QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.134650v1 325/1/56    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Megías, J. Articles by Alcaraz, M. J. Search for Related Content PubMed PubMed Citation Articles by Megías, J. Articles by Alcaraz, M. J.

INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

The Carbon Monoxide-Releasing Molecule Tricarbonyldichlororuthenium(II) Dimer Protects Human Osteoarthritic Chondrocytes and Cartilage from the Catabolic Actions of Interleukin-1β

Department of Pharmacology, Faculty of Pharmacy, University of Valencia, Burjasot, Valencia, Spain (J.M., M.I.G., M.J.A.); Department of Chemistry, Biochemistry, and Molecular Biology, Cardenal Herrera-CEU University, Moncada, Valencia, Spain (M.I.G.); Department of Orthopaedic Surgery and Traumatology, General Hospital, Valencia, Spain (A.B.); and Department of Surgery, Faculty of Medicine, University of Valencia, Spain (F.G.)

Received November 23, 2007; accepted January 11, 2008.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
We have investigated the effects of a carbon monoxide-releasing molecule, tricarbonyldichlororuthenium(II) dimer (CORM-2), on catabolic processes in human osteoarthritis (OA) cartilage and chondrocytes activated with interleukin-1β. In these cells, proinflammatory cytokines induce the synthesis of matrix metalloproteinases (MMPs) and aggrecanases, including members of a disintegrin and metalloproteinase with thrombospondin domain (ADAMTS) family, which may contribute to cartilage loss. CORM-2 down-regulated MMP-1, MMP-3, MMP-10, MMP-13, and ADAMTS-5 in OA chondrocytes, and it inhibited cartilage degradation. These effects were accompanied by increased aggrecan synthesis and collagen II expression in chondrocytes. Our results also indicate that the inhibition of extracellular signal-regulated kinase 1/2 and p38 activation by CORM-2 may contribute to the maintenance of extracellular matrix homeostasis. These observations suggest that CORM-2 could exert chondroprotective effects due to the inhibition of catabolic activities and the enhancement of aggrecan synthesis.

Interleukin (IL)-1β and other proinflammatory cytokines have been detected in OA synovial fluid and chondrocytes. These mediators have been shown to induce MMP and aggrecanase synthesis in chondrocytes in an autocrine/paracrine manner, which may contribute to cartilage loss in OA (Elson et al., 1998; Tetlow et al., 2001). IL-1β is involved in collagenase-mediated cleavage of collagen II, degradation of aggrecan, and the inhibition of gene expression of matrix molecules (Kobayashi et al., 2005). It has also been demonstrated that this cytokine reduces the production of cartilage matrix components such as aggrecan (Gouze et al., 2001) and type II collagen (Goldring et al., 1988).

Carbon monoxide-releasing molecules (CO-RMs) are a new group of drugs able to reproduce the biological actions of CO derived from heme oxygenase-1 (HO-1) activity (for review, see Foresti et al., 2005). Therefore, the vasoactive (Foresti et al., 2004) and cardioprotective (Clark et al., 2003) effects of CO-RMs have been demonstrated. These agents are also able to deliver CO and protect isolated kidneys against cold preservation and ischemia-reperfusion (Sandouka et al., 2006). It is interesting to note that CO-RMs have shown anti-inflammatory effects in some cell lines, including RAW 264.7 macrophages (Sawle et al., 2005), microglia (Bani-Hani et al., 2006), and Caco-2 (Megías et al., 2007). In addition, these compounds can modulate leukocyte-endothelial cell interactions (Urquhart et al., 2007). However, studies on the regulation of chondrocyte or cartilage metabolism by CO-RMs have not yet been reported. Previous observations from our laboratory suggest a beneficial role for HO-1 in OA chondrocytes (Guillen et al., 2007). We postulate that CO-RMs may exert protective effects on these cells. To test this hypothesis, we have investigated whether the CO-RM tricarbonyldichlororuthenium(II) dimer (CORM-2) may modulate catabolic processes in human OA cartilage and chondrocytes activated with IL-1β.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Reagents. IL-1β was from PeproTech EC Ltd. (London, UK). Antibodies against phosphorylated or total extracellular signal-regulated kinase (ERK)1/2, c-Jun NH₂-terminal kinase (JNK), and p38 were from R&D Systems (Minneapolis, MN). The peroxidase-conjugated IgGs were purchased from Dako Denmark A/S (Copenhagen, Denmark). CORM-2, RuCl₃, and other reagents were from Sigma-Aldrich (St. Louis, MO).

