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*LIOTHYRONINE
Endocrinology Vol. 144, No. 6 2480-2488
Copyright © 2003 by The Endocrine Society

Thyroid Hormone Enhances Aggrecanase-2/ADAM-TS5 Expression and Proteoglycan Degradation in Growth Plate Cartilage

Seicho Makihira, Weiqun Yan, Hiroshi Murakami, Masae Furukawa, Toshihisa Kawai, Hiroki Nikawa, Eri Yoshida, Taizo Hamada, Yasunori Okada and Yukio Kato

Departments of Prosthetic Dentistry (S.M., M.F., H.N., T.H.) and Biochemistry (W.Y., E.Y., Y.K.), Hiroshima University Faculty of Dentistry, Minami-ku, Hiroshima 734-8553, Japan; Department of Pediatrics, Osaka City University Medical School (H.M.), Abeno-ku, Osaka 545-8585, Japan; Department of Immunology, The Forsyth Institute (S.M., T.K.), Boston, Massachusetts 02115; and Department of Pathology, Keio University School of Medicine (Y.O.), Shinjuku-ku, Tokyo 160-8582, Japan

Address all correspondence and requests for reprints to: Dr. Seicho Makihira, Department of Immunology, The Forsyth Institute, 140 Fenway, Boston, Massachusetts 02115. E-mail: smakihira{at}forsyth.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Effects of thyroid hormone on proteoglycan degradation in various regions of cartilage were investigated. In propylthiouracil-treated rats with hypothyroidism, proteoglycan degradation in epiphyseal cartilage during endochondral ossification was markedly suppressed. However, injections of T4 reversed this effect of propylthiouracil on proteoglycan degradation. In pig growth plate explants, T3 also induced breakdown of proteoglycan. T3 increased the release of aggrecan monomer and core protein from the explants into the medium. Accordingly, the level of aggrecan monomer remaining in the tissue decreased after T3 treatment, and the monomer lost hyaluronic acid-binding capacity, suggesting that the cleavage site is in the interglobular domain. The aggrecan fragment released from the T3-exposed explants underwent cleavage at Glu373-Ala374, the major aggrecanase-cleavage site. The stimulation of proteoglycan degradation by T3 was less prominent in resting cartilage explants than in growth plate explants and was barely detectable in articular cartilage explants. Using rabbit growth plate chondrocyte cultures, we explored proteases that may be involved in T3-induced aggrecan degradation and found that T3 enhanced the expression of aggrecanase-2/ADAM-TS5 (a disintegrin and a metalloproteinase domain with thrombospondin type I domains) mRNA, whereas we could not detect any enhancement of stromelysin, gelatinase, or collagenase activities or any aggrecanase-1/ADAM-TS4 mRNA expression. We also found that the aggrecanse-2 mRNA level, but not aggrecanase-1, increased at the hypertrophic stage during endochondral ossification. These findings suggest that aggrecanse-2/ADAM-TS5 is involved in aggrecan breakdown during endochondral ossification, and that thyroid hormone stimulates the aggrecan breakdown partly via the enhancement of aggrecanase-2/ADAM-TS5.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTERATIONS in the secretion of thyroid hormone caused by hypothyroidism or hyperthyroidism are associated with dramatic changes in skeletal growth and osseous maturation (1, 2, 3, 4). Complete restoration of growth plate cartilage was demonstrated by administration of thyroid hormone in the propylthiouracil (PTU)-treated rat model of hypothyroidism (5, 6). Thyroid hormone, T3, increases the weight of cartilage explants and the synthesis of cartilage-matrix proteoglycan by chick embryonic chondrocytes (7) as well as the syntheses of collagen and proteoglycan in the primary articular chondrocyte cultures (8). This hormone also stimulates the syntheses of alkaline phosphatase (ALPase) (9, 10) and type X collagen (11, 12, 13, 14), markers for chondrocyte hypertrophy. On the other hand, T4 suppressed cartilage growth by stimulating precocious chondrocyte hypertrophy in rat femur organ culture (15) and chicken chondrocyte cultures (10).

The functions of T3 are directly mediated by three nuclear thyroid receptors (TR), TR{alpha}1, TR{alpha}2, and TRß1, encoded by two genes, TR{alpha} and TRß, respectively (16, 17, 18, 19, 20). TR{alpha}1, TR{alpha}2, and TRß1 are expressed in reserve zone progenitor cells and growth plate chondrocytes at high levels (21). Mice lacking either TR{alpha} alone or both TR{alpha} and TRß exhibit growth retardation and dysgenesis of endochondral ossification (16, 17, 18, 19, 22, 23, 24). Thus, the epiphyseal growth plate is a primary T3 target tissue.

