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Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan

EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO
      Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV356) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV356 neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.
      The mammalian high-temperature requirement A (HtrA) family of serine proteases is defined by a characteristic trypsin-like serine protease domain and one or two C-terminal PDZ domains. Four mammalian HtrA proteins have been identified to date, HtrA1–4. HtrA1 (also called PRSS11) is a ubiquitously expressed extracellular serine protease which contains a signal sequence for secretion, an insulin-like growth factor (IGF)
      The abbreviations used are: IGF
      insulin-like growth factor
      OA
      osteoarthritis
      ECM
      extracellular matrix
      COMP
      cartilage oligomeric matrix protein
      MMP-1
      matrix metalloproteinases-1
      IGD
      G1-interglobular domain
      TIC
      total ion current
      LC-MS/MS
      liquid chromatography-tandem mass spectrometry
      GFP
      green fluorescent protein.
      2The abbreviations used are: IGF
      insulin-like growth factor
      OA
      osteoarthritis
      ECM
      extracellular matrix
      COMP
      cartilage oligomeric matrix protein
      MMP-1
      matrix metalloproteinases-1
      IGD
      G1-interglobular domain
      TIC
      total ion current
      LC-MS/MS
      liquid chromatography-tandem mass spectrometry
      GFP
      green fluorescent protein.
      -binding protein domain, and a Kazal-type serine protease inhibitor domain in addition to the serine protease domain and one C-terminal PDZ domain (
      • Clausen T.
      • Southan C.
      • Ehrmann M.
      ). HtrA1 has been implicated in the progression of several pathologies including age-related macular degeneration, cancer, Alzheimer disease, rheumatoid arthritis, and osteoarthritis (OA) (
      • Dewan A.
      • Liu M.
      • Hartman S.
      • Zhang S.S.
      • Liu D.T.
      • Zhao C.
      • Tam P.O.
      • Chan W.M.
      • Lam D.S.
      • Snyder M.
      • Barnstable C.
      • Pang C.P.
      • Hoh J.
      ,
      • Grau S.
      • Baldi A.
      • Bussani R.
      • Tian X.
      • Stefanescu R.
      • Przybylski M.
      • Richards P.
      • Jones S.A.
      • Shridhar V.
      • Clausen T.
      • Ehrmann M.
      ,
      • Grau S.
      • Richards P.J.
      • Kerr B.
      • Hughes C.
      • Caterson B.
      • Williams A.S.
      • Junker U.
      • Jones S.A.
      • Clausen T.
      • Ehrmann M.
      ,
      • Hu S.I.
      • Carozza M.
      • Klein M.
      • Nantermet P.
      • Luk D.
      • Crowl R.M.
      ,
      • Tsuchiya A.
      • Yano M.
      • Tocharus J.
      • Kojima H.
      • Fukumoto M.
      • Kawaichi M.
      • Oka C.
      ,
      • Yang Z.
      • Camp N.J.
      • Sun H.
      • Tong Z.
      • Gibbs D.
      • Cameron D.J.
      • Chen H.
      • Zhao Y.
      • Pearson E.
      • Li X.
      • Chien J.
      • Dewan A.
      • Harmon J.
      • Bernstein P.S.
      • Shridhar V.
      • Zabriskie N.A.
      • Hoh J.
      • Howes K.
      • Zhang K.
      ,
      • Bowden M.A.
      • Di Nezza-Cossens L.A.
      • Jobling T.
      • Salamonsen L.A.
      • Nie G.
      ,
      • Chien J.
      • Staub J.
      • Hu S.I.
      • Erickson-Johnson M.R.
      • Couch F.J.
      • Smith D.I.
      • Crowl R.M.
      • Kaufmann S.H.
      • Shridhar V.
      ,
      • Baldi A.
      • De Luca A.
      • Morini M.
      • Battista T.
      • Felsani A.
      • Baldi F.
      • Catricalà C.
      • Amantea A.
      • Noonan D.M.
      • Albini A.
      • Natali P.G.
      • Lombardi D.
      • Paggi M.G.
      ). HtrA1 has also been shown to inhibit osteoblast mineralization (
      • Hadfield K.D.
      • Rock C.F.
      • Inkson C.A.
      • Dallas S.L.
      • Sudre L.
      • Wallis G.A.
      • Boot-Handford R.P.
      • Canfield A.E.
      ).
      Expression of HtrA1 has been found to be elevated in articular cartilage in association with OA (
      • Hu S.I.
      • Carozza M.
      • Klein M.
      • Nantermet P.
      • Luk D.
      • Crowl R.M.
      ). In addition, HtrA1 levels are up-regulated in murine cartilage after experimentally induced joint damage (
      • Tsuchiya A.
      • Yano M.
      • Tocharus J.
      • Kojima H.
      • Fukumoto M.
      • Kawaichi M.
      • Oka C.
      ). The physiological role of HtrA1 in OA disease progression as well as in other pathologies is unclear. Preliminary studies using in vitro digestion assays suggest that HtrA1 might be capable of digesting cartilage extracellular matrix (ECM) proteins such as fibromodulin, cartilage oligomeric matrix protein (COMP), fibronectin, decorin, and aggrecan (
      • Tsuchiya A.
      • Yano M.
      • Tocharus J.
      • Kojima H.
      • Fukumoto M.
      • Kawaichi M.
      • Oka C.
      ,

      Ganu, V., Melton, R., Hu, S., Koehn, J., Klein, M., Liebman, J., (2001) Orthopaedic Research Society 47th Annual Meeting, February 25–28, 2001, San Francisco, CA, Vol. 26, p. 266

      ,

      Goldberg, R., Crowl, R., Hu, S., (2000) Orthopaedic Research Society 46th Annual Meeting, March 12–15, 2000, Orlando, FL, Vol. 25, p. 164