Chondrocyte and Explant Culture. Cartilage specimens were obtained from patients with the diagnosis of advanced OA [16 females and 8 males, 72 ± 3 (mean ± S.E.M.) years old] undergoing total knee joint replacement. Diagnosis was based on clinical, laboratory, and radiological evaluation. Samples were obtained under the Institutional Ethical Committee-approved protocol. Cartilage slices were removed from the femoral condyles and tibial plateaus, and they were cut into small pieces. Chondrocytes were isolated by sequential enzymatic digestion: 1 h with 0.1 mg/ml hyaluronidase (Sigma-Aldrich) followed by 12 h with 1 mg/ml collagenase (type IA) (Sigma-Aldrich) at 37°C in DMEM/Ham's F-12 medium (Sigma-Aldrich) containing 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% CO₂ atmosphere. The digested tissue was filtered through a 70-µm nylon mesh, and then it was washed and centrifuged. Cell viability was greater than 95% according to the trypan blue exclusion test. The isolated chondrocytes were seeded at 2.5 x 10⁵ cells/well in six-well plates. Cells were cultured in DMEM/Ham's F-12 medium supplemented with 10% human serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO₂ incubator at 37°C. Chondrocytes in primary culture were allowed to grow until nearly confluence, and then they were incubated with CORM-2 at different concentrations or vehicle for 1 h before stimulation with 100 U/ml IL-1β for different times. CORM-2 was dissolved in dimethyl sulfoxide, and then it was diluted in culture medium [0.1% (v/v)]. Control cells were treated with the same vehicle. Possible cytotoxicity of treatments was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) to formazan. After appropriate stimulation, cells were incubated with 200 µg/ml MTT for 2 h. The medium was then removed, and the cells solubilized in 100 µl of dimethyl sulfoxide to quantitate formazan at 550 nm (Gross and Levi, 1992). For explant cultures, full-thickness pieces of cartilage were removed from the femoral condyles. Slices measuring 2 mm in width x 2 mm in length were dissected from the tissue. Explants were transferred to 24-well plates (10 explants/well) containing DMEM/Ham's F-12 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum, and they were incubated in a humidified 5% CO₂ incubator at 37°C for 2 days before experiments to allow them to stabilize.

Glycosaminoglycan Degradation. Cartilage explants in DMEM/Ham's F-12 medium + 10% fetal bovine serum were labeled with 4 µCi/ml [³⁵S]sulfate for 6 days. The unincorporated radioactivity was removed by extensive washing for 2 days with DMEM/Ham's F-12 medium + 10% fetal bovine serum. Explants were incubated in fresh medium with 100 U/ml IL-1β or IL-1β + CORM-2 for 6 days, with renewal of medium and treatment every other day. The media were collected, and explants were digested with 2 mg/ml papain in 1 mM EDTA, 0.25 mg/ml dithiothreitol, and 20 mM sodium phosphate, pH 6.8, at 56°C for 16 h. Sephadex G25 (GE Healthcare Life Sciences, Barcelona, Spain) chromatography was used to remove unincorporated [³⁵S]sulfate from medium and tissue digests. Radioactivity was measured by liquid scintillation counting. Degradation was expressed as percentage of released radioactivity with respect to total radioactivity.

Proteoglycan Synthesis. Proteoglycan synthesis was quantified by monitoring [³⁵S]sulfate incorporation (Moulharat et al., 2004). After chondrocyte stimulation with 100 U/ml IL-1β or IL-1β + CORM-2 for 24 h, cells were labeled with 2 µCi/ml [³⁵S]sulfate for 24 h. Cells were washed with Hanks' balanced salt solution, and then they were extracted with 4 M guanidinium HCl, 5 mM EDTA, and 5 mM Na acetate, pH 7.2, for 48 h at 4°C. Proteoglycans absorbed on Whatman filter paper (Whatman, Maidstone, UK) were precipitated by cetylpyridinium chloride monohydrate, and radioactivity was measured by liquid scintillation counting. Total radioactivity (medium + cell) was calculated for each well, and the value was normalized with respect to protein content.