During endochondral ossification, chondrocytes undergo a complex series of changes that involve proliferation, proteoglycan synthesis, and hypertrophy, before mineralization. In addition, the proteoglycan level decreases in the hypertrophic zone before bone formation (25, 26, 27). The hypertrophic zone is invaded by capillaries and chondroclasts, which may induce the degradation of proteoglycans. In addition, chondrocytes synthesize various matrix-degrading enzymes, including matrix metalloproteinases (MMP1, -2, -3, -9, and -13) (28, 29, 30), membrane-type MMP (MT-MMP) (31), and aggrecanases [ADAM-TS4 (a disintegrin and a metalloproteinase domain with thrombospondin type I domains) and ADAM-TS5] (32, 33), and these enzymes produced by the chondrocytes may also contribute to proteoglycan degradation during endochondral ossification. Because thyroid hormone modulates the synthetic activities of various chondrocytes, we hypothesized that thyroid hormone would influence the synthesis of proteoglycan-degrading enzymes and aggrecan degradation in the hypertrophic stage. In the present study we investigated the effects of thyroid hormone on the breakdown of proteoglycan in vivo and in vitro, using baby rats with hypothyroidism, rabbit chondrocyte cultures and pig cartilage explants. We further examined the cleavage site of aggrecan core protein of proteoglycan in the explants exposed to thyroid hormone along with the effects of thyroid hormone on the expression of aggrecanases in chondrocyte cultures. The results obtained in this study showed for the first time that thyroid hormone enhances aggrecan degradation via induction of aggrecanase-2 in developing cartilage, and that the mode of the hormone action differs from that of IL-1, which induces various MMPs and aggrecanase-1, but not aggrecanse-2 (34).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
T3, T4, and PTU were purchased from Sigma-Aldrich Corp. (St. Louis, MO). [35S]Sulfate (carrier free) was obtained from the Japan Atomic Energy Institute (Tokyo, Japan). Hyaluronic acid (3.0 x 106 Da) was supplied by Denki Kagaku Kogyo (Tokyo, Japan).

Treatment of rats with PTU
Hypothyroidism was induced in rat pups as described previously (6). Pregnant Sprague Dawley rats were given distilled water with or without 0.02% PTU ad libitum from 3 d before delivery. The day of birth was regarded as d 0. The pups were allowed to suckle freely until they were killed. Epiphyseal cartilage samples were obtained from the femur and tibia at the knee joint on d 10–18. Soft tissue and bone tissue were carefully removed from the samples. Half the PTU-treated rats received daily sc injections of T4 (50 ng/ml body weight in 0.3 mM NaOH) from d 7 until the day before sacrifice.

Pig cartilage explants and rabbit chondrocyte cultures
The growth plate and resting cartilage were obtained from the rib of 7-d-old male pigs. Articular cartilage was also collected from the surface of femur knee joints of the same pigs. The cartilage was minced into small pieces (0.3–1 mm3) and incubated in 10 ml {alpha}MEM supplemented with 10% fetal bovine serum (Mitsubisi-kasei, Tokyo, Japan), 32 U/ml penicillin, and 60 µg/ml kanamycin (medium A) in 100-mm plastic dishes at 37 C under 5% CO2/95% air for 24 h. The minced cartilage was then incubated with 50 µCi/ml [35S]sulfate in medium A for 24 h and washed five times with medium A. The mince was transferred to 16-mm plastic wells (45 ± 6 mg wet weight/well) containing 1 ml {alpha}MEM supplemented with 5% fetal bovine serum and antibiotics and incubated in the presence or absence of T3 (10-8 M). The medium was changed every 3 d. The supernatant from each culture was collected separately every 3 d at time of replacement and saved at -80 C for determinations of radioactivity and the size of proteoglycan.

Rabbit chondrocytes were isolated from the growth plate or resting cartilage of the rib or articular cartilage of femur knee joints of 4-wk-old Japanese White rabbits as described previously (35). The cells were seeded at a density of 5 x 104 cells/6-mm plastic tissue culture dish, 3 x 105 cells/30-mm type I collagen-coated dish, or 1 x 105 cells/60-mm type I collagen-coated dish (Koken, Osaka, Japan) and maintained in medium A.

Determination of uronic acid
Cartilage fragments were homogenized at 4 C in 1 ml 0.9% NaCl/0.2% Triton X-100. The homogenate was then incubated at 37 C for 16 h with 3 mg pronase E (pronase type XIV, Sigma-Aldrich Corp.) in 3 ml 0.05 M Tris-HCl buffer (pH 8.0) containing 1 mM CaCl2, 0.9% NaCl, and 0.2% Triton X-100. Digests of tissues were used for determination of uronic acid (36).