      ). Furthermore, it was recently reported that elevated levels of HtrA1 protein (∼7-fold above normal) are present in synovial fluids obtained from OA patients and that fibronectin fragments generated by HtrA1 cleavage induced the expression of catabolic enzymes such as matrix metalloproteinases-1 (MMP-1) and MMP-3 in synovial fibroblasts (
      • Grau S.
      • Richards P.J.
      • Kerr B.
      • Hughes C.
      • Caterson B.
      • Williams A.S.
      • Junker U.
      • Jones S.A.
      • Clausen T.
      • Ehrmann M.
      ). HtrA1 has also been shown to modulate multiple signaling pathways in vitro. It binds to transforming growth factor-β family proteins including transforming growth factor-β1 and bone morphogenetic proteins 2 and 4 and inhibits signaling mediated by these factors (
      • Oka C.
      • Tsujimoto R.
      • Kajikawa M.
      • Koshiba-Takeuchi K.
      • Ina J.
      • Yano M.
      • Tsuchiya A.
      • Ueta Y.
      • Soma A.
      • Kanda H.
      • Matsumoto M.
      • Kawaichi M.
      ,
      • Tocharus J.
      • Tsuchiya A.
      • Kajikawa M.
      • Ueta Y.
      • Oka C.
      • Kawaichi M.
      ). In addition, HtrA1 has been shown to cleave IGF-binding protein-5 and possibly regulate signaling mediated by IGF (
      • Hou J.
      • Clemmons D.R.
      • Smeekens S.
      ). These findings suggest that the protease HtrA1 may play a physiological role in cartilage during OA.
      Articular cartilage is made up of chondrocytes surrounded by the ECM comprised mainly of the proteoglycan, aggrecan, and type II collagen. During normal homeostasis there is a dynamic balance between anabolic activities such as proteoglycan synthesis as well as catabolic activities in which the ECM is destroyed. When the catabolic activities of proteases, such as MMPs and aggrecanases, offset new matrix synthesis, focal degradation and loss of articular cartilage occurs, resulting in the development of OA. In some in vitro digestion studies, we and others have shown degradation of aggrecan by recombinant HtrA1 (
      • Tsuchiya A.
      • Yano M.
      • Tocharus J.
      • Kojima H.
      • Fukumoto M.
      • Kawaichi M.
      • Oka C.
      ,

      Ganu, V., Melton, R., Hu, S., Koehn, J., Klein, M., Liebman, J., (2001) Orthopaedic Research Society 47th Annual Meeting, February 25–28, 2001, San Francisco, CA, Vol. 26, p. 266

      ,

      Goldberg, R., Crowl, R., Hu, S., (2000) Orthopaedic Research Society 46th Annual Meeting, March 12–15, 2000, Orlando, FL, Vol. 25, p. 164

      ). In the present study we set out to examine the physiological relevance of aggrecan cleavage by HtrA1 in OA disease progression.

      EXPERIMENTAL PROCEDURES

       Cartilage Protein Extraction

      Human cartilage protein extracts were isolated from human femoral head articular cartilage specimens from donors aged 50–82 years which were obtained within 36 h after either autopsy (National Disease Research Interchange, Philadelphia, PA) or total knee replacement surgery (New England Baptist Hospital, Boston, MA). Applicable regulations and guidelines were followed regarding consent issues, protection of human subjects, and donor confidentiality. Protein extracts were prepared from human cartilage specimens (7 OA and 7 age-matched control donors) using 4 m guanidine HCl, 50 mm sodium acetate, pH 5.8, containing complete protease inhibitor mixture (Roche Applied Science) and fractionated by cesium chloride gradient ultracentrifugation. The top ⅓ of the gradient was dialyzed and used for further analysis.

       Adenoviral Infection of Chondrocyte Alginate Bead Cultures

      The adenoviral vector (VQAd5CMV KpA) used in this study is a replication-defective human type-5 adenovirus. cDNAs encoding full-length HtrA1 (wild type and S328A mutant) as well as the truncated mutants (ΔFS with the deletion of amino acids 33–153) were inserted into this vector immediately after the cytomegalovirus promoter and a modified Kozak sequence (CCACC). VQAd5CMV KpA viruses were produced and purified by ViraQuest, Inc. (North Liberty, IA). Titers of viral stocks were determined by plaque assay on 293 cells. Human primary chondrocytes were isolated from cartilage obtained after joint replacement surgery from OA patients with ages ranging from 60 to 80 (New England Baptist Hospital, Boston MA). Briefly, the normal-looking part of the cartilage was removed from the femoral condyles of the diseased joints and sequentially digested with Pronase (0.2%; Roche Applied Science) for 1 h and collagenase P (from Clostridium histolyticum, 0.025%; Roche Applied Science) for 15 h. The digest was filtered through a 70-μm cell strainer (BD Labware, Franklin Lakes, NJ), and cells were washed twice with Dulbecco's modified Eagle's medium/F-12 (Invitrogen). Primary chondrocytes were infected with adenovirus (VQ) encoding wild type or mutant HtrA1 proteins (multiplicity of infection = 25) and then seeded in three-dimensional alginate scaffolds. Efficient infection of chondrocytes (>90%) was confirmed by fluorescent microscopy using adenoviruses encoding green fluorescent protein (GFP). Chondrocyte alginate bead cultures were maintained in Dulbecco's modified Eagle's medium/F-12 with 10% fetal bovine serum (Invitrogen), 50 μg/ml gentamicin, and 50 μg/ml ascorbic acid. Two days later medium was changed to contain 5% and then 2.5% fetal bovine serum for 2 days each. Finally the beads were maintained in similar medium containing 1% Nutridoma-SP serum-free media supplement (Roche Applied Science) (day 1). Five beads were collected on day 21 and dissolved in 55 mm sodium citrate, 150 mm NaCl, 25 mm HEPES, pH 7.0. Sulfated glycosaminoglycan content in the beads was monitored by 1,9-dimethylmethylene blue assay (at pH 1.5) (
      • Enobakhare B.O.
      • Bader D.L.
      • Lee D.A.
      ). Cell viability was assessed by trypan blue staining, total cell counts, or CyQUANT cell proliferation assays (Molecular Probes/ Invitrogen).

       Mass Spectrometry-based Proteomic Analysis

      Proteomic analysis of proteins in human cartilage extracts has been previously described in detail (
      • Wu J.
      • Liu W.
      • Bemis A.
      • Wang E.
      • Qiu Y.
      • Morris E.A.
      • Flannery C.R.
      • Yang Z.
      ). Briefly, cartilage protein extracts isolated as indicated above were separated by SDS-PAGE. Each gel lane was horizontally divided into 32 slices for in-gel tryptic digestion. The digests were separated using an on-line reversed-phase C18 column (5 μm, 200 Å, 75 μm × 10 cm) and analyzed by ion-trap mass spectrometry. Proteins were then identified by data base searching using SpectrumMill (Agilent, Santa Clara, CA). To identify proteins (or degradation fragments) that were released into the media of chondrocyte-seeded alginate cultures infected with HtrA1 or S328A adenoviruses as described above, media collected at day 21 was separated by SDS-PAGE, and each gel lane was divided into 25 slices for in-gel tryptic digestion and analyzed by mass spectrometry in the same way.

