and ePPi in synovial fluids (4–6, 8, 9) were higher in patients with CPPD deposition disease than in OA patients. In some studies, the elevation of ePPi levels in synovial fluids from patients with CPPD compared with OA did not reach statistical significance (7, 10). Consistent with the latter reports, ePPi elaboration by CPPD chondrocytes was higher than that by OA chondrocytes in the present study, but the difference did not reach statistical significance.

The activity of the ePPi-generating enzyme NTPPPH is elevated in conditioned media surrounding CPPD chondrocytes. NTPPPH activity in cartilage extracts (52, 53), in synovial fluids (50, 51), and in cultures of skin fibroblasts (49) from CPPD patients is higher than that in similar preparations from normal controls. Despite the fact that our studies were performed in vitro, our findings regarding NTPPPH activity in conditioned media are consistent with those of these previous studies done mostly in vivo. In addition, studies have generally shown significantly higher NTPPPH activity in CPPD cartilage extracts (52, 53), in CPPD synovial fluids (8, 9), and in cultures of CPPD skin fibroblasts (49) compared with similar preparations from OA patients. In other studies, NTPPPH activity in cartilage extracts (17) and in synovial fluids (10, 17) from CPPD patients was elevated, but values were not significantly higher than in OA samples. In the present study, NTPPPH activity in the media from CPPD chondrocytes was higher than that from OA chondrocytes, but the difference was not significant.

Our results demonstrate that chondrocytes from diseased tissues are subject to many of the regulatory factors that affect ePPi production by normal chondrocytes. In tissue and organ cultures, ePPi accumulation is regulated by growth factors, cytokines, and other bioactive mediators. In particular, TGFβ1 enhances ePPi elaboration by porcine articular chondrocytes (35). In this study, TGFβ1 enhanced, and IGF-1 inhibited, ePPi elaboration by normal human chondrocytes, consistent with the results observed with porcine chondrocytes (26, 35). Similar responses of ePPi levels to growth factors were observed in CPPD and OA chondrocytes. These findings confirm that growth factors, especially TGFβ1, regulate ePPi levels in diseased human chondrocytes. Furthermore, TGFβ1-induced ePPi accumulation in both CPPD and OA chondrocyte cultures was higher than that with normal chondrocytes. TGFβ1 and IGF-1 are present in higher concentrations in OA and CPPD fluids than in normal fluids (54–57). Taken together, these findings indicate that higher sensitivity and higher levels of growth factors in chondrocytes affected with degenerative disease than from normal chondrocytes might cause increased generation of ePPi.

Traditionally, steady-state levels of individual RNA transcripts have been measured by Northern blot and ribonuclease protection assays. RT-PCR has revolutionized the analysis of RNA and has proven to be a very useful method for quantifying even a few molecules of mRNA in tissue samples, although it is considered a semiquantitative technique because of amplification of the RNA amounts. We assessed the quality and quantity of reverse-transcribed cDNA, using primer pairs for CILP, PC-1, ANK, and GAPDH. Our previous study of CILP, PC-1, and GAPDH expression in porcine chondrocytes showed that the results of RT-PCR correlated well with those of Northern blot analysis (26). With regard to NTPPPH expression, we confirmed that CILP mRNA expression paralleled ePPi elaboration in all subsets of chondrocytes and in response to growth factors. These findings are consistent with those of our previous study using porcine chondrocytes (26) and support the notion that CILP might promote CPPD crystal formation by enhancing ePPi production.

In contrast, PC-1 mRNA expression changed little, regardless of the source of chondrocytes or stimulation with growth factors. PC-1 is the predominant NTPPPH involved in mineralization by osteoblasts (32), and PC-1–deficient mice (ttw/ttw) develop hyperossification in early life, which produces progressive ankylosis of peripheral joints (58). Moreover, PC-1 deficiency has been identified recently in a male human infant with idiopathic infantile arterial calcification, in which calcification (hydroxyapatite deposition) in the media of large muscular arteries and smooth muscle cell proliferation occur, associated with periarticular calcification (59). These observations support the contention that PC-1 plays an important role in repressing apatite mineral formation. Increased expression of PC-1 was recently reported as a potential pathogenic factor in knee meniscal fibrocartilage matrix calcification in chondrocalcinosis (60).

