Double‐knockout of ADAMTS‐4 and ADAMTS‐5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis

Objective

To phenotypically characterize ADAMTS‐4– and ADAMTS‐5–double‐knockout mice, and to determine the effect of deletion of ADAMTS‐4 and ADAMTS‐5 on the progression of osteoarthritis (OA) in mice.

Methods

Mice lacking the catalytic domain of ADAMTS‐4 and ADAMTS‐5 were crossed to generate ADAMTS‐4/5–double‐knockout animals. Twelve‐week‐old and 1‐year‐old male and female ADAMTS‐4/5–double‐knockout mice were compared with age‐ and sex‐matched wild‐type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS‐4/5–double‐knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS‐4/5–double‐knockout mice were treated with interleukin‐1 (IL‐1) plus retinoic acid ex vivo, to examine proteoglycan degradation.

Results

There were no genotype‐related phenotype differences between ADAMTS‐4/5–double‐knockout and WT mice through 1 year of age, with the exception that female ADAMTS‐4/5–double‐knockout mice had a lower mean terminal body weight at the 12‐week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS‐4/5–double‐knockout mice compared with WT mice. Following stimulation of cartilage explants with IL‐1 plus retinoic acid, aggrecanase‐mediated degradation in ADAMTS‐4/5–double‐knockout mice was ablated, to a level comparable with that in ADAMTS‐5–knockout mice.

Conclusion

Dual deletion of ADAMTS‐4 and ADAMTS‐5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS‐4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS‐4/5–double‐knockout mice were comparable with those observed with deletion of ADAMTS‐5 alone.

     

Characterization of and osteoarthritis susceptibility in ADAMTS‐4–knockout mice

Objective

To determine the importance of the enzymatic activity of ADAMTS‐4 in normal growth and development and to evaluate the role of ADAMTS‐4 in the progression of osteoarthritis (OA).

Methods

We generated catalytic domain–deleted ADAMTS‐4–transgenic mice and performed extensive gross and histologic analyses of various organs. The mice were challenged by surgical induction of joint instability leading to OA, to determine the importance of the enzymatic activity of ADAMTS‐4 in the progression of the disease. The response of wild‐type (WT) and ADAMTS‐4–knockout (ADAMTS‐4–KO) articular cartilage to interleukin‐1 and retinoic acid challenge in vitro was also evaluated.

Results

ADAMTS‐4–KO mice up to 1 year of age exhibited no gross or histologic abnormalities in 36 tissue sites examined. Despite evidence of ADAMTS‐4 expression and activity in growth plates of WT mice, catalytic silencing of this proteinase caused no abnormalities in skeletal development, growth, or remodeling. There was no effect of ADAMTS‐4 knockout on the progression or severity of OA 4 weeks or 8 weeks after surgical induction of joint instability. Enzymatic cleavage of aggrecan at the TEGE^373–374ARGS site was clearly evident after exposure of articular cartilage from ADAMTS‐4–KO mice to inflammatory cytokines.

Conclusion

Although expression of the ADAMTS‐4 gene has been found in many tissues throughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology. Our study provides evidence that ADAMTS‐4 is the primary aggrecanase in murine growth plates; however, deletion of its enzymatic activity did not affect normal long bone remodeling. Our results also lead to the hypothesis that, in the mouse, ADAMTS‐4 is not the primary enzyme responsible for aggrecan degradation at the TEGE^373–374ARGS site. The elucidation of the relative importance of ADAMTS‐4 in the pathologic process of human OA will require examination of human OA tissues and evidence of disease modification in patients following therapeutic intervention.

     

Release of hyaluronan and hyaladherins (aggrecan G1 domain and link proteins) from articular cartilage exposed to ADAMTS‐4 (aggrecanase 1) or ADAMTS‐5 (aggrecanase 2)

Objective

To determine whether aggrecanase (ADAMTS) activities in articular cartilage can directly lead to the release of hyaluronan (HA) and hyaladherins (aggrecan G1 domain and link proteins), as may occur ex vivo during stimulation of cartilage explants with interleukin‐1 (IL‐1) or retinoic acid or in vivo in synovial joints during aging and joint pathology.