Immunocytochemistry. Chondrocytes in primary culture were allowed to grow until near confluence, and then they were incubated with 100 µM CORM-2 in the presence or absence of 100 U/ml IL-1β for 15 days, with renewal of medium and treatment every 4 days. Cells were fixed with 4% formaldehyde in phosphate-buffered saline for 30 min at 4°C, and collagen II was detected using the type II collagen staining kit (MD Biosciences, Zürich, Switzerland), following the manufacturer's instructions.

Western Blot Analysis. After 15-min stimulation with 100 U/ml IL-1β or IL-1β + 100 µM CORM-2, chondrocytes in primary culture were lysed in 100 µl of buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl, and 25 mM Tris, pH 7.4), and then they were centrifuged at 4°C for 10 min at 10,000g. Proteins (25 µg) in supernatants of cell lysates were separated by 12.5% SDS-polyacrylamide gel electrophoresis, and then they were transferred onto polyvinylidene difluoride membranes (GE Healthcare Life Sciences). Membranes were blocked with 3% bovine serum albumin, and then they were incubated with specific antibodies for 2 h at room temperature. Finally, membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG, and the immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare Life Sciences) using the AutoChemi image analyzer (UVP, Inc., Upland, CA).

Real-Time PCR. Chondrocytes in primary culture were stimulated with 100 U/ml IL-1β or IL-1β + 100 µM CORM-2 for 12 h. Total RNA was extracted using the TRIzol reagent (Invitrogen, Barcelona, Spain) according to the manufacturer's instructions. Reverse transcription was accomplished on 1 µg of total RNA using random primers (TaqMan reverse transcription reagents; Applied Biosystems Spain, Madrid, Spain). PCR reactions were performed using SYBR Green PCR Master Mix (Bio-Rad, Madrid, Spain). Primers were purchased from Superarray Bioscience Corporation (Frederick, MD). PCR assays were performed in duplicate on an iCycler Real-Time PCR Detection System (Bio-Rad) running the following cycling conditions: 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Reaction specificity was determined by melt curve analysis that was performed by heating the plate from 55 to 95°C and measuring SYBR Green I dissociation from the amplicons. Cycle threshold (C_T) values for each gene were corrected using the mean C_T value for β-actin. Relative gene expression was calculated using the C_T method, and values are expressed as -fold change (2^–C_T) relative to the expression values in nonstimulated cells.

Enzyme-Linked Immunosorbent Assay. Chondrocytes in primary culture were stimulated with 100 U/ml IL-1β or IL-1β + CORM-2 for 24 h. Supernatants were harvested, and then they were centrifuged and frozen at –80°C until analysis. Pro-MMP-1, total MMP-3, total MMP-10, and pro-MMP-13 protein were quantified in supernatants by using enzyme-linked immunosorbent assay (ELISA) kits from R&D Systems, with sensitivity of 21.0, 9.0, 4.1, and 7.7 pg/ml, respectively. Aggrecanase activity was measured in supernatants by a sensitive ELISA kit detecting ADAMTS-1, ADAMTS-4, and ADAMTS-5, with sensitivity of 2 pM (MD Biosciences).