Positive CILP immunostaining in both CPPD crystal aggregates and hypertrophic/metaplastic chondrocytes of meniscus from CPPD patients has also been reported (61). Moreover, in our preliminary immunohistochemistry and in situ hybridization studies using hyaline cartilage, CILP expression was strong at the periphery of CPPD crystal deposits (Yamakawa K, et al: unpublished data). These observations and the current report suggest that CILP may be a crucial factor in CPPD crystal formation not only in meniscal fibrocartilage, but also in articular hyaline cartilage.

ANK is another important factor that regulates ePPi levels. ANK is a putative transporter for egress of iPPi from cells. It is reportedly up-regulated in degenerative joint disease (62). Our results confirm that ANK mRNA expression is higher in chondrocytes and cartilage extract from CPPD and OA patients than in normal chondrocytes and cartilage extract. Moreover, chondrocytes and extract from cartilage with both OA and CPPD crystal deposition express higher levels of ANK than do similar samples with OA but no CPPD crystals. In response to growth factors, ANK mRNA expression was increased by TGFβ1, which parallels the effects of TGFβ1 on ePPi accumulation and CILP expression. These findings support the concepts that ANK participates in controlling ePPi elaboration and that up-regulated ANK mRNA expression promotes CPPD crystal formation. Recent reports on mutations of the ANK gene in British (63) and French (64) kindreds of patients with CPPD deposition disease also support the notion of a causative role for ANK in CPPD crystal deposition. No significant link was found between human CILP gene mutation and some familial forms of CPPD deposition disease (65).

Recently, Johnson et al reported that ANK and PC-1 cooperatively regulate both iPPi and ePPi (62). In our current study, ANK expression was closely associated with CILP expression. Coordinated dysregulation of ANK, PC-1, and CILP may result in the increased ePPi levels that promote CPPD crystal formation in cartilage.

There is a discrepancy between elevated ePPi levels and unaltered NTPPPH activities in response to growth factors. Unlike the case in normal chondrocytes, the NTPPPH activity in CPPD and OA chondrocytes did not increase in response to growth factors. This lack of response is consistent with stable mRNA and protein expression of PC-1. The NTPPPH activity in chondrocytes is mostly accounted for by PC-1 (>50%) (66), in contrast to CILP (∼5%) (23). In CPPD and OA cultures and extracts, CILP and ANK protein expression did not differ, nor did they respond to growth factors, although mRNA levels did change. The discrepancy between the mRNA and protein expression may be caused by a regulatory mechanism acting at a posttranscriptional level (alternative splicing, repress translation, etc.), feedback destabilization, or regional differences in the translation rate of the transcript (67, 68). It is also possible that this CILP is so stable that short-term alterations in protein synthesis may not be detectable.

In conclusion, we report that chondrocytes from CPPD patients generate more ePPi and NTPPPH enzyme activity than those from degenerative cartilage without CPPD crystals or from normal chondrocytes. CILP and ANK mRNA expression is also up-regulated in chondrocytes and cartilage extracts from patients with CPPD compared with those from OA patients or from normal cartilage. Furthermore, CILP and ANK mRNA expression and ePPi elaboration are concomitantly stimulated by TGFβ1 and inhibited by IGF-1 in chondrocytes from normal, OA, or CPPD sources. These findings suggest that increased CILP and ANK expression participate in excess accumulation of ePPi and promote CPPD crystal formation in articular cartilage.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Dr. Jeffrey J. Butler (St. Luke's Hospital, Milwaukee, WI) and Dr. James T. Ninomiya (Medical College of Wisconsin) for supplying human cartilage. We also thank Dr. David M. Kingsley (Stanford University School of Medicine, Stanford, CA) for helpful discussion.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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