       Identification of a Novel HtrA1 Cleavage Site within Aggrecan and Generation of VQTV356 Neoepitope Antibody

      The expression vector pET16b was used to express HtrA1 protein and its mutants. Expression was induced at 25 °C using 1 mm isopropyl 1-thio-β-d-galactopyranoside in BL21 (DE3) Escherichia coli strain. Cells overexpressing recombinant proteins were homogenized in a buffer containing 50 mm NaH2PO4, pH 7.5, 300 mm NaCl, 1 m urea. Recombinant proteins were first purified on a nickel-nitrilotriacetic acid column (Qiagen), then on an anion exchange column. Impurities were further removed by size exclusion chromatography. Recombinant aggrecan protein fragments encompassing the G1-interglobular domain (IGD)-G2 (R&D Systems, Minneapolis, MN) or the IGD alone (Millipore, Bedford, MA) were incubated with wild type ΔFS-HtrA1 (amino acids (aa) 157–480) or an active site mutant ΔFS-S328A (aa 157–480) with or without a synthetic peptide agonist (CPII) (
      • Murwantoko
      • Yano M.
      • Ueta Y.
      • Murasaki A.
      • Kanda H.
      • Oka C.
      • Kawaichi M.
      ) (Anaspec, San Jose, CA) at 37 °C for 3 h or overnight. Degradation products were analyzed by SDS-PAGE followed by Coomassie Blue staining and subsequently subjected to N-terminal sequencing (ProSeq Inc., Boxford, MA). Polyclonal neoepitope antibodies were generated against the peptide sequence CGEEDITVQTV (Open Biosystems, Huntsville, AL), which represents the newly formed C terminus of aggrecan after HtrA1 cleavage at VQTV356357TWPD. Antibodies recognizing the spanning sequence (CGEEDITVQTVTWPDMELPLP) were negatively absorbed, and the specific antibody was further affinity-purified. To test specificity of the antibody, purified aggrecan was digested in vitro with excess ΔFS-HtrA1 or HtrA3 (Invitek GmbH, Berlin, Germany) overnight at 37 °C. Digestion products along with undigested aggrecan were deglycosylated and subjected to Western blot analysis as described below using the anti-VQTV356 neoepitope antibody.

       Quantitative Real-time Reverse Transcriptase PCR

      Total cellular RNA from osteoarthritic cartilage from three human donors was isolated using Trizol reagent (Invitrogen) as previously described (
      • Song R.H.
      • Tortorella M.D.
      • Malfait A.M.
      • Alston J.T.
      • Yang Z.
      • Arner E.C.
      • Griggs D.W.
      ). TaqMan analysis was performed using TaqMan Gene Expression Assays specific for HtrA1, HtrA2, HtrA3, and HtrA4 purchased from Applied Biosystems, Inc. (Foster City, CA). Expression levels were normalized to human glyceraldehyde-3-phosphate dehydrogenase, and relative expression was calculated using the ΔΔCT method.

       Western Blot Analysis

      Protein extracts (undigested or digested with excess amounts of ΔFS-HtrA1) or purified aggrecan digested with recombinant HtrA1, ADAMTS-4, or MMP-13 were deglycosylated with chondroitinase ABC (Sigma), keratinase (Seikagaku America, Falmouth, MA), and keratinase II (Seikagaku America, Falmouth, MA). They were then resolved by 4–12% gradient gel electrophoresis, and proteins were transferred onto nitrocellulose membranes (Invitrogen). Nonspecific binding was blocked using 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBS-T). Blots were incubated with the indicated primary antibodies overnight at 4 °C in TBS-T. Secondary antibodies were conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were visualized by the Western Lightning Enhanced Chemiluminescence Substrate (PerkinElmer Life Sciences). To detect HtrA1 protein, a mixture of rabbit anti-HtrA1 (N-terminal) and rabbit anti-HtrA1 (C-terminal) polyclonal antibodies was used (Abgent, San Diego, CA). To detect HtrA1-generated aggrecan neoepitope VQTV356, affinity-purified rabbit anti-VQTV356 polyclonal antibody was used. To detect ADAMTS-4-digested aggrecan, the mouse monoclonal agg-C1/NITEGE371 antibody was used (
      • Chockalingam P.S.
      • Zeng W.
      • Morris E.A.
      • Flannery C.R.
      ). To detect MMP-13-digested aggrecan, the mouse monoclonal anti-IPEN341 neoepitope antibody was used (MD Biosciences, St. Paul, MN).

       Immunohistochemical Detection of HtrA1-generated Aggrecan Neoepitope

      Immunohistochemistry was performed on human femoral head articular cartilage samples obtained from patients undergoing knee replacement surgery (OA1, 65 years old, female; OA2, 75 years old, Caucasian male; OA3, 84 years old, Caucasian female, all from New England Baptist Hospital). Samples were fixed with 10% neutral buffered formalin and embedded in paraffin. Histological sections of 6 μm were cut and mounted onto slides. For epitope unmasking, a deglycosylation solution containing 0.1 unit/ml chondroitinase ABC, 0.1 units/ml keratanase I, and 0.01 unit/ml keratanase II (Seikagaku, Japan) was overlaid onto the tissue sections for 1 h at 37 °C (
      • Chubinskaya S.
      • Mikhail R.
      • Deutsch A.
      • Tindal M.H.
      ). Endogenous peroxidase activity was blocked with peroxidase blocking buffer (Dako, Carpinteria, CA) for 15 min. The sections were then blocked by serum-free protein block solution (Dako) for 30 min. Sections were incubated with 10 μg/ml anti-VQTV356 neoepitope polyclonal antibody or 10 μg/ml normal rabbit control IgG (Pierce) overnight at 4 °C. EnVision horseradish peroxidase-labeled polymer conjugated with anti-rabbit secondary antibodies (Dako) was applied for 1 h at room temperature, with 3,3′-diaminobenzidine used as a chromogen. Nuclei were counterstained with hematoxylin blue (Dako).

      RESULTS

       HtrA1 Protein Is Abundant in Cartilage, and Its Levels Are Highly Elevated in Osteoarthritic Cartilage