Data Analysis. Results are presented as mean ± S.E.M. Statistical analyses were performed using one-way analysis of variance followed by Dunnett's t test for multiple comparisons and unpaired Student's t test for dual comparisons.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Effects on MMPs and Aggrecanases. We determined the effects of CORM-2 on IL-1β-mediated induction of several enzymes relevant to cartilage degradation. After stimulation of OA chondrocytes with IL-1β for 24 h, enhanced levels of MMP-1, MMP-3, MMP-10, and MMP-13 were detected in the medium by ELISA (Fig. 1). CORM-2 significantly reduced the protein levels of these enzymes, whereas the negative control RuCl₃ was ineffective. CORM-2 treatment resulted in a concentration-dependent decrease in MMP-3, MMP-10, and MMP-13 levels, whereas MMP-1 was inhibited at the highest concentration (150 µM). In addition, aggrecanase activity was measured in chondrocyte supernatants as the release of specific aggrecan neoepitopes, detected by ELISA, which was significantly decreased by CORM-2 in a concentration-dependent manner (Fig. 2). We then examined the effects of CORM-2 on mRNA expression of these enzymes. Analysis of mRNA levels by real-time PCR showed that CORM-2 treatment reduced the expression of MMP-10, MMP-13, and ADAMTS-5 mRNA, whereas the reductions in MMP-1, MMP-3, and ADAMTS-4 were not significant (Fig. 3, A and B). The observed effects were not due to cytotoxicity, because cell viability was not significantly modified by 50 to 150 µM CORM-2 either in the absence or presence of IL-1β as determined by the MTT assay (data not shown). As expected, the negative control RuCl₃ at 150 µM used in some assays also showed no cytotoxicity. Taken together, these results suggest that CORM-2 blocks the effects of IL-1β on primary chondrocytes by down-regulation of several matrix-degrading enzymes, thereby preventing cartilage damage.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Effect of CORM-2 on MMPs protein levels released by human OA chondrocytes. Cells in primary culture were stimulated with 100 U/ml IL-1β for 24 h in the presence or absence of CORM-2 or the negative control RuCl3, and the levels of pro-MMP-1, total MMP-3, total MMP-10, and pro-MMP-13 protein were measured in supernatants by ELISA. Values are expressed as nanograms of MMP protein per milligram of cell protein. Data are mean ± S.E.M. of independent cultures with cells from seven different donors. *, p < 0.05; **, p < 0.01, with respect to IL-1β; ##, p < 0.01, with respect to nonstimulated cells.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Effect of CORM-2 on aggrecanase activity released by human OA chondrocytes. Cells in primary culture were stimulated with 100 U/ml IL-1β for 24 h in the presence or absence of CORM-2 or the negative control RuCl3, and aggrecanase activity was measured in supernatants by ELISA. Data are expressed as mean ± S.E.M. of independent cultures with cells from seven different donors. **, p < 0.01, with respect to IL-1β; ##, p < 0.01, with respect to nonstimulated cells.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effect of CORM-2 on mRNA expression of MMPs and aggrecanases. Cells in primary culture were stimulated with 100 U/ml IL-1β for 12 h in the presence or absence of 100 µM CORM-2. mRNA expression was determined by real-time PCR. Data are expressed as mean ± S.E.M. of independent cultures with cells from three different donors. Metalloproteinases are shown in A, and metalloproteinases and aggrecanases are shown in B. *, p < 0.05, with respect to IL-1β.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effect of CORM-2 on glycosaminoglycan degradation in OA explants (A) or glycosaminoglycan synthesis in OA chondrocytes in primary culture (B). Explants or cells were stimulated with 100 U/ml IL-1β in the presence or absence of CORM-2 or the negative control RuCl3, and glycosaminoglycan degradation or synthesis was measured by radiometric procedures, as indicated under Materials and Methods. Data are expressed as mean ± S.E.M. of independent cultures with explants or cells from three different donors. **, p < 0.01, with respect to IL-1β. ##, p < 0.01, with respect to nonstimulated cells.

Effects on Collagen II Expression. To evaluate the influence of CORM-2 on collagen II, we carried out immunocytochemical analyses. Figure 5 shows that OA chondrocytes in culture exhibit a high level of collagen II expression. In contrast, IL-1β stimulation results in a dramatic reduction of this protein with respect to basal incubations. It is interesting to note that 100 µM CORM-2 treatment maintained the expression of this extracellular matrix component in cells in basal conditions, and it restored collagen II expression in chondrocytes stimulated with IL-1β.

