(ADAMTS9) Expression by Chondrocytes During Endochondral Ossiﬁcation＊

Arch Histol Cytol, 72 (3): 175-185 (2009)

A disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS9) expression by chondrocytes during endochondral ossiﬁcation＊

Kanae Kumagishi1, Keiichiro Nishida1, Tomoichiro Yamaai2, Ryusuke Momota1, Shigeru Miyaki3, Satoshi Hirohata4, Ichiro Naito1, Hiroshi Asahara3, Yoshifumi Ninomiya4, and Aiji Ohtsuka1

Departments of 1Human Morphology, 2Oral Function and Anatomy, and 4Molecular Biology and Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama; and 3Department of Regenerative Biology and Medicine, National Research Institute for Child Health and Development, Tokyo, Japan

S u m m a r y . A d i s i n t e g r i n a n d m e t a l l o p r o t e i n a s e chondrocyte phenotypes, respectively. We found the gene with thrombospondin motifs 9 (ADAMTS9) is known expression of ADAMTS9 by ATDC5 cells as a dual mode, to influence aggrecan degradation in endochondral both before the expression of type X collagen and after ossification, but its role has not been well understood. hypertrophic differentiation. The immunoreactivity of In the present study, in vitro gene expression of ADAMTS9 ADAMTS9 was observed in chondrocytes of proliferative was investigated by RT-PCR in ATDC5 cells in which and hypertrophic zones in the growth plate. The experimentally chondrogenic differentiation had been population of ADAMTS9 positive cells decreased with age. induced. We also investigated the protein localization The results of the present study suggest that ADAMTS9 and gene expression pattern of ADAMTS9 in the tibia might have a role in aggrecan cleavage around the growth plate cartilage of male mice in a day 1 neonate, chondrocytes to allow chondrocyte proliferation and 7-week-old young adult, and a 12-week-old adult by hypertrophy. immunohistochemistry and in situ hybridization and compared the results with the expression of proliferating cell nuclear antigen (PCNA) and type X collagen for Introduction the identification of proliferative and hypertrophic In the process of endochondral ossification, undifferentiated chondrocytes undergo cell proliferation Received November 13, 2009 and differentiation to become mature chondrocytes in the growth plate. They produce abundant extracellular *This work was supported by a Grant for Child Health and matrices, mainly composed of aggrecan and type II Development (20A-3) from the Ministry of Health, Labour collagen (Tsumaki et al., 1996). When they differentiate and Welfare (K.N.) and a grant-in-aid for Scientific Research to hypertrophic chondrocytes, they generate type X from the JSPS (20390399 to S.H.). collagen and alkaline phosphatase (ALPase). With the cell death of the hypertrophic chondrocytes, the Address for correspondence: Keiichiro Nishida, M.D, Ph.D. extracellular matrix is mineralized, and replaced by bone (Associate Professor), Department of Human Morphology, after vascular invasion. Matrix degrading enzymes, such Okayama University Graduate School of Medicine, Dentistry as matrix metalloproteinases (MMPs) or aggrecanases and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, are known to play important roles during these processes Okayama 700-8558, Japan (Clare et al., 1995; Mandal et al., 2003). A disintegrin Phone: +81-86-235-7089, Fax: +81-86-235-7095 and metalloproteinase with thrombospondin motifs E-mail: [email protected] (ADAMTS) is a family of metalloproteinase, and some 176 K. Kumagishi et al.:

of them have been characterized by their aggrecanase and fixed on day 1 neonate, at 7 weeks, or at 12 weeks by activity which is responsible for aggrecan degradation perfusion via the left ventricle with 4% paraformaldehyde in the endochondral ossification and also in joint in a 0.1 M phosphate buffer (pH7.4) at room temperature. inflammatory diseases (Malfait et al., 2002; Kevorkian et After separation of the hind limbs, their skin was removed al., 2004; Naito et al., 2007). and the hind limbs were trimmed around the knee joint. The structures of ADAMTSs are homologous to a They were immersed in the same fixative at 4℃ overnight disintegrin and metalloproteinase (ADAM) proteins, but and then decalcified by 10% EDTA in a 0.1 M phosphate they also contain at least 1 C-terminal thrombospondin buffer (pH 7.5) for 1 week by daily refreshing with type 1 (TSP1) repeat and are secreted rather than shaking. After decalcification, specimens were dehydrated membrane bound (Porter et al., 2005). ADAMTS1, using graded concentrations of an ethanol series prior 4, 5, 8, 9, 15 have been classified into three groups to embedding in Paraplast plus (Sigma, St Louis, MO, by their capacity to degrade aggrecan molecules as USA). Serial sections (7 μm thickness) were cut and follows: aggrecanase-1 group includes ADAMTS1 mounted on APS coated slides. and 4; aggrecanase-2 group includes ADAMTS5; and aggrecanase-3 group includes ADAMTS8, 9, and 15. Cell cultures Of these, ADAMTS4 and 5 exhibit the most powerful aggrecanase activities (Abbaszade et al., 1999; Tortorella The ATDC5, a mouse chondrosarcoma cell line, was et al., 1999, 2001; Rogerson et al., 2008). ADAMTSs obtained from the Riken Cell Bank (Tsukuba). Cells were and the MMPs reportedly play important roles in the cultured in a maintenance medium consisting of a 1:1 metabolism in the extracellular matrix of the cartilage mixture of Dulbecco’s modified eagle (DME) and Ham’s during the development and the progress of joint F12 medium containing 5% fetal bovine serum (FBS) as diseases (Martel-Pelletier et al., 2001; Arner et al., 2002; described earlier (Shukunami et al., 1996). The ATDC5 Chockalingam et al., 2004; Pásztói et al., 2009). Although cells were plated at 6.4 ×104 cells /cm2 in a 6-well plate ADAMTSs are especially believed to be involved in the with DMEM and chondrogenesis was induced by insulin cellular growth, differentiation, and maturation of the (10 μg/ml, Nacalai Tesque, Kyoto). The medium was growth plate chondrocytes, their roles in the endochondral replaced every other day. Cells were maintained at 37℃ ossification have not been completely elucidated in a humidified atmosphere of 5% CO2 in air. (Demircan et al., 2005; Mitani et al., 2006; Hatipoglu et al., 2009a; Yaykasli et al., 2009). Reverse transcription-polymerase chain reaction Thus, the present study was designed to investigate (RT-PCR) the gene expression and protein localization pattern of ADAMTS9 in the growth plate cartilage of the mouse For the determination of ADAMTS9 and type X collagen tibia from neonate to 12 weeks of age using in situ expression in ATDC5 cells, total RNA was prepared hybridization and immunohistochemistry. ADAMTS9 by using an RNeasy Lipid Tissue Mini Kit (QIAGEN, mRNA expression was also confirmed by RT-PCR during Hilden, Germany). The cDNA of ATDC5 cells at 0, 8, 14, the insulin-induced chondrogenesis of ATDC5 cells. 22, 28, and 35 days were reverse transcribed from 1 μg Our results indicated that ADAMTS9 is expressed by of the total RNA by a Quanti Tect Reverse Transcription chondrocytes and might be involved in the process of Kit (QIAGEN, Hilden, Germany). A PCR procedure was endochondral ossification by supporting the chondrocyte performed according to Demircan’s method (Demircan et proliferentiation and hypertrophy with matrix degradation. al., 2005). Primer sequences are listed in Table 1. An RT- PCR analysis was performed using a Light Cycler Rapid Thermal Cycling system (Roche Diagnostics, Laval, Canada). The optimal cycle number was determined Materials and Methods empirically in each case to maximize the sensitivity of quantifications. PCR products were electroporated in Animals and tissue preparation 1.5% agarose gels containing ethidium bromide. All experiments were undertaken according to the Guidelines and Regulations for the Animal Immunohistochemistry Experimentation of Okayama University. Male ICR mice were used in this study. Newborn mice were maintained Sections were irradiated on ice with a 360 W microwave for up to 12 weeks. to activate antigens after deparaffinization. Irradiation was The mice were sacrificed under general anesthesia, performed using three cycles of two min irradiation with ADAMTS9 in growth plate chondrocytes 177

15 sec intervals for cooling down by using a microwave. Results Polyclonal rabbit antibodies directed against ADAMTS9 (diluted to 1:200, Abcam, Cambridge, UK), proliferating In vitro gene expression of ADAMTS9 and type X cell nuclear antigen (PCNA) (diluted to 1:200, Santa Cruz collagen during insulin-induced chondrogenesis in Biotechnology, Inc., CA, USA), and type X collagen ATDC5 cells (diluted to 1:200, Santa Cruz Biotechnology, Inc., CA, USA) were used with Histofine® Simple Stain Max-PO The gene expression of ADAMTS9 was detected by (Nichirei, Tokyo). To reduce non-specific background PCR for comparison with the gene expression of type X signals, sections were blocked with a normal serum. To collagen in insulin-induced chondrogenesis of ATDC5 block endogenous peroxidase activity, 1 mM sodium cells up to 35 days (Fig. 1). The gene expression of type azide was added to a color reaction medium containing X collagen was observed after 22 days of treatment. On 3-3’-diaminobenzidine (DAB); this reaction was enhanced the other hand, the gene expression of ADAMTS9 showed by adding 20 mM nickel chloride. After immunoreactions were completed, sections were counterstained with methylgreen.