      Fresh cartilage samples were extracted, and low buoyant density proteins were separated from aggrecan by cesium chloride gradient ultracentrifugation. Using a semiquantitative mass spectrometry-based proteomic approach, we identified several hundred cartilage proteins, some of which are differentially expressed in OA cartilage versus age-matched control cartilage (
      • Wu J.
      • Liu W.
      • Bemis A.
      • Wang E.
      • Qiu Y.
      • Morris E.A.
      • Flannery C.R.
      • Yang Z.
      ). Fig. 1A shows the relative abundance of HtrA1 in seven OA cartilage specimens (as reflected by the total ion current (TIC) values specific to HtrA1-derived peptides) compared with several other cartilage proteins. TIC value is a measurement that has been shown to closely correlate with protein concentration even in complex proteomes such as human serum (
      • Chelius D.
      • Zhang T.
      • Wang G.
      • Shen R.F.
      ). HtrA1 was found to be among the most abundant of matrix proteins, which included proline- and arginine-rich end leucine-rich repeat protein (PRELP), decorin, fibronectin, cartilage link protein 1, COMP, cartilage intermediate layer protein (CILP), and chondroadherin. Due at least in part to the detection limitations of mass spectrometry as well as to their generally low levels of abundance, this study detected only five proteases: HtrA1, MMP-3, MMP-2, lysosomal aspartyl protease, and MMP-1. HtrA1 was at least 10-fold more abundant than any of these other detected proteases (Fig. 1A). The TIC values generated by peptides unique to HtrA1 were compared between human cartilage extract samples (7 age-matched control versus 7 OA samples). Significantly increased levels of HtrA1 protein (∼7.8-fold) were found in OA cartilage specimens with an average TIC value of 1.8E+09 versus an average TIC of 2.3E+08 detected in age-matched samples (p = 0.0097) (Fig. 1B). Although the TIC values generated by mass spectrometry are semiquantitative, this result is consistent with previous findings by Hu et al., which showed increased HtrA1 mRNA expression of a similar magnitude in OA cartilage (
      • Hu S.I.
      • Carozza M.
      • Klein M.
      • Nantermet P.
      • Luk D.
      • Crowl R.M.
      ). Furthermore, Western blot analysis indicated an up-regulation of intact HtrA1 protein in OA cartilage protein extracts as compared with age-matched control cartilage (Fig. 1C). The likely source of HtrA1 within the articular joint appears to be the chondrocytes, as human primary chondrocytes express 15-fold more HtrA1 as compared with human synoviocytes as determined by quantitative real-time reverse transcription PCR (data not shown).
      Figure thumbnail gr1
      FIGURE 1HtrA1 is an abundant protein in OA cartilage. A, Human OA articular cartilage extracts (n = 7 specimens) were analyzed by LC-MS/MS. HtrA1 was one of the most abundant cartilage proteins identified. Bars represent average values of TIC for some selected proteins in all seven OA samples. 1, PRELP (proline- and arginine-rich end leucine-rich repeat protein); 2, decorin; 3, fibronectin; 4, cartilage link protein 1; 5, COMP; 6, CILP (cartilage intermediate layer protein); 7, vimentin; 8, chondroadherin; 9, HtrA1; 10, MMP-3; 11, MMP-2; 12, lysosomal aspartyl protease; 13, MMP-1. B, HtrA1 protein levels are elevated in OA cartilage. Proteins in age-matched control and OA human cartilage extracts (n = 7 specimens/group) were analyzed by LC-MS/MS. The TIC of the unique peptides derived from HtrA1 in each sample approximates its relative abundance. White bars represent HtrA1 TICs from age-matched samples, and filled bars show HtrA1 TICs from OA samples. The average TIC value detected in the OA samples was 1.8E+09, whereas the average TIC detected in the age-matched samples was 2.3E+08 (p = 0.0097). C, detection of HtrA1 protein in cartilage extract. Western blot (IB) analysis of age-matched (A-M) and OA cartilage extracts using an HtrA1-specific antibody showed HtrA1 was up-regulated in OA cartilage and ran at the expected size of 51 kDa (full-length).

       Overexpression of Proteolytically Active Forms, but Not Inactive Mutants of HtrA1, Reduces Proteoglycan Content in Chondrocyte Alginate Cultures

      Freshly isolated human primary chondrocytes were suspended in alginate beads to allow long term culturing and formation of a cartilage-like matrix structure in the scaffold. Chondrocytes were infected with adenoviruses overexpressing either GFP or wild type HtrA1 or its mutants before seeding into alginate. Fig. 2A shows four adenoviral constructs encoding wild type and mutant HtrA1. All constructs were efficiently expressed in human chondrocytes, and the proteins were secreted into the culture medium, as illustrated in the Western blot performed using anti-HtrA1 antibodies (Fig. 2B). A control virus, VQ-GFP, was used to determine infection efficiency of the VQ adenovirus in human primary chondrocyte alginate culture. Four weeks post-infection, greater than 80% of the cells in the alginate culture were still expressing GFP as observed by fluorescent microscopy (data not shown). Adenoviral overexpression of wild type HtrA1 or a truncated proteolytically active mutant (ΔFS) in chondrocyte alginate bead cultures reduced total proteoglycan content in the inter-territorial area by 80% after 3 weeks of culture (Fig. 2C). Adenoviral overexpression of GFP or proteolytically inactive HtrA1 mutants (S328A or ΔFS-S328A) had no or little effect on proteoglycan content. None of the adenoviral constructs used in these studies affected cell viability as measured by trypan blue staining, total cell counts, or CyQUANT cell proliferation assays (data not shown).
      Figure thumbnail gr2
      FIGURE 2Proteoglycan content in three-dimensional chondrocyte/alginate cultures is dramatically reduced by HtrA1 overexpression. A, three-dimensional chondrocyte/alginate cultures were infected with wild type HtrA1, proteolytically inactive S328A, which contains a Ser to Ala mutation in the serine protease domain, or two HtrA1 truncations, ΔFS-HtrA1 and ΔFS-S328A. The follistatin (FS) domain is a combination of the IGF-binding protein (IGFBP) and the Kazal-type serine protease inhibitor (KI) domains. B, Western blot analysis using anti-HtrA1 antibodies shows all four constructs are efficiently expressed proteins in human chondrocytes. Note that less wild type HtrA1 protein was detected than the mutant form (S328A), potentially because of its autocatalytic activity (
      • Hu S.I.
      • Carozza M.
      • Klein M.
      • Nantermet P.
      • Luk D.
      • Crowl R.M.
      ). C, alginate beads were collected on day 21. Sulfated glycosaminoglycan (GAG) content in the beads was monitored by 1,9-dimethylmethylene blue assay (
      • Enobakhare B.O.
      • Bader D.L.
      • Lee D.A.
      ) (at pH 1.5). Results shown are the mean ± S.E. of the measured glycosaminoglycan levels for each treatment group (3 wells, 5 beads from each well; **, p < 0.01 as compared with GFP control). Chondrocyte alginate cultures from three human donors were used in three independent experiments. The results of a representative experiment are shown.