View larger version (70K): [in this window] [in a new window]   Fig. 5. Effect of 100 µM CORM-2 on collagen II expression in OA chondrocytes. Cells were incubated with CORM-2 in the presence or absence of 100 U/ml IL-1β for 15 days. Chondrocytes were fixed and stained with a monoclonal antibody against human collagen II, as indicated under Materials and Methods. Original magnification, 200x.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Effect of CORM-2 on MAPK phosphorylation in human OA chondrocytes stimulated with 100 U/ml IL-1β for 15 min in the absence or presence of 100 µM CORM-2. The expression of phosphorylated and total ERK1/2, JNK, and p38 in the cellular fraction was analyzed by Western blotting. Relative expression of phosphorylated and total protein bands was calculated after densitometric analysis. The immunoblot is representative of three independent experiments. Data are expressed as mean ± S.E.M. *, p < 0.05; **, p < 0.01, with respect to IL-1β. ##, p < 0.01, with respect to nonstimulated cells.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In OA, extracellular matrix depletion is the consequence of decreased synthesis and increased catabolic activity of proteolytic enzymes. Cartilage destruction is a complex process involving a wide range of proteinases. Loss of aggrecan precedes collagen degradation in OA (Nagase and Kashiwagi, 2003), and both processes are central pathophysiological features contributing to cartilage erosion during the progression of degenerative joint diseases (Caterson et al., 2000). The presence of proinflammatory cytokines such as IL-1β in OA joints would lead to the induction of a number of catabolic enzymes, including collagenases and aggrecanases (Goldring and Goldring, 2004). There is clear evidence that MMP-1 (collagenase-1) and MMP-13 (collagenase-3) play an important role in the degradation of collagen II in the extracellular matrix (Billinghurst et al., 1997; Wu et al., 2002). It has also been reported that MMP-13 is the major collagenase in OA cartilage (Bau et al., 2002; Burrage et al., 2006). The present findings demonstrate the ability of CORM-2 to inhibit the production of both enzymes, with a higher effect on MMP-13.

The activation of collagenolytic MMPs depends on proteolysis by serine proteinases and other MMPs, which may be a rate-limiting step in cartilage collagenolysis (Milner et al., 2001). In this respect, it is interesting to note the role of MMP-3 (stromelysin 1) and MMP-10 (stromelysin 2) in activating the proforms of collagenases (Murphy et al., 1987; Knäuper et al., 1996), leading to a significant increase in cartilage collagenolysis (Barksby et al., 2006). Our studies have shown the inhibitory effects of CORM-2 on both enzymes, indicating that this agent may act at different levels in the cascade of reactions leading to collagen degradation.

It is becoming apparent that aggrecan breakdown would be dependent mainly on the activity of aggrecanases such as ADAMTS-4 and ADAMTS-5 (Malfait et al., 2002), although some MMPs such as MMP-13 are also able to degrade aggrecan (Burrage et al., 2006). ADAMTS-5 is the most strongly expressed aggrecanase in OA cartilage (Bau et al., 2002), and it plays a crucial role in catalyzing proteoglycan degradation induced by IL-1 (Stanton et al., 2005). Aggrecanase-mediated aggrecan degradation is an early feature of OA that may provide the basis to cartilage protection as the aggrecan macromolecule protects the collagen fibrillar structure from the proteolytic attack by collagenases (Malfait et al., 2002). Our data indicate that CORM-2 treatment reduces aggrecanase activity and ADAMTS-5 gene expression in human OA chondrocytes.

Herein, we have described for the first time the protective effects of a member of the CO-RM family on human OA cartilage. We have shown that CORM-2 inhibits the degradation of aggrecan but that it increases its synthesis and collagen II expression. As a result, the maintenance of aggrecan and collagen II content by CORM-2 would result in cartilage protection, avoiding the loss of tissue function. In addition, our results suggest that these actions may be mediated through the down-regulation of enzymes that target both collagen II and aggrecan, major components of extracellular matrix.

Our results indicate that CO released by CORM-2 can reproduce the protective effects of HO-1 induction in OA chondrocytes (Guillen et al., 2007). Nevertheless, whether this exogenous source of CO could be comparable with endogenous CO levels derived from HO-1 activity is not known. These limitations have been discussed in a recent study using CORM-3 (Urquhart et al., 2007). Concerning CORM-2 effects, it is known that this agent can induce HO-1 (Sawle et al., 2005; Megías et al., 2007). Nevertheless, HO-1 up-regulation by CORM-2 in chondrocytes stimulated with IL-1β is weak (Guillen et al., 2005), suggesting that this mechanism does not make a significant contribution to the observed protective effects.