Methods

Bovine articular cartilage discs (live or freeze‐killed) were cultured in the presence of IL‐1 or were incubated in digestion buffer containing recombinant human ADAMTS‐4 (rHuADAMTS‐4; aggrecanase 1) or rHuADAMTS‐5 (aggrecanase 2). Culture media, digestion supernatants, and tissue extracts were assayed for sulfated glycosaminoglycan (sGAG) content and analyzed by Western blotting to detect aggrecanase‐generated G1 domain (using neoepitope monoclonal antibody AGG‐C1/anti‐NITEGE³⁷³) and link proteins (using monoclonal antibody 8‐A‐4), as well as by quantitative enzyme‐linked immunosorbent assays to detect aggrecanase‐generated G1 domain (G1‐NITEGE³⁷³) and HA.

Results

IL‐1 treatment of live cartilage explants induced a time‐dependent release of sGAG, aggrecanase‐generated G1 domain (G1‐NITEGE³⁷³), and HA into the culture media. Exposure of live or freeze‐killed articular cartilage discs to rHuADAMTS‐4 or rHuADAMTS‐5 resulted in a dose‐ and time‐dependent release of sGAG and hyaluronan from the tissue, accompanied by a concomitant release of functionally intact hyaladherins (aggrecan G1‐NITEGE³⁷³ and link proteins).

Conclusion

Coincident with aggrecanolysis, aggrecanase activities in articular cartilage may actuate the release of HA and associated hyaladherins, thereby further compromising the integrity of the cartilage matrix during degenerative joint diseases such as osteoarthritis.

     

Cytokine‐induced increases in ADAMTS‐4 messenger RNA expression do not lead to increased aggrecanase activity in ADAMTS‐5–deficient mice

Objective

To compare the regulation of aggrecanase messenger RNA (mRNA) and enzyme activity by proinflammatory cytokines in primary mouse chondrocytes.

Methods

Primary chondrocytes were isolated from knee epiphyses of 6–8‐day‐old mice and cultured as monolayers. The cells were incubated with tumor necrosis factor α (TNFα), oncostatin M (OSM), or interleukin‐6 (IL‐6)/soluble IL‐6 receptor, and mRNA levels were measured by quantitative polymerase chain reaction at various time points. To measure aggrecanase activity, the cells were incubated with cytokine in the presence of exogenous aggrecan, and substrate cleavage was measured using antibodies to neoepitopes.

Results

Expression of both ADAMTS‐4 and ADAMTS‐5 mRNA was up‐regulated by TNFα and OSM. ADAMTS‐5 mRNA expression was also up‐regulated by IL‐6. Treatment of wild‐type mouse chondrocytes with each of the 3 cytokines increased cleavage of aggrecan at Glu³⁷³↓³⁷⁴Ala and Glu¹⁶⁷⁰↓¹⁶⁷¹Gly; in chondrocytes lacking ADAMTS‐5 activity, there was negligible cleavage at either site despite increased expression of ADAMTS‐4 mRNA in the presence of TNFα or OSM. None of the cytokines substantially altered mRNA expression of ADAMTS‐1 or ADAMTS‐9.

Conclusion

Despite substantial increases in the expression of ADAMTS‐4 mRNA induced by TNFα and OSM, these cytokines induced little if any increase in aggrecanolysis in ADAMTS‐5–deficient mouse chondrocytes. Our data show a poor correlation between the level of cytokine‐induced ADAMTS‐4 mRNA expression and the level of aggrecan‐degrading activity in cultured chondrocytes.