Measurement of ALPase activity
Cartilage fragments or cell layers were homogenized with a glass homogenizer in 0.9% NaCl/0.2% Triton X-100 at 0 C and centrifuged for 15 min at 12,000 x g. The ALPase activity of the supernatant, which contained 95% of the total activity, was assayed in 0.5 M Tris-HCl buffer (pH 9.5) supplemented with 0.5 mM p-nitrophenyl phosphate (pNP) and 0.5 mM MgCl2. The mixture was incubated at 37 C for 15 or 30 min, and the reaction was stopped by the addition of 0.25 vol 1 M NaOH. Hydrolysis of pNP was monitored as change in A410 in a spectrometer (Hitachi, Hialeah, FL), with para-nitrophenol used as a standard. One unit was defined as the activity catalyzing hydrolysis of 1 µmol pNP/30 min·mg protein-1.

Relative hydrodynamic sizes of proteoglycans
After 12 d of incubation, the [35S]sulfate-treated cartilage was washed five times with {alpha}MEM and overlaid with 1.0 ml 50 mM Tris-HCl buffer (pH 8.0) containing 4 M guanidine-HCl, 0.36 mM pepstatin A, 10 mM EDTA, 10 mM N-ethylmaleimide, and 1 mM phenylmethylsulfonylfluoride (buffer A), followed by incubation for 24 h at 4 C on a shaker. The supernatant was subjected to centrifugation (4,000 x g for 15 min) to separate insoluble components, and stored at -80 C until it was analyzed. The precipitate was washed with water and then digested with 1 mg/ml pronase E as described previously (37). It is noteworthy that the level of [35S]sulfate incorporation into 4 M guanidine-insoluble material was negligibly low (data not shown).

A portion (0.9 ml) of the cultured supernatant was mixed with an equal volume of 8 M guanidine-HCl in water and 0.1 ml buffer A containing 0.5 mg cartilage proteoglycans. The 4 M guanidine extract (0.9 ml) was also mixed with 0.1 ml buffer A containing 0.5 mg cartilage proteoglycan. Samples were applied to a Sepharose CL-2B column (1.0 x 95 cm) that had been equilibrated with buffer A.

Alternatively, a portion (0.9 ml) of the 4 M guanidine extract was mixed with 100 mM Tris-HCl (pH 7.0), 0.1 M NaCl, and protease inhibitors (buffer B) containing 5 mg hyaluronic acid (3.0 x 106 Da). The solution (1 vol) was further mixed with 3 vol 95% ethanol containing 1.3% potassium acetate and incubated at 4 C for 6 h. The suspension was centrifuged at 20,000 x g for 30 min at 4 C. The precipitate was solubilized in 0.9 ml buffer B and incubated at 4 C for 16 h. The medium was mixed with an equal volume of buffer B containing 5 mg hyaluronic acid (1.9 x 106 Da), 100 mM Tris-HCl (pH 7.0), 0.1 M NaCl, and protease inhibitors and incubated at 4 C for 16 h. The samples were applied to a Sepharose CL-2B column (1.0 x 95 cm) that was equilibrated in buffer B (37, 38).

NH2-teminal sequencing of an aggrecan fragment
Cartilage mince was incubated for 2–14 d in 0.2 ml {alpha}MEM supplemented with 5% fetal bovine serum in the presence of T3. The explants were washed five times with {alpha}MEM and overlaid with 1.0 ml 50 mM Tris-HCl buffer (pH 8.0) containing 4 M guanidine-HCl with protease inhibitors.

A portion (6 ml) of the medium was dried, after which the dried medium and tissues were extracted by 4 M guanidine-HCl containing protease inhibitors. Samples were taken for CsCl gradient centrifugation under dissociative conditions (starting density, 1.47 g/ml). D1 through D4 fractions were dialyzed sequentially against water containing protease inhibitors, then digested with chondroitinase ABC (4 U/125 µg protein) and keratinase (0.2 U/120 µg protein) (39).

The samples digested with chondroitinase ABC and keratinase were applied to a Sepharose CL-6B column (1.0 x 95 cm) that was equilibrated in 100 mM Tris-HCl (pH 7.0) buffer containing 0.1 M NaCl and protease inhibitors. Fractions 4–6 were analyzed by SDS-PAGE and transferred to polyvinylidene difluoride membrane for NH2-terminal sequencing.

Determinations of collagenase, gelatinase, and stromelysin activities and the level of tissue inhibitor of metalloproteinases (TIMP)
Rabbit growth plate chondrocytes were seeded at 3 x 105 cells/30-mm collagen-coated dish and maintained in medium A. Eleven days after seeding, the cells were incubated with or without 10-8 M T3 or 5 ng/ml IL-1 for 48 h in 2 ml {alpha}MEM supplemented with 0.01% BSA.

Gelatinase activity was determined using conditioned medium in the presence of 1 mM 4-aminophenylmercuric acetate (an activator of latent MMPs) against heat-denatured 14C-acetylated collagen (type I) as a substrate as previously described (40).