In situ hybridization The cDNA clone used for ADAMTS9 ribo probe synthesis was a generous gift from Dr. Suneel S. Apte. Ribo probes for type X collagen were prepared as described previously (Webster et al., 2003). Labeled transcripts of mouse ADAMTS9 and type X collagen were synthesized either with T7 or Sp6 RNA polymerase with a digoxigenin labeled RNA labeling kit (Roche Diagnostics, Laval, Canada). Transcripts in sense orientation revealed no speciﬁc hybridization pattern. In situ hybridization was performed according to the method by Meyer et al. (1997) with minor modifications. Sections were deparaffinized and digested with 20 μg/ml proteinase K (Sigma) for 10 min at room temperature. They were incubated with acetic Fig. 1. The gene expression of ADAMTS9 on insulin- anhydride (Triethanolamin, HCl in DW before use and induced chondrogenesis of ATDC5 cells up to 35 days. then acetic anhydride was added) with vigorous agitation The gene expression of type X collagen was used as a for 15 min at room temperature. After sections were marker of hypertrophic chondrocytes. The gene expression treated with riboprobes in a buffer, they were incubated of type X collagen was observed after 22 days of treatment. ℃ on slides at 55 overnight. Signals were visualized by a The gene expression of ADAMTS9 showed two peaks: the colorimetric reaction with nitroblue tetrazolium chloride first peak was found from 8 to 14 days after induction, (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) and the second peak was found from 28 to 35 days after ℃ at 4 for 4 days. The sections were counterstained with induction. methylgreen after the color reaction.

Table 1. Primers for RT-PCR Oligonucleotide sequence (5’- -3’) Accession No. ADAMTS9 Forward GGA CAA GTG AAG GAC ATC C NM-175314 Reverse TAG GCA GAG GAT ACC GAA GG (Jungers et al., 2005) Type X Forward GCA GCA TTA CGA CCC AAG AT NM-009925 Collagen Reverse TCT GTG AGC TCC ATG ATT GC (Nakajima et al., 2009) GAPDH Forward CAA CTG CCA ACG TGT CAG TGG Reverse AAG GTG GAG GAG TGG GTG TCG 178 K. Kumagishi et al.:

Fig. 2. HE staining (A) and immunohistochemistry for ADAMTS9 (B), type X collagen (C), and proliferating cell nuclear antigen (PCNA, D) in the tibia growth plate cartilage of a normal 7-week- old young adult mouse. Immunoreactions for signals for PCNA and type X collagen were restricted to chondrocytes of the proliferative and hypertrophic zones, respectively, while ADAMTS9 (arrows) was found in chondrocytes of the proliferative (PR) and hypertrophic (HY) zones. To increase the sensitivity of color reaction, nickel choride was added to the color reaction medium containing 3-3’-diaminobenzide (DAB). Bar = 50 μm (D)

double peaks: the first peak of the gene expression of chondrocytes in both proliferative and hypertrophic zones ADAMTS9 was found from 8 to 14 days, and the second (Fig. 2B). The signal of immunoreaction for ADAMTS9 peak was found from 28 to 35 days after induction. The was more intense in chondrocytes of the hypertorophic gene expression of ADAMTS9 at the ﬁrst peak was more zone than in those of the proliferative zone. intense than that at the second peak. Comparison of the gene expression and the Comparison of immunoreactivities of ADAMTS9, immunoreactivities of ADAMTS9 in the tibia of type X collagen, and PCNA in 7 week-old adult mice neonatal mice in vivo In the growth plate cartilage of the 7-week-old mouse The gene expression of ADAMTS9 was observed in tibia, signals for PCNA were observed in chondrocytes chondrocytes of proliferative and hypertrophic zones of the proliferative zone in which chondrocytes were in the growth plate cartilage of normal neonates but not organized into distinct columns (Fig. 2D). A positive in chondrocytes of the reserve zone (Fig. 3A, C). This immunoreaction for type X collagen was found only in expression pattern corresponds closely to the ascending chondrocytes of the hypertrophic zone, which contained expression of ADAMTS9 found by PCR in ATDC5 cells greatly enlarged cells (Fig. 2C). Cells in the reserve 8 days after the induction of chondrogenesis by insulin zone did not show any signals for PNCA nor for type (Fig. 1). By immunohistochemistry, the localization of X collagen. The widths of the positive cells for both ADAMTS9 positive cells also showed a pattern similar PCNA and type X collagen in young adult mice were to the gene expression pattern mentioned above (Fig. 3 B, narrower than those of neonates ( data not shown). D). Immunoreactivity for ADAMTS9 was observed in ADAMTS9 in growth plate chondrocytes 179