To date, new strategies in cartilage protection focused on MMP inhibitors has not resulted in clinical benefit (Murphy and Lee, 2005). Therefore, the inhibition of ADAMTS-4/ADAMTS-5 has been proposed as a new pharmacological target to develop cartilage-protecting agents (Malfait et al., 2002). It is possible that compounds such as CO-RMs, which are able to control both degradative pathways, could provide better protection. These observations support the interest of further studies on this class of agents.

Activation of MAPK signaling pathways seems to mediate IL-1β-dependent regulation of extracellular matrix components and MMPs expression in human articular chondrocytes (Fan et al., 2007). In particular, ERK1/2 is a negative regulator of chondrogenesis and chondrocyte differentiation (Yoon et al., 2002). Our results show that CORM-2 inhibits ERK1/2 and p38 phosphorylation, which plays an important role in MMPs induction, and in the down-regulation of aggrecan and collagen II expression. Interestingly, this study using primary human chondrocytes from OA patients and not cell lines suggests that the inhibition of ERK1/2 and p38 activation by CORM-2 may contribute to the maintenance of the chondrocytic phenotype and extracellular matrix homeostasis.

The ability of CO to bind metal centers in metalloproteins is known (Roberts et al., 2004), which may result in direct regulation of MAPK phosphorylation-dephosphorylation processes through possible interactions with protein phosphatase 2C (Boczkowski et al., 2006). In addition, CO could inhibit NADPH oxidase (Boczkowski et al., 2006) and increase mitochondrial reactive oxygen species production (Piantadosi, 2002), leading to the indirect modulation of MAPK signaling. Further studies would be necessary to determine the mechanisms responsible for MAPK inhibition by CORM-2 in chondrocytes. The ability of CO to react with transition metals could also result in the inhibition of metalloproteins relevant to cartilage degradation such as MMPs (Desmard et al., 2005).

In summary, CORM-2 exhibits protecting effects on cartilage metabolism through the depression of catabolic activities and the stimulation of glycosaminoglycan synthesis. These findings could affect the development of new therapies for the protection or repair of cartilage in degenerative joint diseases.

	Footnotes

 
This work was supported by Grants SAF2004-03835 (Ministerio de Educación y Ciencia-Fondo Europeo de Desarrollo Regional) and ACOMP2007/066 (Generalitat Valenciana). J.M. thanks Spanish Ministerio de Educación y Ciencia for a fellowship.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.134650.

ABBREVIATIONS: OA, osteoarthritis; MMP, matrix metalloproteinase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin domain; IL, interleukin; CO-RM, carbon monoxide-releasing molecule; HO-1, heme oxygenase-1; CORM-2, tricarbonyldichlororuthenium(II) dimer; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; DMEM, Dulbecco's modified Eagle's medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PCR, polymerase chain reaction; C_T, cycle threshold; ELISA, enzyme-linked immunosorbent assay; MAPK, mitogen-activated protein kinase.

Address correspondence to: Dr. María José Alcaraz, Department of Pharmacology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andres Estelles s/n, 46100 Burjasot, Valencia, Spain. E-mail: maria.j.alcaraz{at}uv.es

	References

Top Abstract Materials and Methods Results Discussion References

 

Bani-Hani MG, Greenstein D, Mann BE, Green CJ, and Motterlini R (2006) Modulation of thrombin-induced neuroinflammation in BV-2 microglia by carbon monoxide-releasing molecule 3. J Pharmacol Exp Ther 318: 1315–1322.[Abstract/Free Full Text]

Barksby HE, Milner JM, Patterson AM, Peake NJ, Hui W, Robson T, Lakey R, Middleton J, Cawston TE, Richards CD, et al. (2006) Matrix metalloproteinase 10 promotion of collagenolysis via procollagenase activation: implications for cartilage degradation in arthritis. Arthritis Rheum 54: 3244–3253.[CrossRef][Medline]

Bau B, Gebhard PM, Haag J, Knorr T, Bartnik E, and Aigner T (2002) Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro. Arthritis Rheum 46: 2648–2657.[CrossRef][Medline]

Billinghurst RC, Dahlberg L, Ionescu M, Reiner A, Bourne R, Rorabeck C, Mitchell P, Hambor J, Diekmann O, Tschesche H, et al. (1997) Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest 99: 1534–1545.[Medline]