Stromelysin activity in the culture medium was measured with [3H]carboxy-methylated transferring assay (41) in the presence of 2 mM diisopropylfluorophosphate and 10 mM N-ethylmaleimide to inhibit serine and cysteine proteinases. Enzymic activity was expressed as units per milliliter, with one enzyme unit being defined as the amount of enzyme hydrolyzing 1 µg substrate/min at 37 C.

TIMP-1 was determined by sandwich enzyme immune-assay (42).

Real-time quantitative RT-PCR analysis
Sixteen days after chondrocyte cultures became confluent, total RNA was extracted with Tri-reagent (Sigma-Aldrich Corp.). First strand cDNA was synthesized using Omniscript Reverse Transcriptase (QIAGEN, Chatsworth, CA) with total RNA (1 µg). Genomic DNA that might contaminate the RNA samples was digested with deoxyribonuclease I (Ambion, Inc., Austin, TX) before RT. A PCR product of rabbit aggrecanase was generated from the total RNA preparation by RT-PCR using a pair of degenerated primers based on the nucleotide sequence of human aggrecanase-1/ADAM-TS4 (accession no. 148213) or aggrecanase-2/ADAM-TS5 (accession no. 142099). Sequences for primers used in these analyses were as follows: 5'-GACCTTCCGTGAAGAGCAGTGT-3' and 5'-CCTGGCAGGTGAGTTTGCAT-3' for aggrecanase-1 cDNA amplification, and 5'-ATGACCATGAGGAGCACTACGA-3' and 5'-GGAGAACATATGGTCCCAACGT-3' for aggrecanase-2.

Real-time quantitative RT-PCR analyses for aggrecanase were performed using an ABI PRISM 7700 sequence detection system instrument and software (PE Applied Biosystems, Foster City, CA). First strand cDNA was synthesized using Omniscript Reverse Transcriptase with total RNA (1 µg). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as an internal standard to control variability in amplification due to differences in the starting total RNA concentrations. Sequences for all primers used in these analyses were as follows: 5'-GACCTTCCGTGAAGAGCAGTGT-3' and 5'-CCTGGCAGGTGAGTTTGCAT-3' for aggrecanase-1 cDNA amplification; 5'-ATGACCATGAGGAGCACTACGA- 3' and 5'-GGAGAACATATGGTCCCAACGT-3' for aggrecanase-2; and 5'-GCTTCACCACCTTCTTGATG-3' for GAPDH, as previously described (43). The sequences of TaqMan fluorogenic probes used were 5'-6FAM-CCTACAACCACCGAACCGACCTCTTCAA-TAMRA-3', 5'-6FAM- TGCCCACATAAATCCTCCCGAGTAAACA-TAMRA-3', 5'-6FAM-ACGTCCGACCGTGACCGCAATAAGTA-TAMRA-3', 5'-6FAM-TGCACCACCTGATAGCCGAAATCCACAC-TAMRA-3', and 5'-6VIC-TGCCGCCTGGAGAAAGCTGCTAAGTA-TAMRA-3' for agrecanase-1, aggrecanase-2, and GAPDH, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of thyroid hormone on uronic acid content of epiphyseal cartilage during endochondral ossification in baby rats in vivo
Treatment of mother rats with PTU caused retardation of growth of their pups, and daily administrations of T4 to the pups from d 7 reversed this growth retardation (Table 1Go) as expected from previous studies (6). However, there had been no previous studies on the effect of thyroid hormone on cartilage-proteoglycan degradation. Using these pups, we examined the effect of thyroid state on the proteoglycan (uronic acid) content of epiphyseal cartilage. The cartilage samples were obtained from the femur and tibia at the knee joints. In the epiphyseal cartilage of normal pups, proteoglycan degradation, indicated by a marked decrease in the uronic acid content, occurred between d 13 and 18 (Table 1Go). The proteoglycan degradation was accompanied by a 4-fold increase in ALPase activity, suggesting that the proteoglycan degradation was associated with endochondral ossification.


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  		Table 1. Effects of treatments of rats with PTU alone or PTU plus T4 on the uronic acid content and ALPase activity in developing cartilage at the knee joint

 
However, in PTU-treated pups, neither reduction of uronic acid nor induction of ALPase was observed between d 13 and 18, and injections of T4 completely reversed these effects of PTU. It is noteworthy that the injections of T4 did not reduce the uronic acid level before the enhancement of endochondral ossification (d 10–13; Table 1Go). These findings suggest that thyroid hormone is essential for proteoglycan degradation in developing limbs, and that this effect depends on the stage of chondrocytes.