       Identification of Aggrecan as a Potential HtrA1 Substrate in the Context of Cartilage Matrix Formed in Chondrocyte Alginate Cultures

      The decrease of total proteoglycan in the alginate beads may be caused partly by an inhibitory effect of HtrA1 on proteoglycan synthesis as HtrA1 may block anabolic signaling mediated by growth factors such as transforming growth factor-β and IGF-1 in chondrocytes (
      • Oka C.
      • Tsujimoto R.
      • Kajikawa M.
      • Koshiba-Takeuchi K.
      • Ina J.
      • Yano M.
      • Tsuchiya A.
      • Ueta Y.
      • Soma A.
      • Kanda H.
      • Matsumoto M.
      • Kawaichi M.
      ,
      • Hou J.
      • Clemmons D.R.
      • Smeekens S.
      ). In addition, active HtrA1 protease may directly cleave aggrecan and other matrix proteins in the cartilage-like alginate cultures, leading to the release of degradation products into the media. This study was designed to focus on the capability of HtrA1 to digest matrix proteins.
      During the culturing of the alginate beads, media were replaced every 2 days. The amounts of proteoglycan released into the media in a 2-day time frame were very low, and they were barely detectable using the dimethylmethylene blue assay. We employed a mass spectrometry-based approach to identify proteins or fragments including aggrecan degradative products in the culture media. Conditioned media from chondrocyte alginate beads that overexpressed wild type HtrA1 or the mutant S328A (collected on day 21) were subjected to SDS-PAGE and analyzed by nano-LC-MS/MS followed by peptide sequence searches against the NCBI non-redundant human protein data base. Proteins differentially released into the conditioned media in wild type versus S328A-overexpressing cultures were assumed to be either a direct result of cleavage by HtrA1 or an indirect result of degradation of the ECM due to the overexpression of HtrA1. Among the proteins in conditioned media that were unique to alginate bead cultures overexpressing active HtrA1, we found degraded products of aggrecan with various molecular weights (Fig. 3). These results are consistent with previous reports which described in vitro digestion of aggrecan by recombinant HtrA1 (
      • Tsuchiya A.
      • Yano M.
      • Tocharus J.
      • Kojima H.
      • Fukumoto M.
      • Kawaichi M.
      • Oka C.
      ,

      Ganu, V., Melton, R., Hu, S., Koehn, J., Klein, M., Liebman, J., (2001) Orthopaedic Research Society 47th Annual Meeting, February 25–28, 2001, San Francisco, CA, Vol. 26, p. 266

      ,

      Goldberg, R., Crowl, R., Hu, S., (2000) Orthopaedic Research Society 46th Annual Meeting, March 12–15, 2000, Orlando, FL, Vol. 25, p. 164

      ). Our proteomic results also suggest that HtrA1 is capable of cleaving aggrecan in the context of cartilage matrix.
      Figure thumbnail gr3
      FIGURE 3Mass spectrometry identified aggrecan as a potential HtrA1 substrate in the context of cartilage matrix formed in chondrocyte alginate cultures. Three-dimensional chondrocyte/alginate cultures overexpressing wild type HtrA1 or S328A were cultured for 21 days. Culture media were replaced every 2 days. Conditioned media collected on day 21 was separated by SDS-PAGE, subjected to in-gel tryptic digestion, and analyzed by LC-MS/MS. Multiple degradation products of aggrecan (asterisks) of varying molecular weights were detected in conditioned media from alginate cultures overexpressing wild type HtrA1 (black circles) but not S328A (gray circles).

       Identification of a Novel HtrA1-specific Cleavage Site within the Aggrecan Core Protein

      To identify an HtrA1-susceptible cleavage site within the aggrecan core protein, recombinant aggrecan protein constructs encompassing the IGD (Fig. 4A) or G1-IGD-G2 (Fig. 4B) domains were incubated with purified ΔFS-HtrA1 (amino acids 157–480) or ΔFS-S328A recombinant proteins with or without the CPII peptide agonist. ΔFS-HtrA1 exhibits similar protease activity as full-length HtrA1 in vitro using aggrecan as substrate (data not shown) and is a commonly used form of the purified protein (
      • Grau S.
      • Richards P.J.
      • Kerr B.
      • Hughes C.
      • Caterson B.
      • Williams A.S.
      • Junker U.
      • Jones S.A.
      • Clausen T.
      • Ehrmann M.
      ,
      • Tsuchiya A.
      • Yano M.
      • Tocharus J.
      • Kojima H.
      • Fukumoto M.
      • Kawaichi M.
      • Oka C.
      ,
      • Murwantoko
      • Yano M.
      • Ueta Y.
      • Murasaki A.
      • Kanda H.
      • Oka C.
      • Kawaichi M.
      ). We have used this truncated protein throughout the remainder of this report. CPII is a synthetic peptide comprising the terminal hexapeptide derived from the C-terminal propeptide of type II collagen, which has previously been shown to stimulate the protease activity of HtrA1 by ∼3-fold using bovine serum albumin as the substrate (
      • Murwantoko
      • Yano M.
      • Ueta Y.
      • Murasaki A.
      • Kanda H.
      • Oka C.
      • Kawaichi M.
      ). It has been shown that HtrA1 PDZ domain interacts with the C-terminal propeptide of procollagen type II and III (Col2a1-C-Pro and Col3a1-C-Pro) with high affinity (
      • Murwantoko
      • Yano M.
      • Ueta Y.
      • Murasaki A.
      • Kanda H.
      • Oka C.
      • Kawaichi M.
      ). Elevated levels of Col2a1-C-Pro (7.6-fold) has been observed in osteoarthritic cartilage as a result of increased collagen synthesis in an potential attempt at repair (
      • Nelson F.
      • Dahlberg L.
      • Laverty S.
      • Reiner A.
      • Pidoux I.
      • Ionescu M.
      • Fraser G.L.
      • Brooks E.
      • Tanzer M.
      • Rosenberg L.C.
      • Dieppe P.
      • Robin Poole A.
      ). Incubation of recombinant aggrecan with ΔFS-HtrA1 (but not ΔFS-S328A) for 3 h resulted in distinct cleavage products as detected by SDS-PAGE followed by Coomassie Blue staining (Fig. 4, A and B). Interestingly, this cleavage was strongly enhanced by the addition of CPII. Overnight incubation (16 h) of ΔFS-HtrA1/CPII with aggrecan resulted in complete digestion of aggrecan constructs (data not shown). The ΔFS-S328A mutant alone (Fig. 4B) or ΔFS-S328A plus CPII (data not shown) caused no digestion of aggrecan proteins. N-terminal sequencing of the aggrecan degradative fragments identified the HtrA1-specific cleavage site as VQTV356357TWPD within the IGD of aggrecan (Fig. 4, A and B). A polyclonal neoepitope antibody was raised against the newly formed C termini VQTV356 of aggrecan as described under “Experimental Procedures.” Western blot analysis of in vitro ΔFS-HtrA1-digested purified human aggrecan using anti-VQTV356 demonstrated that this antibody does not react with undigested human aggrecan, but it specifically recognizes the newly formed C terminus after HtrA1-mediated cleavage (Fig. 4C). The HtrA1-specific cleavage site within the interglobular domain lies between a known MMP-susceptible site and a known aggrecanase site. As expected, Western blot analysis confirmed this, as the size of HtrA1-generated VQTV356 neoepitope (∼60 kDa) lies in between the MMP-generated DIPEN341 (∼50 kDa) and the aggrecanase-generated TEGE373 neoepitopes (∼72 kDa) (Fig. 4C).
      Figure thumbnail gr4
      FIGURE 4HtrA1 cleaves aggrecan within the IGD. Recombinant aggrecan protein fragments (black arrows) encompassing the IGD (A) or G1-IGD-G2 (B) were incubated with purified ΔFS-HtrA1, ΔFS-S328A, or ΔFS-HtrA1 with CPII for 3 h. Digestion products (gray arrow) were visualized by SDS-PAGE followed by Coomassie Blue staining. N-terminal sequencing identified the HtrA1 cleavage site as VQTV356357TWPD. The white arrows represent HtrA1 proteins, and U represents undigested aggrecan proteins. C, a neoepitope antibody, which recognizes HtrA1-digested aggrecan at VQTV356, was developed. Purified full-length human aggrecan, undigested (first lane) or HtrA1-digested (second lane), was analyzed by Western blot. Anti-VQTV356 solely recognizes HtrA1-digested aggrecan but not the intact aggrecan. The size of the VQTV356 neoepitope lies between the size of aggrecanase-specific digestion products, detected by the agg-c1/TEGE373 neoepitope antibody (third lane), and the MMP-specific digestion products, detected by the IPEN341 neoepitope antibody (fourth lane).