Fig. 3. Patterns of the gene expression and the localization of ADAMTS9 in neonatal mice tibia. A, C: Expression of the ADAMTS9 mRNA detected by in situ hybridization. B, D: Localization of ADAMTS9 protein detected by immunohistochemistry. The gene expression of ADAMTS9 was observed in chondrocytes of the proliferative (PR) and hypertrophic (HY) zones but not in chondrocytes of the reserve (RV) zone in the growth plate cartilage. By immunohistochemistry, the localization of ADAMTS9 positive cells showed a pattern similar to the gene expression pattern. Arrowheads indicate gene expression for ADAMTS9. Arrows indicate protein localization for ADAMTS9. Bars = 100 μm (A), 50 μm (C) 180 K. Kumagishi et al.:

Fig. 4. Patterns of the gene expression and localization of ADAMTS9 in normal 7-week-old young adult mouse tibia. A, C: Expression of ADAMTS9 mRNA detected by in situ hybridization. B, D: Localization of ADAMTS9 protein detected by immunohistochemistry. The gene expression of ADAMTS9 was observed in chondrocytes of both the proliferative (PR) and hypertrophic (HY) zones but not in chondrocytes of the reserve (RV) zone. The endochondral ossiﬁcation was still continuous at this age although the zone of proliferative chondrocytes was not so thick as that of neonates. The pattern of immunoreactivities of ADAMTS9 was similar to the gene expression pattern of ADAMTS9. Arrowheads indicate the gene expression for ADAMTS9. Arrows indicate protein localization for ADAMTS9. Bars = 100 μm (A), 50 μm (C) ADAMTS9 in growth plate chondrocytes 181

Fig. 5. Patterns of the expression and localization of ADAMTS9 in normal 12-week-old adult mouse tibia. A, C: Expression of ADAMTS9 mRNA detected by in situ hybridization. B, D: Localization of ADAMTS9 protein detected by immunohistochemistry. Positive hybridization signals for ADAMTS9 were observed in chondrocytes of the hypertrophic (HY) zone in the growth plate cartilage. Immunoreactivity for ADAMTS9 showed a pattern similar to the ADAMTS9 gene expression. Arrowheads indicate the gene expression for ADAMTS9. Arrows indicate protein localization for ADAMTS9. Bars = 100 μm (A), 50 μm (C)

found by PCR of insulin-induced chondrogenesis of Comparison of the gene expression and the ATDC5 cells after day 28 (Fig. 1). The pattern of immunoreactivities of ADAMTS9 in the tibia of young immunoreactivities of ADAMTS9 was similar to the gene adult mice in vivo at 7 weeks of age expression pattern of ADAMTS9, as mentioned above The gene expression of ADAMTS9 was observed in (Fig. 4 B, D). chondrocytes of both the proliferative and hypertrophic zones but not in chondrocytes of the reserve zone in Comparison of the gene expression and the normal 7-week-old adult mice (7 weeks, Fig. 4A, C). The immunoreactivities of ADAMTS9 in the tibia of endochondral ossiﬁcation was still continuous at this age 12-week-old adult mice in vivo although the zone of immature chondrocytes was not so thick as that of 0-d or 1-week old mice. This expression Positive hybridization signals for ADAMTS9 were was in accord with the second peak of ADAMTS9 observed in chondrocytes of hypertrophic zone in the 182 K. Kumagishi et al.:

growth plate cartilage in 12-week-old adult mice (12 100% hypertrophic zones, but this was only slightly weeks, Fig. 5 A, C). There were fewer small chondrocytes expressed in chondrocytes of the 31–70% proliferative that could still be observed at the top of the growth plate zone in the growth plate cartilage. On the other hand, the at 12 weeks; however, these were immunohistochemically population of ADAMTS9-positive cells in 12-week-old negative for both PCNA and ADAMTS9 while the adult mice were 11–30% hypertrophic zone in the growth immunoreactivity for ADAMTS9 showed a pattern plate cartilage. No positive immunoreaction was seen in similar to the ADAMTS9 gene expression pattern chondrocytes of the reverse zone. The signal intensity mentioned above (Fig. 5B, D), the strength of the of both the gene expression and the immunoreactivities immunohistochemical signals did decrease with age. of ADAMTS9 decreased with age. Undifferentiated mesenchymal cells, osteoclasts, and osteoblasts expressed no ADAMTS9 signal at any age. Age related differences in the localization of ADAMTS9 Age-related differences in immunoreactivity for ADAMTS9 were investigated in the growth plate Discussion cartilage of the tibia from neonates to 12-week-old adult mice (Fig. 3 – 5). The population of ADAMTS9- The ADAMTS family consists of 19 members, and positive cells in normal neonates in both zones of the ADAMTS1, 4, 5, 8, 9, and 15 are capable of cleaving an growth plate cartilage were as follows (Fig. 6): 71 – aggrecan molecule (Caterson et al., 2000; Porter et al., 100% positive cells in the proliferative chondrocyte zone, 2005; Hatipoglu et al., 2009b). Their aggrecanase activity and 71 – 100% in the hypertrophic chondrocyte zone. is known to responsible for aggrecan degradation in joint Both zones of the growth plate cartilage of neonates inﬂammatory diseases (Nagase et al., 2003; Kevorkian et were localized ubiquitously. The immunoreaction for al., 2004; Demircan et al., 2005; Bondeson et al., 2008). ADAMTS9 in 7-week-old young adults exhibited the It has also been reported that expressions of ADAMTS1, localized pattern mostly in chondrocytes of the 71 – 4, and 5 were found in the growth plate of the rat tibia (Jungers et al., 2005; Mitani et al., 2006), although there were no morphological difference between wild type and targeted mutant mice against ADAMTS1, 4, or 5 genes (Glasson et al., 2004; Little et al., 2005; Stanton et al., 2005). In the present study, we demonstrated for the ﬁrst time that ADAMTS9 is expressed at mRNA and protein levels in the mouse tibia growth plate. The present study investigated the gene expression of ADAMTS9 in vitro after the induction of chondrogenesis by insulin in ATDC5 cells and found that the gene expression of ADAMTS9 was exhibited two peaks, the first peak being observed from day 8 to day 14 after treatment, and the second peak being observed from day 28 to day 35. ADAMTS9-negative ATDC5 during the first week of cell culture might correspond to the chondrocytes of the reserve zone. As type X collagen expression, a marker for the hypertrophic chondrocyte, was detected after day 22, chondrocytes at the first Fig. 6. Summary of the expression and localization of peak correspond to the proliferative zone, and those ADAMTS9 at the protein level in the mouse growth plate of the second peak to the hypertrophic phenotype. cartilage. The left panel shows the layer of the growth plate The gene expression of the first peak was significantly cartilage; RV: reserve zone, PR: proliferative zone, HY: more intense than that of the second peak, suggesting hypertrophic zone. The reserve zone and proliferative zone a higher expression of ADAMTS9 by chondrocytes in are not identified in the growth plate of 12-week-old mice. the proliferative zone than the hypertrophic zone. It The population of ADAMTS-positive cells were expressed as: is considered that ADAMTS9 may provide space for –: 0%, ±: 0–10%, ＋: 11–30%, ＋＋: 31–70%, ＋＋＋: 70– the migration of proliferating chondrocytes as well as 100% expanding chondrocytes during terminal differentiation. ADAMTS9 in growth plate chondrocytes 183