Effects of thyroid hormone on proteoglycan catabolism in pig cartilage explants
To examine the effect of thyroid hormone on proteoglycan degradation in vitro, pig cartilage explants labeled with [35S]sulfate for 24 h were cultured in the presence or absence of T3 for 3–12 d, and 35S-labeled proteoglycans were released from the explants into medium. We used pig cartilage explants because the determination of the molecular size of degrading proteoglycans required a large amount of cartilage tissue. In the absence of T3, 65% of the 35S-labeled proteoglycans were released from the growth plate explants in 6 d (Fig. 1AGo). In contrast, only 20–30% of the 35S-labeled proteoglycans were released into the medium from explants of resting cartilage and articular cartilage by d 12 (Fig. 1AGo). T3 increased by 92% the release of 35S-labeled proteoglycans in the growth plate cultures in 6 d. However, using this assay, we could not detect any T3 stimulation of the release of proteoglycans from resting or articular cartilage explants (Fig. 1AGo).



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  		Figure 1. Effects of T3 on the release of proteoglycans from cartilage explants. A, Pig cartilage explants were labeled with [35S]sulfate for 24 h, washed, and then incubated for 3–12 d in medium with 5% fetal bovine serum in the presence ({blacksquare}, {bullet}, and {blacktriangleup}) or absence ({square}, {circ}, and {triangleup}) of T3 (10-8 M) for 12 d. The level of 35S-labeled macromolecules on d 0 was 44,197 ± 6,863 cpm/mg wet weight in growth plate explants ({bullet} and {circ}), 23,371 ± 2,657 cpm/mg wet weight in articular cartilage explants ({blacksquare} and {square}), and 23,024 ± 5,443 cpm/mg wet weight in resting cartilage explants ({blacktriangleup} and {triangleup}). Values are the average ± SD for three cultures. B and C, Sepharose CL-2B chromatography of 35S-labeled proteoglycans from growth plate explants previously exposed to [35S]sulfate, washed, and then chased in the presence (solid line) or absence (dotted line) of 10-8 M T3. Portions of the medium on d 6 (upper panel) and the cartilage extract on d 12 (lower panel) were applied to a column of Sepharose CL-2B that had been equilibrated in 4 M guanidine-HCl/50 mM Tris-HCl, pH 8.0, with protease inhibitors as described in Materials and Methods. Fractions (1.5 ml/fraction) were collected, and aliquots (0.5 ml) of the fractions were used to determine the radioactivity. Radioactivity was measured in a scintillation counter (Beckman, Fullerton, CA). The V0 and total volume (Vt) were determined with high molecular weight hyaluronic acid and phenol red, respectively.

 
The size of [35S]proteoglycan monomer in the medium and tissue fractions of the growth plate explants was analyzed by Sepharose CL-2B chromatography under dissociative solvent conditions. The average size of the [35S]proteoglycan monomer released into the medium was smaller than that of the monomer remaining in the explants (Fig. 1Go, B and C). These observations suggested that products of the monomer released into the medium were degraded compared with the monomer in the explants. The level of 35S-labeled proteoglycans in the medium was higher in T3-treated cultures than in T3-free cultures (Fig. 2BGo). In contrast, the level of 35S-labeled proteoglycans in the tissue fraction of T3-free cultures was higher than that in T3-treated cultures (Fig. 1CGo).



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  		Figure 2. Analysis of the sizes of proteoglycans by gel exclusion chromatography under associative solvent conditions. Portions of the medium in d 6 cultures of growth plate (A), articular cartilage (C), and resting cartilage (D) and the tissue extract of d 12 cultures of growth plate (B) maintained in the presence ({bullet}) or absence ({circ}) of 10-8 M T3 were mixed with hyaluronic acid and applied to a column of Sepharose CL-2B that had been equilibrated with buffer containing 0.1 M Tris-HCl (pH 7.0), 0.1 M NaCl, and protease inhibitors as described in Materials and Methods. Fractions (1.5 ml/fraction) were collected, and aliquots (0.5 ml) of the fractions were used to determine the radioactivity. Radioactivity was measured in a Beckman scintillation counter. The V0 and Vt were determined with high molecular weight hyaluronic acid and phenol red, respectively.

 
Next, the size of 35S-labeled proteoglycans released into the medium was analyzed by gel exclusion chromatography on Sepharose CL-2B in the presence of exogenous hyaluronic acid under associative solvent conditions. Under these conditions, the intact monomer aggregates with hyaluronic acid and is eluted at void volume (V0; Fig. 2Go, shaded area). As shown in Fig. 2Go, A and B, less than 25% of 35S-labeled proteoglycans from the medium fraction of growth plate explants were eluted at V0 (Fig. 2AGo), whereas the majority of 35S-labeled proteoglycans from the tissue fraction of growth plate were eluted at V0 (Fig. 2BGo). Therefore, most of the monomer released into the medium seemed to lose its hyaluronic acid-binding capacity.

T3 stimulated the release of 35S-labeled proteoglycans that lacked the hyaluronic acid-binding activity from the explants of the growth plate (Fig. 3AGo) and resting cartilage (Fig. 3DGo), but it showed a marginal effect in the cultures of articular cartilage (Fig. 3CGo). The stimulation was more prominent in the growth plate explants than in the resting cartilage explants. Because gel exclusion chromatography of 35S-labeled proteoglycans can detect their degradation with a lower threshold than does the determination of the proteoglycan content alone, the former method, but not the latter one, revealed the thyroid hormone stimulation of proteoglycan degradation in resting cartilage (Fig. 1AGo vs. Fig. 2DGo).