Boczkowski J, Poderoso JJ, and Motterlini R (2006) CO-metal interaction: vital signaling from a lethal gas. Trends Biochem Sci 31: 614–621.[CrossRef][Medline]

Burrage PS, Mix KS, and Brinckerhoff CE (2006) Matrix metalloproteinases: role in arthritis. Front Biosci 11: 529–543.[Medline]

Caterson B, Flannery CR, Hughes CE, and Little CB (2000) Mechanisms involved in cartilage proteoglycan catabolism. Matrix Biol 19: 333–344.[CrossRef][Medline]

Clark JE, Naughton P, Shurey S, Green CJ, Johnson TR, Mann BE, Foresti R, and Motterlini R (2003) Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule. Circ Res 93: e2–e8.[Abstract/Free Full Text]

Desmard M, Amara N, Lanone S, Motterlini R, and Boczkowski J (2005) Carbon monoxide reduces the expression and activity of matrix metalloproteinases 1 and 2 in alveolar epithelial cells. Cell Mol Biol (Noisy-le-grand) 51: 403–408.[Medline]

Elson CJ, Mortuza FY, Perry MJ, Warnock MG, Webb GR, and Westacott CI (1998) Cytokines and focal loss of cartilage in osteoarthritis. Br J Rheumatol 37: 106–107.[Free Full Text]

Fan Z, Soder S, Oehler S, Fundel K, and Aigner T (2007) Activation of interleukin-1 signaling cascades in normal and osteoarthritic articular cartilage. Am J Pathol 171: 938–946.[Abstract/Free Full Text]

Foresti R, Hammad J, Clark JE, Johnson TR, Mann BE, Friebe A, Green CJ, and Motterlini R (2004) Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule. Br J Pharmacol 142: 453–460.[CrossRef][Medline]

Foresti R, Shurey C, Ansari T, Sibbons P, Mann BE, Johnson TR, Green CJ, and Motterlini R (2005) Reviewing the use of carbon monoxide-releasing molecules (CO-RMs) in biology: implications in endotoxin-mediated vascular dysfunction. Cell Mol Biol (Noisy-le-grand) 51: 409–423.[Medline]

Goldring MB (2000) The role of the chondrocyte in osteoarthritis. Arthritis Rheum 43: 1916–1926.[CrossRef][Medline]

Goldring MB, Birkhead J, Sandell LJ, Kimura T, and Krane SM (1988) Interleukin 1 suppresses expression of cartilage-specific types II and IX collagens and increases types I and III collagens in human chondrocytes. J Clin Invest 82: 2026–2037.[Medline]

Goldring SR and Goldring MB (2004) The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin Orthop Relat Res (427 Suppl): S27–S36.[CrossRef][Medline]

Gouze JN, Bordji K, Gulberti S, Terlain B, Netter P, Magdalou J, Fournel-Gigleux S, and Ouzzine M (2001) Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin-1beta-mediated effects in rat chondrocytes. Arthritis Rheum 44: 351–360.[CrossRef][Medline]

Gross SS and Levi R (1992) Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem 267: 25722–25729.[Abstract/Free Full Text]

Guillen MI, Megías J, Gomar F, and Alcaraz MJ (2007) Haem oxygenase-1 regulates catabolic and anabolic processes in osteoarthritic chondrocytes. J Pathol, in press.

Guillen MI, Valverde C, and Alcaraz MJ (2005) Protective role of heme oxygenase-1 in human osteoarthritic chondrocytes. Osteoarthritis Cartilage 13 (Suppl. A): S145.

Knäuper V, Murphy G, and Tschesche H (1996) Activation of human neutrophil procollagenase by stromelysin 2. Eur J Biochem 235: 187–191.[Medline]

Kobayashi M, Squires GR, Mousa A, Tanzer M, Zukor DJ, Antoniou J, Feige U, and Poole AR (2005) Role of interleukin-1 and tumor necrosis factor alpha in matrix degradation of human osteoarthritic cartilage. Arthritis Rheum 52: 128–135.[CrossRef][Medline]

Malfait AM, Liu RQ, Ijiri K, Komiya S, and Tortorella MD (2002) Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage. J Biol Chem 277: 22201–22208.[Abstract/Free Full Text]