We also investigated age-dependent differences in the aggrecanases for endochondral ossification. On the other gene expression and protein localization of ADAMTS9 hand, no phenotype of targeted mutants of ADAMTS9 during growth of the tibia from neonates to 12-week- has been reported yet. The aggrecanolytic activity of old adult, in comparison with the distribution of PCNA- ADAMTS9 weaker than that of ADAMTS4/5 might and type X collagen-positive chondrocytes. The results have an advantage in the relatively slow progression of showed that the expression and localization of ADAMTS9 cartilage growth. In addition to the results of our finding was broadly observed in chondrocytes of both the on the gene expression of ADAMTS9 in growth plate proliferative and hypertrophic zones in the growth plate chondrocytes, targeted gene silencing of ADAMTS9 cartilage in neonates and 7-week-old young adult mice in vitro would add to the functional importance of this (Fig. 2 – 5). In 12-week-old adult mice, the expression aggrecanase in the process of endochondral ossification. of ADAMTS9 was restricted to chondrocytes of the hypertrophic zone in the growth plate cartilage. There were fewer small chondrocytes still observed at the top Acknowledgements of the growth plate at 12 weeks; however, they were We thank Dr. Suneel S. Apte (Department of Biomedical immunohistochemically negative for both PCNA and Engineering, Lerner Research Institute, Cleveland Clinic ADAMTS9. These results suggest that the ADAMTS9 Foundation) for providing the cDNA clone. expression might not depend on the shape of the chondrocyte but on its developmental stage. Our findings also showed that, although the localization pattern of PCNA overlapped with that of ADAMTS9 in neonates, the localization of PCNA was a little broader than that of References ADAMTS9 at 12 weeks. The fact that signals for PCNA were found only in chondrocytes of the proliferative Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross zone in neonates and at 7 weeks, but not at 12 weeks, OH, Link JR, Ellis DM, Tortorella MD, Pratta MA, might indicate that the growth activity of the growth plate Hollis JM, Wynn R, Duke JL, George HJ, Hillman may decrease around 12 weeks of age in mice. Because MC Jr, Murphy K, Wiswall BH, Copeland RA, PCNA is known as a cell cycle related factor (Christov Decicco CP, Bruckner R, Nagase H, Itoh Y, Newton et al., 1975), the overlapped expression of PCNA and RC,Magolda RL, Trzaskos JM, Burn TC: Cloning and ADAMTS9 might indicate that the role of ADAMTS9 is characterization of ADAMTS11, an aggrecanase from not only one of the aggrecanase but also a factor related to the ADAMTS family. J Biol Chem 274: 23443-23450 cell proliferation. (1999). It was reported that ADAMTS1, 4, 5, 9 have Arner EC: Aggrecanase-mediated cartilage degradation. aggrecanase activities (Clark et al., 2000; Arner et Curr Opin Pharmacol 2: 322-329 (2002). al., 2002) while ADAMTS9 has a relatively weaker Bondeson J, Wainwright S, Hughes C, Caterson B: aggrecanase activity than the others (Stanton et al., 2005; The regulation of the ADAMTS4 and ADAMTS5 Zeng et al., 2006; Koo et al., 2007). Mitani et al. (2006) aggrecanases in osteoarthritis: a review. Clin Exp investigated the expression patterns of ADAMTS1, 4, and Rheumatol 26: 139-145 (2008). 5 in the rat mandibular condylar cartilage, and articular Caterson B, Flannery CR, Hughes CE, Little CB: and growth plate cartilage and concluded that ADAMTS5 Mechanisms involved in cartilage proteoglycan might mainly contribute to the extracellular matrix catabolism. Matrix Biol 19: 333-344 (2000). metabolism in the growth plate and condylar cartilage Chakraborti S, Mandal M, Das S, Mandal A, Chakraborti during growth. In contrast ADAMTS1 and ADAMTS4 T: Regulation of matrix metalloproteinases: an may be involved in the extracellular matrix turnover in overview. Mol Cell Biochem 253: 269-285 (2003). the articular cartilage in relation to ADAMTSs specific Chockalingam PS, Zeng W, Morris EA, Flannery CR: substrates such as aggrecan, brevican, and versican. Release of hyaluronan and hyaladherins (aggrecan In the present study, the gene expression and protein G1 domain and link proteins) from articular localization of ADAMTS9 showed patterns similar to cartilage exposed to ADAMTS-4 (aggrecanase 1) or those of ADAMTS5. In addition, it was reported that ADAMTS-5 (aggrecanase 2). Arthritis Rheum 50: there were no genotype-related phenotype differences 2839-2848 (2004). between ADAMTS4/5 double-knockout and wild type Christov K, Thomas C, Sandritter W: DNA measurements mice up to 1 year of age (Majumdar et al., 2007). These on cell nuclei of normal, proliferating and neoplastic results suggest that ADAMTS4/5 are not indispensable thyroid tissues in rats. Neoplasma 22: 285-294 (1975). 184 K. Kumagishi et al.:

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