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  		Figure 3. Determination of cleavage site in aggrecan in T3-exposed explants. Proteins in fraction 7 were separated by SDS-PAGE under nonreducing conditions, transferred to a polyvinylidene difluoride membrane, then stained with Coomassie Brilliant Blue. The N-terminal amino acid sequence of band B was determined using an automatic protein sequencer (476A, PE Applied Biosystems).

 
Determination of the N-terminal sequence of an aggrecan fragment released into the medium from T3-exposed explants
Proteoglycans released in the medium from pig growth plate explants were extracted under dissociative solvent conditions and digested with chondroitinase ABC and keratinase. The aggrecan core protein and its fragments were applied to a Sepharose column. The aggrecan core protein (A) and its fragments (B) were eluted in fractions 4–6 and then subjected to SDS-PAGE (Fig. 4Go). The lower band (B) was more abundant in T3-treated culture than in T3-free culture (Fig. 4Go). The N-terminal sequence of band B was ARGSVIL (Fig. 4Go), which can be cleaved by aggrecanase-1 or aggrecanase-2, but not by MMPs (34).



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  		Figure 4. Effects of T3 on the release of proteoglycans from the cell matrix layer in rabbit chondrocyte cultures from the growth plate (A), resting cartilage (B), and surface region of articular cartilage (C). Cultures were labeled with [35S]sulfate for 24 h, washed, and then incubated for 0–14 d in medium with 5% fetal bovine serum (A–C) in the presence ({bullet}) or absence ({circ}) of T3 (10-7 M). Cells of each day were cultured for totally 8–22 d. Values are the average ± SD for four cultures. *, P < 0.05; **, P < 0.01.

 
Effects of thyroid hormone on the release of proteoglycans into the medium of rabbit chondrocyte cultures
To examine whether the thyroid hormone stimulation can be observed in other systems besides pig cartilage explants along with the effect of thyroid hormone on the synthesis of proteoglycan-degrading enzymes, we used rabbit chondrocyte cultures, because this culture system has been characterized and widely used (28, 35, 43, 44, 45). Rabbit growth plate and resting and articular chondrocytes in confluent culture were labeled with [35S]sulfate and then chased in the presence of 5% fetal bovine serum for 2–14 d. The time required for 50% of the 35S-labeled proteoglycans to be released from the matrix in cultures from rib growth plate chondrocytes was 5 d (Fig. 4AGo). This estimated half-life (5 d) of cartilage proteoglycan was shorter than that (8–9 d) in cultures of resting chondrocytes of the same ribs and articular cartilage from the same rabbits (Fig. 4Go, B and C).

T3 stimulated the release of 35S-labeled proteoglycans into the medium by rabbit cultured growth plate chondrocytes (Fig. 4AGo), but no significant increase in the displacement of 35S-labeled proteoglycans was observed upon treatment with T3 in cultures of rabbit resting and articular chondrocytes (Fig. 4Go, B and C). These findings also suggest that thyroid hormone stimulation of proteoglycan degradation is particularly prominent in chondrocytes involved in endochondral ossification, and that the proteoglycan-degrading activity of growth plate chondrocytes is greater than that of resting and articular chondrocytes.

Effects of T3 on collagenase, gelatinase, and stromelysin activities and the level of TIMP released into medium by chondrocytes
Collagenase, gelatinase, and stromelysin can degrade cartilage proteoglycan at neutral pH (46). Furthermore, these MMPs cleave the link protein and the hyaluronic acid-binding region of the proteoglycan core protein (47). Thus, it was of interest to examine the effects of T3 on the release of MMPs and TIMP by chondrocytes. T3 at 10-7 M had little effect on the release of collagenase, gelatinase, stromelysin, and TIMP-1 (Table 2Go). On the other hand, IL-1 markedly increased the releases of these enzymes into the medium, and it decreased the release of TIMP-1 (Table 2Go). The threshold of detection of the enzyme activities in the assay may be too high to fully show the effect of thyroid hormone, but Table 2Go indicates that the effect of thyroid hormone on the MMPs/TIMP-1 is far less than that of IL-1.