Megías J, Busserolles J, and Alcaraz MJ (2007) The carbon monoxide-releasing molecule CORM-2 inhibits the inflammatory response induced by cytokines in Caco-2 cells. Br J Pharmacol 150: 977–986.[CrossRef][Medline]

Milner JM, Elliott SF, and Cawston TE (2001) Activation of procollagenases is a key control point in cartilage collagen degradation: interaction of serine and metalloproteinase pathways. Arthritis Rheum 44: 2084–2096.[CrossRef][Medline]

Moulharat N, Lesur C, Thomas M, Rolland-Valognes G, Pastoureau P, Anract P, De Ceuninck F, and Sabatini M (2004) Effects of transforming growth factor-beta on aggrecanase production and proteoglycan degradation by human chondrocytes in vitro. Osteoarthr Cartil 12: 296–305.[CrossRef][Medline]

Murphy G, Cockett MI, Stephens PE, Smith BJ, and Docherty AJ (1987) Stromelysin is an activator of procollagenase. A study with natural and recombinant enzymes. Biochem J 248: 265–268.[Medline]

Murphy G and Lee MH (2005) What are the roles of metalloproteinases in cartilage and bone damage? Ann Rheum Dis 64: iv44–iv47.[Abstract/Free Full Text]

Nagase H and Kashiwagi M (2003) Aggrecanases and cartilage matrix degradation. Arthritis Res Ther 5: 94–103.[Medline]

Piantadosi CA (2002) Biological chemistry of carbon monoxide. Antioxid Redox Signal 4: 259–270.[CrossRef][Medline]

Roberts GP, Youn H, and Kerby RL (2004) CO-sensing mechanisms. Microbiol Mol Biol Rev 68: 453–473.[Abstract/Free Full Text]

Sandouka A, Fuller BJ, Mann BE, Green CJ, Foresti R, and Motterlini R (2006) Treatment with CO-RMs during cold storage improves renal function at reperfusion. Kidney Int 69: 239–247.[CrossRef][Medline]

Sandy JD (2006) A contentious issue finds some clarity: on the independent and complementary roles of aggrecanase activity and MMP activity in human joint aggrecanolysis. Osteoarthr Cartil 14: 95–100.[Medline]

Sawle P, Foresti R, Mann BE, Johnson TR, Green CJ, and Motterlini R (2005) Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages. Br J Pharmacol 145: 800–810.[CrossRef][Medline]

Stanton H, Rogerson FM, East CJ, Golub SB, Lawlor KE, Meeker CT, Little CB, Last K, Farmer PJ, Campbell IK, et al. (2005) ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature 434: 648–652.[CrossRef][Medline]

Tetlow LC, Adlam DJ, and Woolley DE (2001) Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes. Arthritis Rheum 44: 585–594.[CrossRef][Medline]

Urquhart P, Rosignoli G, Cooper D, Motterlini R, and Perretti M (2007) Carbon monoxide-releasing molecules modulate leukocyte-endothelial interactions under flow. J Pharmacol Exp Ther 321: 656–662.[Abstract/Free Full Text]

Wu W, Billinghurst RC, Pidoux I, Antoniou J, Zukor D, Tanzer M, and Poole AR (2002) Sites of collagenase cleavage and denaturation of type II collagen in aging and osteoarthritic articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 and matrix metalloproteinase 13. Arthritis Rheum 46: 2087–2094.[CrossRef][Medline]

Yoon YM, Kim SJ, Oh CD, Ju JW, Song WK, Yoo YJ, Huh TL, and Chun JS (2002) Maintenance of differentiated phenotype of articular chondrocytes by protein kinase C and extracellular signal-regulated protein kinase. J Biol Chem 277: 8412–8420.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. I. Guillen, J. Megias, V. Clerigues, F. Gomar, and M. J. Alcaraz The CO-releasing molecule CORM-2 is a novel regulator of the inflammatory process in osteoarthritic chondrocytes Rheumatology, September 1, 2008; 47(9): 1323 - 1328. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.134650v1 325/1/56    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Megías, J. Articles by Alcaraz, M. J. Search for Related Content PubMed PubMed Citation Articles by Megías, J. Articles by Alcaraz, M. J.