       HtrA1-digested Aggrecan Is More Abundant in Osteoarthritic Cartilage Than in Age-matched Control Cartilage

      To further evaluate the physiological relevance of HtrA1-mediated cleavage of aggrecan within cartilage and in the pathophysiology of OA disease progression, we performed Western blot analysis on human cartilage protein extracts isolated from OA and age-matched donors using the anti-VQTV356 neoepitope antibody. HtrA1-digested aggrecan was present in all seven OA cartilage extracts tested, whereas age-matched control donors demonstrated little or no cleavage of aggrecan by HtrA1 (Fig. 5A). To assess the relative contribution of HtrA1 digestion of aggrecan to aggrecanase activity in OA cartilage, comparable enzyme-linked immunosorbent assays were performed. The aggrecanase-specific NITEGE373 neoepitope was present at levels about 20-fold over the VQTV356 cleavage product (based on nanomolar concentrations) in 7 osteoarthritic cartilage extract samples (see supplemental Fig. S1). These data suggest that cleavage of aggrecan by HtrA1 is also a physiologically relevant event that could contribute to extensive aggrecan degradation in OA.
      Figure thumbnail gr5
      FIGURE 5HtrA1-digested aggrecan fragment is more abundant in osteoarthritic cartilage. A, human protein extracts isolated from cartilage from seven patients diagnosed with OA and seven age-matched donors as described above were subjected to Western blot analysis using anti-VQTV356 neoepitope antibody. HtrA1-generated aggrecan neoepitope (∼82 kDa) was found to be abundant in all OA samples tested, whereas it was barely detected in age-matched non-OA samples. The control (C) represents conditioned media from cartilage explants treated with HtrA1. Treatment of human cartilage explant with active site mutant HtrA did not generate the VQTV356 fragment. Coomassie Blue staining of gels (bottom) shows equal protein loading. B, OA cartilage protein extracts were incubated with excess exogenous HtrA1 in vitro (lane 3). The VQTV356 fragment present in OA cartilage (∼82 kDa, lane 2) was converted to a size comparable with in vitro HtrA1-digested aggrecan (∼60 kDa, lane 1).
      The molecular size (∼82 kDa) of the aggrecan fragment containing VQTV356 in osteoarthritic protein extracts (Fig. 5A) was somewhat larger than that of the VQTV356-positive fragment generated by in vitro cleavage of purified aggrecan (Fig. 4C). This is probably not due to a secondary N-terminal HtrA1 cleavage site, as further digestion of MMP-digested aggrecan with HtrA1 does not decrease the size of the MMP-specific DIPEN341 fragment (data not shown). It is possible, however, that HtrA1-digested aggrecan, in the context of the extracellular matrix, is complexed with an unknown protein, which retards its migration on SDS-PAGE and is removed by cleavage of aggrecan with excess exogenous HtrA1 in vitro. In fact, Western blot analysis showed further digestion of OA cartilage protein extracts with excess exogenous HtrA1 in vitro converted the 82-kDa VQTV356 immunoreactive band to one comparable with in vitro HtrA1-digested aggrecan (∼60 kDa) (Fig. 5B).
      To examine the possible contribution of other HtrA family members in the cleavage of aggrecan in OA, we examined the expression levels of HtrA2, HtrA3, and HtrA4 in human OA articular cartilage. HtrA2 and HtrA4 were expressed at very low or undetectable levels, respectively, as measured by real-time quantitative PCR and, therefore, presumably do not significantly contribute to osteoarthritis disease progression (Fig. 6A). HtrA3 was expressed at about 3-fold less than HtrA1 (Fig. 6A) and is known to have similar anti-anabolic functions (
      • Tocharus J.
      • Tsuchiya A.
      • Kajikawa M.
      • Ueta Y.
      • Oka C.
      • Kawaichi M.
      ). Our results showed incubation of aggrecan with HtrA3 did not generate the VQTV356 neoepitope (Fig. 6B); however, these data do not exclude the possibility that HtrA3 may contribute to matrix degradation during OA.
      Figure thumbnail gr6
      FIGURE 6Expression analysis of HtrA2, HtrA3, and HtrA4 in human osteoarthritic cartilage. A, relative expression levels of HtrA family members, HtrA1, HtrA2, HtrA3, and HtrA4, in human osteoarthritic cartilage were determined. Quantitative real time reverse transcription-PCR showed HtrA2 and HtrA4 are expressed at very low levels or was not detected (ND), respectively. HtrA3 was expressed 3-fold less than HtrA1. Expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). These data represent the average protein expression levels in cartilage isolated from three individual human donors. B, HtrA1- and HtrA3-digested aggrecan was analyzed by Western blot. HtrA1, not HtrA3, digests aggrecan at VQTV356 as shown by the presence of the immunoreactive band. UD represents undigested aggrecan as a control.

       Immunostaining for HtrA1-generated Aggrecan Neoepitope in Human Cartilage

      We then examined the localization of HtrA1-mediated aggrecan cleavage in human osteoarthritic cartilage by immunohistochemistry (Fig. 7). Immunostaining for the VQTV356 neoepitope was detected mainly in the surface layer of the cartilage (Fig. 7, A–C), where matrix degradation typically initiates in OA. Whereas more concentrated staining was observed in the surface layer where extensive loss of chondrocyte cells and matrix proteins was evident (Fig. 7, G–I, Safranin O staining), the staining also extends into the deeper zones of the cartilage. A diffuse staining pattern was observed; however, intense staining was also detected in the chondrocyte lacunae.
      Figure thumbnail gr7
      FIGURE 7Immunohistochemical detection of HtrA1-generated aggrecan neoepitope in human arthritic cartilage. Localization of HtrA1-cleaved aggrecan fragments in human femoral head cartilage with OA was analyzed by immunohistochemistry (counterstained with hematoxylin). Serial sections of cartilage samples (A, D, and G; B, E, and H; C, F, and I are sections from three different cartilage samples) were stained with either affinity-purified anti-VQTV356 IgG (A–C) or rabbit normal IgG as control (D–F). Safranin O staining of the sections (G–I) shows loss of proteoglycan in the surface layer of the cartilage. Note the diffuse brown VQTV356 staining in the matrix as well as more intense staining of the chondrocyte lacunae in A–C. Staining of chondrocyte lacunae is visible in the deeper layers of cartilage in OA samples. Bar = 200 μm.