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  		Table 2. Effects of T3 on collagenase, gelatinase, and stromelysin activities and the level of TIMP released into the medium by chondrocytes

 
Effects of T3 on aggrecanase mRNA levels
To examine whether T3 enhances the synthesis of aggrecanase-1 or -2, we incubated rabbit growth plate chondrocytes in the presence or absence of T3. Real-time quantitative RT-PCR analysis showed that in the cultures, 10-7 M T3 enhanced aggrecanase-2 mRNA expression within 12 h, with this effect reaching a plateau at 24 h at a concentration of 10-7 M (Fig. 5Go, A and B). On the other hand, the level of aggrecanase-1 mRNA was very low compared with that of aggrecanase-2, and T3 at any concentrations had little or no effect on aggrecanase-1 mRNA expression under the same culture conditions mentioned above (data not shown). T3 (10-7M) enhanced aggrecanase-2 mRNA expression in resting chondrocytes but not in articular chondrocytes (Fig. 5CGo). Because the basal aggrecanase-2 mRNA level was lower in resting chondrocytes than in growth plate chondrocytes (data not shown), T3 stimulation of aggrecanase-2 could be observed in resting chondrocytes even though hormone stimulation of proteoglycan degradation in resting chondrocytes was less than that in growth plate chondrocytes.



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  		Figure 5. A, Time course of the enhancement of aggrecanase-2 mRNA expression by T3 in rabbit growth plate chondrocyte cultures. On d 16, cells were exposed to T3 at 10-7 M for 12–48 h in serum-free {alpha}MEM. The findings of real-time quantitative RT-PCR analysis of the aggrecanase abundance were normalized to that of the GAPDH mRNA. B, Effects of increasing concentrations of aggrecanase on the aggrecanase-2 mRNA expression in growth plate chondrocyte cultures. Cells were exposed various concentrations of T3 in serum-free {alpha}MEM for 24 h. C, Effects of T3 on the aggrecanase-2 mRNA expression in resting and articular chondrocyte cultures. Cells were exposed to T3 at 10-7 M in serum-free {alpha}MEM for 24 h. Values are the average ± SD for three cultures.

 
Expression of aggrecanase-2 mRNA during chondrocyte differentiation
Rabbit growth plate chondrocytes in culture undergo proliferation, matrix formation, hypertrophy, and calcification as they do in vivo (43). The cultures in our study became confluent on d 6. The Indian hedgehog (Ihh) and PTH/PTHrP receptor mRNA levels started to increase on d 14 and then decreased on d 22. Osteopontin and type X collagen started to increase on d 14 and reached a plateau on d 22 (Fig. 6Go) (43). On d 18, chondrocytes were spherical and surrounded by a refractile matrix. Calcification took place on d 26 (43). Based on these observations, we regarded cells from d 6–10 as the proliferating stage, from d 14–18 as the prehypertrophic (matrix-forming) stage, and from d 22–26 as the hypertrophic stage (43). In this culture system, aggrecanase-2 mRNA was low in the proliferating and prehypertrophic stages and increased in the hypertrophic stage (Fig. 6Go). The aggrecanase-1 mRNA level was very low throughout all stages (data not shown).



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  		Figure 6. The changes in aggrecanase-2, Ihh, PTH/PTHrP receptor, osteopontin, and type X collagen mRNA levels in rabbit growth plate chondrocyte cultures at various stages of differentiation. Chondrocytes were cultured for 6, 10, 14, 18, 22, and 26 d, and the aggrecanase-2 mRNA level was determined by real-time quantitative RT-PCR analysis. The changes in Ihh, PTH/PTHrP receptor, osteopontin and type X collagen mRNA levels were shown according to the data in previous studies with similar rabbit chondrocyte cultures (43 ). Each symbol stands for a relative mRNA expression level compared with a maximum level: -, less than 40%; +, less than 60%; ++, less than 80%; and +++, less than 100%. Values are the average ± SD for three cultures.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Aggrecan plays a crucial role in maintaining cartilage resiliency and may serve as an inhibitor of mineralization (48). Chondrocytes usually maintain a balance between the synthesis and breakdown of aggrecan (49, 50). However, this balance is destroyed in the hypertrophic zone. MMP-2, -3, and -9 have been suggested to be involved in proteoglycan degradation in cartilage (28, 29, 30, 51), but thyroid hormone had little effect on collagenase, gelatinase, and stromelysin activities or the TIMP-1 level in growth plate cultures, whereas IL-1 increased these enzymatic activities and decreased the TIMP-1 level. Thus, additional enzymes must be involved in the stimulation of proteoglycan breakdown by thyroid hormone, and these enzymes must potentially have the ability to cleave aggrecan at Glu373-Ala374 within the interglobular domain.

