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An aggrecanase other than ADAMTS-4 or ADAMTS-5 cleaves ...Fig. 1. Immunostaining of the FREEE 1467...
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A B C D A C D B An aggrecanase other than ADAMTS-4 or ADAMTS-5 cleaves in the aggrecan CS-rich region in vivo + 1 Rogerson, F M; 1 Golub S B; 1 Boehm E; 1 Fosang, A J + 1 University of Melbourne Department of Paediatrics & Murdoch Childrens Research Institute, Melbourne, Australia ABSTRACT INTRODUCTION Aggrecan is responsible for the weight-bearing properties of articular cartilage and its loss by proteolytic degradation is a hallmark of arthritis. The function of aggrecan lies in its abundant chondroitin sulfate (CS) modifications that ultimately draw water into cartilage tissue. In pathology, aggrecan catabolism is mediated principally by aggrecanases, which belong to the ADAMTS family of metalloproteinases. ADAMTS- 4 and ADAMTS-5 have been identified as the major aggrecanases in a number of species and they cleave aggrecan at Glu 373 ↓ 374 Ala, resulting in loss of the entire CS-rich region. The mechanisms involved in aggrecan turnover in normal tissue remain largely unknown. In mice deficient in both ADAMTS-4 and ADAMTS-5 activities (TS-4/TS-5 ∆-cat) there are no obvious defects in joint morphology or skeletal development (1), suggesting that neither enzyme has a role in resorbing aggrecan during endochondral bone formation. We have discovered an activity in femoral head cartilage of TS-4/TS-5 ∆-cat mice that cleaves predominantly at the Glu 1467 ↓ 1468 Gly aggrecanase site and also at the Glu 1279 ↓ 1280 Gly aggrecanase site (1). We do not know if this activity is an in vitro phenomenon or if it has a role in vivo. To help answer this question we used immunohistochemistry to detect aggrecan cleavage at the Glu 1279 ↓ 1280 Gly and Glu 1467 ↓ 1468 Gly sites in mouse knee cartilage. METHODS Knee joints dissected from wildtype and TS-4/TS-5 ∆-cat mice at 3 and 6-weeks of age were fixed in 4% paraformaldehdye overnight, then decalcified in 7% (w/v) EDTA for 5 days prior to embedding in paraffin and cutting 5 μM sections. Endogenous peroxidase activity was quenched with 3% H2O2 for 30 min. Antigen retrieval with 10 mM citric acid, pH 6.0 containing 0.05% (v/v) Tween 20, for 30 min at 60ºC preceded treatment with 0.2% hyaluronidase for 1 h at 37ºC to enhance antibody penetration. Non-specific binding was blocked with 1.5% (v/v) normal goat serum + 1% (w/v) BSA for 1 h. The section were incubated with either α-FREEE 1467 (0.2 μg/mL), α-SSELE 1279 (1 μg/mL) primary antibodies overnight at 4ºC, then incubated with biotinylated α-rabbit IgG for 1 h, followed by the ABC Reagent (Vectastain) for 30 min. The sections were developed with diaminobenzidine and counterstained with haematoxylin. Negative controls included: 1) incubation in the absence of primary antibody, and 2) preincubation of primary antibody with a 500-fold excess of the peptide antigen for 1 h on ice. This work was approved by the institutional animal ethics committee. RESULTS SECTION In wild-type mice strong pericellular staining for the FREEE 1467 neoepitope was observed around hypertrophic chondrocytes at the bottom of the growth plate (Fig. 1A), at the periphery of the secondary centre of ossification (Fig. 1B) and in the meniscus (not shown). Staining was observed at both 3 and 6 weeks of age. In TS-4/TS-5 ∆-cat mice, at 6 weeks of age, very similar staining was observed in the growth plate (Fig 1C) and at the periphery of the secondary centre of ossification (Fig. 1D), and was very similar in intensity. Interestingly, at 3 weeks of age this pattern of staining was seen around the secondary centre of ossification but not in the growth plate (not shown). Pre- incubation with excess FREEE 1467 peptide completely abolished this staining (inset in Fig. 1A), demonstrating its specificity. In wild-type mice strong pericellular