      DISCUSSION

      The extracellular matrix of articular cartilage is comprised mainly of proteoglycans (∼20% by weight) and type II collagen fibers (∼5%) in addition to water (75%). Balanced synthesis and turnover of matrix components is required to maintain a functional cartilage structure. Both insufficient matrix protein synthesis and excessive degradation can lead to pathological changes often seen in degenerative conditions such as OA. Thus, in addition to structural macromolecules, many other proteins, for example growth factors, cytokines, and proteases, also play important roles in maintaining cartilage homeostasis. Dysregulated expression of these proteins may thereby also contribute to pathological sequelae.
      Mass spectrometry technology affords opportunities to semiquantitatively profile the cartilage proteome. Analysis of total protein extracts of OA cartilage specimens identified HtrA1 as the most abundant protease that was detectable. Among all the identified proteins, HtrA1 was one of the most abundant, with a level approaching some structural proteins such as link protein and COMP (link protein and COMP are less than 10-fold more abundant than HtrA1). Due to the fact that HtrA1 expression is induced during OA and given the relatively high abundance of HtrA1 found in cartilage, it is likely that HtrA1 activity(ies) play a contributing role in OA pathogenesis. Indeed, our results indicate that HtrA1 contributes to proteoglycan destruction by degrading aggrecan in vivo, a hallmark feature of OA disease progression.
      In an effort to gain insight into the physiological function of HtrA1 in cartilage, we also employed mass spectrometry to identify potential HtrA1 substrates within the context of the ECM made by human chondrocytes embedded in alginate beads. Aggrecan degradation products were found to be differentially released into conditioned media from wild type HtrA1-overexpressing chondrocyte/alginate cultures. These data support previous findings identifying aggrecan as a potential substrate (
      • Tsuchiya A.
      • Yano M.
      • Tocharus J.
      • Kojima H.
      • Fukumoto M.
      • Kawaichi M.
      • Oka C.
      ,

      Ganu, V., Melton, R., Hu, S., Koehn, J., Klein, M., Liebman, J., (2001) Orthopaedic Research Society 47th Annual Meeting, February 25–28, 2001, San Francisco, CA, Vol. 26, p. 266

      ,

      Goldberg, R., Crowl, R., Hu, S., (2000) Orthopaedic Research Society 46th Annual Meeting, March 12–15, 2000, Orlando, FL, Vol. 25, p. 164

      ). Here, we extended these findings by identifying the HtrA1-specific cleavage site as VQTV356357TWPD within the interglobular domain of the aggrecan core protein.
      Using a newly generated neoepitope antibody, we demonstrated HtrA1-digested aggrecan was present in human osteoarthritic cartilage and localized to areas of severe cartilage degradation (Fig. 7). Our data correlate with a previous study which employed a mouse experimental arthritis model and found much higher levels of HtrA1 protein in regions of the cartilage surface where most chondrocytes were dead and cartilage was severely damaged with massive depletion of glycosaminoglycans. As arthritis progressed in this model, chondrocytes in all layers and particularly those showing characteristic morphology of hypertrophic chondrocytes became positive for HtrA1 production (
      • Tsuchiya A.
      • Yano M.
      • Tocharus J.
      • Kojima H.
      • Fukumoto M.
      • Kawaichi M.
      • Oka C.
      ). Interestingly, a proportion of the aggrecan G1 fragment containing the HtrA1-generated VQTV356 neoepitope was also released from cartilage explants when treated with exogenous HtrA1 (Fig. 5A), similar to what was observed with the comparable aggrecan G1 fragment containing the aggrecanase-generated NITEGE373 neoepitope (
      • Chockalingam P.S.
      • Zeng W.
      • Morris E.A.
      • Flannery C.R.
      ).
      Analysis of cartilage protein extracts showed that HtrA1-digested aggrecan is more prevalent in osteoarthritic cartilage compared with control cartilage from age-matched patients (Fig. 5). Our results also showed that HtrA1 protease activity accounted for a considerable proportion of digested aggrecan present in osteoarthritic cartilage. Quantitatively, the VQTV356 neoepitope was present at about 5% that of the level of NITEGE373 neoepitope in osteoarthritic cartilage protein extracts tested. Considering the prominence and highly active state of aggrecanases in osteoarthritic cartilage comparatively, the potential contribution of HtrA1 in cartilage matrix degradation in OA may also be significant. Because of this, the VQTV356 neoepitope antibody has potential use as a biomarker for osteoarthritic disease.
      The HtrA1-specific cleavage site at VQTV356357TWPD lies between a known aggrecanase site at TEGE373374ARGS and an MMP cleavage site at DIPEN341342FFGV. Digestion of aggrecan at these sites within the IGD are considered critical events, which occur during the extensive cartilage degradation in OA (
      • Lohmander L.S.
      • Neame P.J.
      • Sandy J.D.
      ,
      • Sandy J.D.
      • Flannery C.R.
      • Neame P.J.
      • Lohmander L.S.
      ). Cleavage within the IGD releases the highly glycosylated and hydrated aggrecan C terminus (which gives cartilage its compressibility and stiffness) from the cartilage matrix. Loss of this region of aggrecan from the ECM results in severe weakening and erosion of cartilage in osteoarthritic joints. HtrA1-mediated aggrecan digestion within the IGD may represent another damaging cleavage event during OA progression. Reportedly aggrecan is not the only substrate that can be cleaved by HtrA1 in cartilage matrix (
      • Tsuchiya A.
      • Yano M.
      • Tocharus J.
      • Kojima H.
      • Fukumoto M.
      • Kawaichi M.
      • Oka C.
      ). Also it is likely that HtrA1 may contribute to OA disease progression by antagonizing signaling mediated by anabolic factors such as transforming growth factor-β (
      • Oka C.
      • Tsujimoto R.
      • Kajikawa M.
      • Koshiba-Takeuchi K.
      • Ina J.
      • Yano M.
      • Tsuchiya A.
      • Ueta Y.
      • Soma A.
      • Kanda H.
      • Matsumoto M.
      • Kawaichi M.
      ) and IGF-1 (
      • Hou J.
      • Clemmons D.R.
      • Smeekens S.
      ).