Aggrecan is known to be cleaved within the interglobular domain, and two major sites of cleavage have been identified in this region at Asn341-Phe342 and Glu373-Ala374 (52). Although several MMPs (MMP-1, -2, -3, -7, -8, -9, and -13) (53) cleave aggrecan at Asn341-Phe342, they are not responsible for the observed cleavage at Glu373-Ala374 (54). A novel proteolytic activity, termed aggrecanase, has been hypothesized to be responsible for the cleavage at Glu373-Ala374. IL-1 induced the release of aggrecan fragments cleaved at Asn341-Phe342 and Glu373-Ala374 into the medium (52). In recent studies aggrecanase-1 and -2, members of the ADAM-TS family, were purified and cloned, and it was demonstrated that they cleaved aggrecan at Glu373-Ala374 (32, 33, 55). In the arthritic cartilage samples, aggrecanase-1 was expressed at 2- to 3-fold higher level than aggrecanase-2, but in normal rib cartilage, aggrecanase-1 was expressed at a low level, with aggrecanase-2 being expressed at an approximately 4-fold higher level (33). Our findings indicated that aggrecanse-2, but not aggrecanse-1, was up-regulated in the hypertrophic stage, suggesting that during endochondral ossification, but not during inflammation, the contribution of aggrecanase-2 to aggrecan degradation is greater than that of aggrecanase-1. Aggrecanase-2 mRNA expression in growth plate cartilage was further enhanced by T3 in a dose- and time-dependent manner, whereas we could not detect any thyroid hormone enhancement of aggrecanse-1 expression. These observations suggest that T3 stimulates proteoglycan degradation at least partly via the induction of aggrecanse-2.

Vascular invasion induces resorption of the cartilage proteoglycan matrix (28, 29). The results in the present study showed, however, that growth plate chondrocytes are also involved in proteoglycan breakdown, and that high proteoglycan-degrading activity is a marker of hypertrophic chondrocytes. We previously reported that hypertrophic chondrocytes in culture induce deposits of calcium phosphate appatite in the matrix, whereas the chondrocytes remain viable (45, 56). Electron microscopic examination of epiphyseal cartilage revealed that all hypertrophic chondrocytes, including the terminal chondrocytes adjacent to the regions of mineralization and vascular invasion, have intact organelles (57). Thus, proteoglycan breakdown cannot be accounted for by degeneration of hypertrophic chondrocytes. On the other hand, the degradation of proteoglycans in cartilage explants is blocked by actinomycin D, cycloheximide, and deoxyglucose (58), suggesting that the proteoglycan-degrading activity of chondrocytes depends on some new synthesis of RNA, protein, and ATP. Therefore, the expression of aggrecanase-2 induced by T3 may play a role in the proteoglycan degradation by hypertrophic chondrocytes.

We showed here that the degradation of aggrecan core protein synthesized by growth plate chondrocytes was induced in the presence of T3. The degraded product of aggrecan in T3-treated cultures lacked hyaluronic acid-binding activity, implying that the G1 domain was cleaved from aggrecan core protein. This effect of thyroid hormone to induce breakdown of aggrecan was consistently observed in both rabbit growth plate chondrocyte cultures and pig growth plate explants. Thyroid hormone also induced proteoglycan breakdown in epiphyseal cartilage of PTU-treated rats with hypothyroidism in vivo.

Previous studies have shown that thyroid hormone increases proteoglycan synthesis by chondrocytes in culture (7, 8). However, the effect of thyroid hormone on proteoglycan synthesis in growth plate chondrocyte cultures was less than that of IGF-I or TGFß. Unlike thyroid hormone, neither IGF-I nor TGFß increased proteoglycan breakdown in growth plate chondrocyte cultures (Kato, Y., unpublished observation). In human skin fibroblast cultures, thyroid hormone inhibits glycosaminoglycan accumulation in time- and dose-dependent manners (59, 60). Thus, thyroid hormone seems to play a unique role in both the synthesis and degradation of proteoglycan. During amphibian metamorphosis, for example, thyroid hormone causes bone remodeling and resorption of the tail. This metamorphosis-promoting action may be accounted for partly by the hormone stimulation of proteoglycan degradation observed in the present study.

We conclude that thyroid hormone plays a crucial role in the control of proteoglycan degradation by growth plate chondrocytes. The mode of the thyroid hormone action on aggrecan breakdown differed from that of IL-1, which induces various MMPs and aggrecanase-1, but not aggrecanse-2 (34), suggesting that the development- and inflammation-dependent degradation of aggrecan uses different sets of proteases. This novel action of thyroid hormone on aggrecanase-2 and aggrecan degradation may be important in gaining insight into the role of thyroid hormone in skeletal development.


   Acknowledgments
 
We thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.


   Footnotes
 
This work was supported by a grant from the Ministry of Education, Science, Sports, and Culture of Japan (12557156) and by NIDCR Grant DE-1455.

Abbreviations: ADAM-TS, A disintegrin and a metalloproteinase domain with thrombospondin type I domains; ALPase, alkaline phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ihh, Indian hedgehog; MMP, matrix metalloproteinases; MT-MMP, membrane-type matrix metalloproteinase; pNP, p-nitrophenyl phosphate; PTU, propylthiouracil; TIMP, tissue inhibitor of metalloproteinases; TR, thyroid receptor; V0, void volume; Vt, total volume.

Received July 23, 2002.

Accepted for publication February 26, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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