staining for the SSELE 1279 neoepitope was observed around hypertrophic chondrocytes at the bottom of the growth plate (Fig. 2A) and at the periphery of the secondary centre of ossification (Fig. 2B). Staining was observed at both 3 and 6 weeks of age. However, in TS-4/TS-5 ∆-cat mice this pericellular staining was absent in both the growth plate (Fig. 2C) and at the periphery of the secondary centre of ossification (Fig. 2D). Pre- incubation with excess SSELE 1279 peptide abolished this staining (inset in Fig. 2A), demonstrating its specificity. ACKNOWLEDGMENT We thank Johnson & Johnson Pharmaceutical Research & Development, L.L.C. for the ADAMTS-4 and ADAMTS-5 floxed mice. Fig. 1. Immunostaining of the FREEE 1467 neoepitope (arrowed) in wild- type growth plate (A) and 2º centre of ossification (B), and in TS-4/TS-5 ∆-cat growth plate (C) and 2º centre of ossification (D). Scale bar = 50 μM. Fig. 2. Immunostaining of the SSELE 1279 neoepitope (arrowed) in wild- type growth plate (A) and 2º centre of ossification (B), and in TS-4/TS-5 ∆-cat growth plate (C) and 2º centre of ossification (D). Scale bar = 50 μM. DISCUSSION The mechanisms of aggrecan turnover in normal cartilage are poorly understood. A proportion of aggrecan in human articular cartilage lacks the C-terminal G3 domain, and this proportion increases with age (2). Here we provide evidence for an enzyme that could contribute to C- terminal truncation by cleaving aggrecan at Glu 1467 ↓ 1468 Gly. We previously reported an activity in mouse femoral head cartilage that cleaved at this site but not at the pathogenic Glu 373 ↓ 374 Ala aggrecanase site (1). The enzyme cleaving aggrecan at Glu 1467 ↓ 1468 Gly therefore truncates aggrecan but does not destroy its function. Given that FREEE 1467 immunostaining was strongest around hypertrophic chondrocyte, this enzyme might contribute to aggrecan cleavage in regions where cartilage is resorbed in preparation for bone deposition. REFERENCES 1. Rogerson FM et al. Arthritis Rheum 2008; 58: 1664-1673. 2. Dudhia J et al. Biochem.J. 1996; 313: 933-940. Paper No. 263 • ORS 2011 Annual Meeting
Transcript of An aggrecanase other than ADAMTS-4 or ADAMTS-5 cleaves ...Fig. 1. Immunostaining of the FREEE 1467...
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An aggrecanase other than ADAMTS-4 or ADAMTS-5 cleaves in the aggrecan CS-rich region in vivo
+1Rogerson, F M; 1Golub S B; 1Boehm E; 1Fosang, A J +1University of Melbourne Department of Paediatrics & Murdoch Childrens Research Institute, Melbourne, Australia
ABSTRACT INTRODUCTION Aggrecan is responsible for the weight-bearing properties of articular cartilage and its loss by proteolytic degradation is a hallmark of arthritis. The function of aggrecan lies in its abundant chondroitin sulfate (CS) modifications that ultimately draw water into cartilage tissue. In pathology, aggrecan catabolism is mediated principally by aggrecanases, which belong to the ADAMTS family of metalloproteinases. ADAMTS-4 and ADAMTS-5 have been identified as the major aggrecanases in a number of species and they cleave aggrecan at Glu373↓374Ala, resulting in loss of the entire CS-rich region. The mechanisms involved in aggrecan turnover in normal tissue remain largely unknown. In mice deficient in both ADAMTS-4 and ADAMTS-5 activities (TS-4/TS-5 ∆-cat) there are no obvious defects in joint morphology or skeletal development (1), suggesting that neither enzyme has a role in resorbing aggrecan during endochondral bone formation. We have discovered an activity in femoral head cartilage of TS-4/TS-5 ∆-cat mice that cleaves predominantly at the Glu1467↓1468Gly aggrecanase site and also at the Glu1279↓1280Gly aggrecanase site (1). We do not know if this activity is an in vitro phenomenon or if it has a role in vivo. To help answer this question we used immunohistochemistry to detect aggrecan cleavage at the Glu1279↓1280Gly and Glu1467↓1468Gly sites in mouse knee cartilage.