      Acknowledgments

      We thank Eric Fortier, Paul J. Yaworsky, Maya Arai, Diane Peluso, Richard T. Sheldon, Julio Tejada, Ning Li, Mei Zhang, Weilan Zeng, Amanda Bemis, and Jiang Wu for technical assistance and helpful suggestions. We also thank Wayne Stochaj and colleagues for HtrA1 protein expression and purification.

      Supplementary Material

      REFERENCES

        • Clausen T.
        • Southan C.
        • Ehrmann M.
        Mol. Cell. 2002; 10: 443-455
        • Dewan A.
        • Liu M.
        • Hartman S.
        • Zhang S.S.
        • Liu D.T.
        • Zhao C.
        • Tam P.O.
        • Chan W.M.
        • Lam D.S.
        • Snyder M.
        • Barnstable C.
        • Pang C.P.
        • Hoh J.
        Science. 2006; 314: 989-992
        • Grau S.
        • Baldi A.
        • Bussani R.
        • Tian X.
        • Stefanescu R.
        • Przybylski M.
        • Richards P.
        • Jones S.A.
        • Shridhar V.
        • Clausen T.
        • Ehrmann M.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 6021-6026
        • Grau S.
        • Richards P.J.
        • Kerr B.
        • Hughes C.
        • Caterson B.
        • Williams A.S.
        • Junker U.
        • Jones S.A.
        • Clausen T.
        • Ehrmann M.
        J. Biol. Chem. 2006; 281: 6124-6129
        • Hu S.I.
        • Carozza M.
        • Klein M.
        • Nantermet P.
        • Luk D.
        • Crowl R.M.
        J. Biol. Chem. 1998; 273: 34406-34412
        • Tsuchiya A.
        • Yano M.
        • Tocharus J.
        • Kojima H.
        • Fukumoto M.
        • Kawaichi M.
        • Oka C.
        Bone. 2005; 37: 323-336
        • Yang Z.
        • Camp N.J.
        • Sun H.
        • Tong Z.
        • Gibbs D.
        • Cameron D.J.
        • Chen H.
        • Zhao Y.
        • Pearson E.
        • Li X.
        • Chien J.
        • Dewan A.
        • Harmon J.
        • Bernstein P.S.
        • Shridhar V.
        • Zabriskie N.A.
        • Hoh J.
        • Howes K.
        • Zhang K.
        Science. 2006; 314: 992-993
        • Bowden M.A.
        • Di Nezza-Cossens L.A.
        • Jobling T.
        • Salamonsen L.A.
        • Nie G.
        Gynecol. Oncol. 2006; 103: 253-260
        • Chien J.
        • Staub J.
        • Hu S.I.
        • Erickson-Johnson M.R.
        • Couch F.J.
        • Smith D.I.
        • Crowl R.M.
        • Kaufmann S.H.
        • Shridhar V.
        Oncogene. 2004; 23: 1636-1644
        • Baldi A.
        • De Luca A.
        • Morini M.
        • Battista T.
        • Felsani A.
        • Baldi F.
        • Catricalà C.
        • Amantea A.
        • Noonan D.M.
        • Albini A.
        • Natali P.G.
        • Lombardi D.
        • Paggi M.G.
        Oncogene. 2002; 21: 6684-6688
        • Hadfield K.D.
        • Rock C.F.
        • Inkson C.A.
        • Dallas S.L.
        • Sudre L.
        • Wallis G.A.
        • Boot-Handford R.P.
        • Canfield A.E.
        J. Biol. Chem. 2008; 283: 5928-5938
      1. Ganu, V., Melton, R., Hu, S., Koehn, J., Klein, M., Liebman, J., (2001) Orthopaedic Research Society 47th Annual Meeting, February 25–28, 2001, San Francisco, CA, Vol. 26, p. 266

      2. Goldberg, R., Crowl, R., Hu, S., (2000) Orthopaedic Research Society 46th Annual Meeting, March 12–15, 2000, Orlando, FL, Vol. 25, p. 164

        • Oka C.
        • Tsujimoto R.
        • Kajikawa M.
        • Koshiba-Takeuchi K.
        • Ina J.
        • Yano M.
        • Tsuchiya A.
        • Ueta Y.
        • Soma A.
        • Kanda H.
        • Matsumoto M.
        • Kawaichi M.
        Development. 2004; 131: 1041-1053
        • Tocharus J.
        • Tsuchiya A.
        • Kajikawa M.
        • Ueta Y.
        • Oka C.
        • Kawaichi M.
        Dev. Growth Differ. 2004; 46: 257-274
        • Hou J.
        • Clemmons D.R.
        • Smeekens S.
        J. Cell Biochem. 2005; 94: 470-484
        • Enobakhare B.O.
        • Bader D.L.
        • Lee D.A.
        Anal. Biochem. 1996; 243: 189-191
        • Wu J.
        • Liu W.
        • Bemis A.
        • Wang E.
        • Qiu Y.
        • Morris E.A.
        • Flannery C.R.
        • Yang Z.
        Arthritis Rheum. 2007; 56: 3675-3684
        • Murwantoko
        • Yano M.
        • Ueta Y.
        • Murasaki A.
        • Kanda H.
        • Oka C.
        • Kawaichi M.
        Biochem. J. 2004; 381: 895-904
        • Song R.H.
        • Tortorella M.D.
        • Malfait A.M.
        • Alston J.T.
        • Yang Z.
        • Arner E.C.
        • Griggs D.W.
        Arthritis Rheum. 2007; 56: 575-585
        • Chockalingam P.S.
        • Zeng W.
        • Morris E.A.
        • Flannery C.R.
        Arthritis Rheum. 2004; 50: 2839-2848
        • Chubinskaya S.
        • Mikhail R.
        • Deutsch A.
        • Tindal M.H.
        J. Histochem. Cytochem. 2001; 49: 1165-1176
        • Chelius D.
        • Zhang T.
        • Wang G.
        • Shen R.F.
        Anal. Chem. 2003; 75: 6658-6665
        • Nelson F.
        • Dahlberg L.
        • Laverty S.
        • Reiner A.
        • Pidoux I.
        • Ionescu M.
        • Fraser G.L.
        • Brooks E.
        • Tanzer M.
        • Rosenberg L.C.
        • Dieppe P.
        • Robin Poole A.
        J. Clin. Invest. 1998; 102: 2115-2125
        • Lohmander L.S.
        • Neame P.J.
        • Sandy J.D.
        Arthritis Rheum. 1993; 36: 1214-1222
        • Sandy J.D.
        • Flannery C.R.
        • Neame P.J.
        • Lohmander L.S.
        J. Clin. Invest. 1992; 89: 1512-1516