     

Granulin‐epithelin precursor binds directly to ADAMTS‐7 and ADAMTS‐12 and inhibits their degradation of cartilage oligomeric matrix protein

Objective

To determine 1) whether a protein interaction network exists between granulin‐epithelin precursor (GEP), ADAMTS‐7/ADAMTS‐12, and cartilage oligomeric matrix protein (COMP); 2) whether GEP interferes with the interactions between ADAMTS‐7/ADAMTS‐12 metalloproteinases and COMP substrate, including the cleavage of COMP; 3) whether GEP affects tumor necrosis factor α (TNFα)–mediated induction of ADAMTS‐7/ADAMTS‐12 expression and COMP degradation; and 4) whether GEP levels are altered during the progression of arthritis.

Methods

Yeast two‐hybrid, in vitro glutathione S‐transferase pull‐down, and coimmunoprecipitation assays were used to 1) examine the interactions between GEP, ADAMTS‐7/ADAMTS‐12, and COMP, and 2) map the binding sites required for the interactions between GEP and ADAMTS‐7/ADAMTS‐12. Immunofluorescence cell staining was performed to visualize the subcellular localization of GEP and ADAMTS‐7/ADAMTS‐12. An in vitro digestion assay was employed to determine whether GEP inhibits ADAMTS‐7/ADAMTS‐12–mediated digestion of COMP. The role of GEP in inhibiting TNFα‐induced ADAMTS‐7/ADAMTS‐12 expression and COMP degradation in cartilage explants was also analyzed.

Results

GEP bound directly to ADAMTS‐7 and ADAMTS‐12 in vitro and in chondrocytes, and the 4 C‐terminal thrombospondin motifs of ADAMTS‐7/ADAMTS‐12 and each granulin unit of GEP mediated their interactions. Additionally, GEP colocalized with ADAMTS‐7 and ADAMTS‐12 on the cell surface of chondrocytes. More importantly, GEP inhibited COMP degradation by ADAMTS‐7/ADAMTS‐12 in a dose‐dependent manner through 1) competitive inhibition through direct protein–protein interactions with ADAMTS‐7/ADAMTS‐12 and COMP, and 2) inhibition of TNFα‐induced ADAMTS‐7/ADAMTS‐12 expression. Furthermore, GEP levels were significantly elevated in patients with either osteoarthritis or rheumatoid arthritis.

Conclusion

Our observations demonstrate a novel protein–protein interaction network between GEP, ADAMTS‐7/ADAMTS‐12, and COMP. Furthermore, GEP is a novel specific inhibitor of ADAMTS‐7/ADAMTS‐12–mediated COMP degradation and may play a significant role in preventing the destruction of joint cartilage in arthritis.

     

Cartilage degradation biomarkers predict efficacy of a novel,highly selective matrix metalloproteinase 13 inhibitor in a dog model of osteoarthritis: Confirmation by multivariate analysis that modulation of type ii collagen and aggrecan degradation peptides parallels pathologic changes

Objective

To demonstrate that the novel highly selective matrix metalloproteinase 13 (MMP‐13) inhibitor PF152 reduces joint lesions in adult dogs with osteoarthritis (OA) and decreases biomarkers of cartilage degradation.

Methods

The potency and selectivity of PF152 were evaluated in vitro using 16 MMPs, TACE, and ADAMTS‐4 and ADAMTS‐5, as well as ex vivo in human cartilage explants. In vivo effects were evaluated at 3 concentrations in mature beagles with partial medial meniscectomy. Gross and histologic changes in the femorotibial joints were evaluated using various measures of cartilage degeneration. Biomarkers of cartilage turnover were examined in serum, urine, or synovial fluid. Results were analyzed individually and in combination using multivariate analysis.

Results

The potent and selective MMP‐13 inhibitor PF152 decreased human cartilage degradation ex vivo in a dose‐dependent manner. PF152 treatment of dogs with OA reduced cartilage lesions and decreased biomarkers of type II collagen (type II collagen neoepitope) and aggrecan (peptides ending in ARGN or AGEG) degradation. The dose required for significant inhibition varied with the measure used, but multivariate analysis of 6 gross and histologic measures indicated that all doses differed significantly from vehicle but not from each other. Combined analysis of cartilage degradation markers showed similar results.