METHODS Knee joints dissected from wildtype and TS-4/TS-5 ∆-cat mice at 3 and 6-weeks of age were fixed in 4% paraformaldehdye overnight, then decalcified in 7% (w/v) EDTA for 5 days prior to embedding in paraffin and cutting 5 µM sections. Endogenous peroxidase activity was quenched with 3% H2O2 for 30 min. Antigen retrieval with 10 mM citric acid, pH 6.0 containing 0.05% (v/v) Tween 20, for 30 min at 60ºC preceded treatment with 0.2% hyaluronidase for 1 h at 37ºC to enhance antibody penetration. Non-specific binding was blocked with 1.5% (v/v) normal goat serum + 1% (w/v) BSA for 1 h. The section were incubated with either α-FREEE1467 (0.2 µg/mL), α-SSELE1279 (1 µg/mL) primary antibodies overnight at 4ºC, then incubated with biotinylated α-rabbit IgG for 1 h, followed by the ABC Reagent (Vectastain) for 30 min. The sections were developed with diaminobenzidine and counterstained with haematoxylin. Negative controls included: 1) incubation in the absence of primary antibody, and 2) preincubation of primary antibody with a 500-fold excess of the peptide antigen for 1 h on ice. This work was approved by the institutional animal ethics committee.
RESULTS SECTION In wild-type mice strong pericellular staining for the FREEE1467 neoepitope was observed around hypertrophic chondrocytes at the bottom of the growth plate (Fig. 1A), at the periphery of the secondary centre of ossification (Fig. 1B) and in the meniscus (not shown). Staining was observed at both 3 and 6 weeks of age. In TS-4/TS-5 ∆-cat mice, at 6 weeks of age, very similar staining was observed in the growth plate (Fig 1C) and at the periphery of the secondary centre of ossification (Fig. 1D), and was very similar in intensity. Interestingly, at 3 weeks of age this pattern of staining was seen around the secondary centre of ossification but not in the growth plate (not shown). Pre-incubation with excess FREEE1467 peptide completely abolished this staining (inset in Fig. 1A), demonstrating its specificity. In wild-type mice strong pericellular staining for the SSELE1279 neoepitope was observed around hypertrophic chondrocytes at the bottom of the growth plate (Fig. 2A) and at the periphery of the secondary centre of ossification (Fig. 2B). Staining was observed at both 3 and 6 weeks of age. However, in TS-4/TS-5 ∆-cat mice this pericellular staining was absent in both the growth plate (Fig. 2C) and at the periphery of the secondary centre of ossification (Fig. 2D). Pre-incubation with excess SSELE1279 peptide abolished this staining (inset in Fig. 2A), demonstrating its specificity. ACKNOWLEDGMENT We thank Johnson & Johnson Pharmaceutical Research & Development, L.L.C. for the ADAMTS-4 and ADAMTS-5 floxed mice.
Fig. 1. Immunostaining of the FREEE1467 neoepitope (arrowed) in wild-type growth plate (A) and 2º centre of ossification (B), and in TS-4/TS-5 ∆-cat growth plate (C) and 2º centre of ossification (D). Scale bar = 50 µM.
Fig. 2. Immunostaining of the SSELE1279 neoepitope (arrowed) in wild-type growth plate (A) and 2º centre of ossification (B), and in TS-4/TS-5 ∆-cat growth plate (C) and 2º centre of ossification (D). Scale bar = 50 µM. DISCUSSION The mechanisms of aggrecan turnover in normal cartilage are poorly understood. A proportion of aggrecan in human articular cartilage lacks the C-terminal G3 domain, and this proportion increases with age (2). Here we provide evidence for an enzyme that could contribute to C-terminal truncation by cleaving aggrecan at Glu1467↓1468Gly. We previously reported an activity in mouse femoral head cartilage that cleaved at this site but not at the pathogenic Glu373↓374Ala aggrecanase site (1). The enzyme cleaving aggrecan at Glu1467↓1468Gly therefore truncates aggrecan but does not destroy its function. Given that FREEE1467 immunostaining was strongest around hypertrophic chondrocyte, this enzyme might contribute to aggrecan cleavage in regions where cartilage is resorbed in preparation for bone deposition. REFERENCES 1. Rogerson FM et al. Arthritis Rheum 2008; 58: 1664-1673. 2. Dudhia J et al. Biochem.J. 1996; 313: 933-940.
Paper No. 263 • ORS 2011 Annual Meeting