Conclusion

This highly selective MMP‐13 inhibitor exhibits chondroprotective effects in mature animals. Biomarkers of cartilage degradation, when evaluated in combination, parallel the joint structural changes induced by the MMP‐13 inhibitor. These data support the potential therapeutic value of selective MMP‐13 inhibitors and the use of a set of appropriate biomarkers to predict efficacy in OA clinical trials.

     

Low molecular weight isoforms of the aggrecanases are responsible for the cytokine‐induced proteolysis of aggrecan in a porcine chondrocyte culture system

Objective

The major proteases responsible for aggrecan turnover in articular cartilage are the aggrecanases (ADAMTS‐4 and ADAMTS‐5). Although several studies have demonstrated C‐terminal truncation of these aggrecanases, the mechanism and importance of this processing are poorly understood. The objective of this study was to further investigate ADAMTS‐4 and ADAMTS‐5 C‐terminal truncation in a porcine model in vitro culture system.

Methods

Chondrocyte–agarose cultures with well‐established extracellular matrices were treated with or without interleukin‐1 (IL‐1), for a variety of different culture time periods. Cultures were analyzed for release of sulfated glycosaminoglycan, aggrecanase‐generated interglobular domain (IGD)–aggrecan cleavage, and the presence of ADAMTS‐4 and ADAMTS‐5 isoforms. Inhibition of aggrecanase activity with monoclonal antibodies, tissue inhibitor of metalloproteinases 3 (TIMP‐3), and cycloheximide pretreatment were used to identify ADAMTS isoforms involved in IGD–aggrecan catabolism.

Results

Multiple isoforms, including possible zymogens, of ADAMTS‐4 and ADAMTS‐5 were sequestered within the extracellular matrix formed by 3‐week chondrocyte–agarose cultures. IL‐1 exposure induced production of a low molecular weight (37 kd) isoform of ADAMTS‐4. This isoform was capable of degrading exogenous aggrecan at the IGD–aggrecanase site, was inhibited by TIMP‐3, was blocked after preincubation with an antibody to a sequence in the catalytic domain of ADAMTS‐4, and required de novo synthesis in the presence of IL‐1 for its generation.

Conclusion

In porcine chondrocyte–agarose cultures, a 37‐kd ADAMTS‐4 isoform appears to be the major matrix protease responsible for the IGD–aggrecanase activity detected in response to exposure to IL‐1. This conclusion contradicts that of recent studies of transgenic knockout mice and highlights the need to determine the roles of the different aggrecanase(s) in human disease.

     

ADAMTS‐9 is synergistically induced by interleukin‐1β and tumor necrosis factor α in OUMS‐27 chondrosarcoma cells and in human chondrocytes

Objective

To compare induction of the aggrecanases (ADAMTS‐1, ADAMTS‐4, ADAMTS‐5, ADAMTS‐8, ADAMTS‐9, and ADAMTS‐15) by interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) in chondrocyte‐like OUMS‐27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.

Methods

OUMS‐27 cells were stimulated for different periods of time and with various concentrations of IL‐1β and/or TNFα. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL‐1β and/or TNFα. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real‐time polymerase chain reaction and Northern blotting. ADAMTS‐9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL‐1β–stimulated OUMS‐27 cells was investigated.

Results

IL‐1β increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL‐1β stimulation was greater in chondrocytes than in fibroblasts. The combination of IL‐1β and TNFα had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS‐9 protein was also induced in IL‐1β–stimulated OUMS‐27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 up‐regulation in OUMS‐27 cells.

Conclusion

ADAMTS9 is an IL‐1β– and TNFα‐inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS‐9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS‐9 may have a pathologic role in arthritis.

     

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n‐3 fatty acids

Objective

To determine if n‐3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n‐6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage.

Methods

The metabolic profile of human OA cartilage was determined at the time of harvest and after 24‐hour exposure to n‐3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin‐1 (IL‐1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]).

Results

Supplementation with n‐3 PUFA (but not other fatty acids) reduced, in a dose‐dependent manner, the endogenous and IL‐1–induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS‐4, MMP‐13, and MMP‐3 (but not TIMP‐1, ‐2, or ‐3) was also specifically abolished with n‐3 PUFA supplementation. In addition, n‐3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5‐lipoxygenase, 5‐lipoxygenase–activating protein, tumor necrosis factor α, IL‐1α, and IL‐1β) without affecting the expression of message for several other proteins involved in normal tissue homeostasis.

Conclusion

These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n‐3 PUFA, but not to other classes of fatty acids.

     

Association between the abnormal expression of matrix‐degrading enzymes by human osteoarthritic chondrocytes and demethylation of specific CpG sites in the promoter regions

Objective

To investigate whether the abnormal expression of matrix metalloproteinases (MMPs) 3, 9, and 13 and ADAMTS‐4 by human osteoarthritic (OA) chondrocytes is associated with epigenetic “unsilencing.”

Methods

Cartilage was obtained from the femoral heads of 16 patients with OA and 10 control patients with femoral neck fracture. Chondrocytes with abnormal enzyme expression were immunolocalized. DNA was extracted, and the methylation status of the promoter regions of MMPs 3, 9, and 13 and ADAMTS‐4 was analyzed with methylation‐sensitive restriction enzymes, followed by polymerase chain reaction amplification.

Results

Very few chondrocytes from control cartilage expressed the degrading enzymes, whereas all clonal chondrocytes from late‐stage OA cartilage were immunopositive. The overall percentage of nonmethylated sites was increased in OA patients (48.6%) compared with controls (20.1%): 20% versus 4% for MMP‐13, 81% versus 47% for MMP‐9, 57% versus 30% for MMP‐3, and 48% versus 0% for ADAMTS‐4. Not all CpG sites were equally susceptible to loss of methylation. Some sites were uniformly methylated, whereas in others, methylation was generally absent. For each enzyme, there was 1 specific CpG site where the demethylation in OA patients was significantly higher than that in controls: at −110 for MMP‐13, −36 for MMP‐9, −635 for MMP‐3, and −753 for ADAMTS‐4.

Conclusion

This study provides the first evidence that altered synthesis of cartilage‐degrading enzymes by late‐stage OA chondrocytes may have resulted from epigenetic changes in the methylation status of CpG sites in the promoter regions of these enzymes. These changes, which are clonally transmitted to daughter cells, may contribute to the development of OA.

     

Matrix‐assisted laser desorption ionization–imaging mass spectrometry: A new methodology to study human osteoarthritic cartilage

Objective

Information about the distribution of proteins and the modulation that they undergo in the different phases of rheumatic pathologies is essential to understanding the development of these diseases. We undertook this study to demonstrate the utility of mass spectrometry (MS)–based molecular imaging for studying the spatial distribution of different components in human articular cartilage sections.

Methods

We compared the distribution of peptides and proteins in human control and osteoarthritic (OA) cartilage. Human control and OA cartilage slices were cut and deposited on conductive slides. After tryptic digestion, we performed matrix‐assisted laser desorption ionization–imaging MS (MALDI‐IMS) experiments in a MALDI–quadrupole time‐of‐flight mass spectrometer. Protein identification was undertaken with a combination of multivariate statistical methods and Mascot protein database queries. Hematoxylin and eosin staining and immunohistochemistry were performed to validate the results.

Results

We created maps of peptide distributions at 150‐μm raster size from control and OA human cartilage. Proteins such as biglycan, prolargin, decorin, and aggrecan core protein were identified and localized. Specific protein markers for cartilage oligomeric matrix protein and fibronectin were found exclusively in OA cartilage samples. Their distribution displayed a stronger intensity in the deep area than in the superficial area. New tentative OA markers were found in the deep area of the OA cartilage.

Conclusion

MALDI‐IMS identifies and localizes disease‐specific peptides and proteins in cartilage. All the OA‐related peptides and proteins detected display a stronger intensity in the deep cartilage. MS‐based molecular imaging is demonstrated to be an innovative method for studying OA pathology.