Molecular Cloning and Biological Activity of a Novel Lysyl Oxidase-related Gene Expressed in Cartilage*

We cloned a cDNA encoding a novel lysyl oxidase-related protein, named LOXC, by suppression subtractive hybridization between differentiated and calcified ATDC5 cells, a clonal mouse chondrogenic EC cell line. The deduced amino acid sequence of mouse LOXC consists of 757 amino acids and shows 50% identity with that of mouse lysyl oxidase. Northern blot analysis showed a distinct hybridization band of 5.4 kilobases, and Western blot analysis showed an immunoreactive band at 82 kilodaltons. Expression of LOXC mRNA was detected in osteoblastic MC3T3-E1 cells and embryonic fibroblast C3H10T1/2 cells, whereas none of NIH3T3 fibroblasts and myoblastic C2C12 cells expressed LOXC mRNA in vitro. Moreover, the LOXC mRNA and protein levels dramatically increased throughout a process of chondrogenic differentiation in ATDC5 cells. In vivo, LOXC gene expression was localized in hypertrophic and calcified chondrocytes of growth plates in adult mice. The conditioned media of COS-7 cells transfected with the full-length LOXC cDNA showed the lysyl oxidase activity in both type I and type II collagens derived from chick embryos, and these activities of LOXC were inhibited by β-aminopropionitrile, a specific inhibitor of lysyl oxidase. Our data indicate that LOXC is expressed in cartilage in vivo and modulates the formation of a collagenous extracellular matrix.

We cloned a cDNA encoding a novel lysyl oxidaserelated protein, named LOXC, by suppression subtractive hybridization between differentiated and calcified ATDC5 cells, a clonal mouse chondrogenic EC cell line. The deduced amino acid sequence of mouse LOXC consists of 757 amino acids and shows 50% identity with that of mouse lysyl oxidase. Northern blot analysis showed a distinct hybridization band of 5.4 kilobases, and Western blot analysis showed an immunoreactive band at 82 kilodaltons. Expression of LOXC mRNA was detected in osteoblastic MC3T3-E1 cells and embryonic fibroblast C3H10T1/2 cells, whereas none of NIH3T3 fibroblasts and myoblastic C2C12 cells expressed LOXC mRNA in vitro. Moreover, the LOXC mRNA and protein levels dramatically increased throughout a process of chondrogenic differentiation in ATDC5 cells. In vivo, LOXC gene expression was localized in hypertrophic and calcified chondrocytes of growth plates in adult mice. The conditioned media of COS-7 cells transfected with the fulllength LOXC cDNA showed the lysyl oxidase activity in both type I and type II collagens derived from chick embryos, and these activities of LOXC were inhibited by ␤-aminopropionitrile, a specific inhibitor of lysyl oxidase. Our data indicate that LOXC is expressed in cartilage in vivo and modulates the formation of a collagenous extracellular matrix.
Endochondral bone formation is a multistep programmed event in skeletal development. Undifferentiated mesenchymal chondroprogenitor cells differentiate into chondrocytes through a cellular condensation process. Such chondrocytes surround themselves with an abundant layer of extracellular matrix, including type II, IX, and XI collagens, that is characteristic of cartilage (1,2). These cells go through sequential processes of proliferation and maturation and then change their genetic program to be converted into hypertrophic and calcified chondrocytes, expressing type X collagen. These events are under the regulatory control of a variety of growth and differentiation modulating factors, including bone morphogenetic proteins (3,4), fibroblast growth factors (5,6), parathyroid hormone-related peptide (7)(8)(9), and Indian hedgehog (10). It is also clear that the components of extracellular matrix in cartilage play important roles in modulating and maintaining the phenotype of chondrocytes. During a process of hypertrophic conversion and calcification of chondrocytes, mineralization of extracellular matrix occurs before these chondrocytes are replaced by bone tissues. However, the molecular mechanisms underlying these sequential events remain largely unknown.
We previously reported that the clonal mouse cell line, ATDC5, enables the monitoring of the multistep chondrogenic differentiation in a single culture (11)(12)(13)(14). When cultured in the presence of insulin, ATDC5 cells form cartilaginous nodules through cellular condensation. When the formation of cartilage nodules is completed, the cells are then converted to type X collagen-expressing hypertrophic chondrocytes, following the process of mineralization. By taking advantage of the fact that these chondroprogenitor-like cells undergo sequential differentiation in a synchronous manner, we compared mRNAs expressed in hypertrophic and calcified ATDC5 cells, expressing type X collagen, with those in differentiated ATDC5 cells, before expressing type X collagen, by suppression subtractive hybridization, and we isolated a novel cDNA clone encoding a lysyl oxidase-related protein expressed in cartilage as well as ATDC5 cells.
RNA Extraction and Suppression Subtractive Hybridization-Poly(A) ϩ RNA was isolated from differentiated (day 21) and calcified (day 42) ATDC5 cells by a single-step method as described previously (17) and analyzed by suppression subtractive hybridization according to the manufacturer's instructions (PCR-Select cDNA Subtractions Kit, CLONTECH Laboratories, Inc., Palo Alto, CA). The cDNA fragment of * This work was supported by Grant 0123 from the Japan Orthopaedics and Traumatology Foundation, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  ϳ600-base pairs (bp) 1 expressed at a high level in calcified ATDC5 cells was identified and subcloned into pCR 2.1 vector (Invitrogen Co., San Diego, CA).
cDNA Library Construction and Isolation of Mouse LOXC cDNA-The oligo(dT)-primed cDNA library from poly(A) ϩ RNA of calcified ATDC5 cells was constructed in ZAP Express vector (Stratagene, La Jolla, CA), and 1 ϫ 10 6 plaques were screened with the cDNA fragment as a probe (12,16). Plaques were transferred to the membranes (137-mm nylon membrane, PerkinElmer Life Sciences), and the 600-bp fragment was 32 P-labeled (BcaBEST labeling kit, Takara, Otsu, Japan), and hybridization was performed as described previously (12). The membranes were washed to a final stringency of 0.1ϫ SSPE (3 M NaCl, 197 mM NaH 2 PO 4 , 25 mM EDTA), and 0.1% SDS at 55°C. The nucleotide sequence was determined with ALFred DNA Sequencer (Amersham Pharmacia Biotech).
Northern Analysis-ATDC5 cells, C3H10T1/2 cells, MC3T3-E1 cells, NIH3T3 cells, and C2C12 cells were plated in 6-multiwell plastic plates and cultured as described above. Total RNA from various cell lines cultured in vitro as well as poly(A) ϩ RNA from rib cartilage of 3-weekold ICR mice were isolated and analyzed by Northern hybridization as described previously (12,17). Briefly, total RNA (20 g) and poly(A) ϩ RNA (2 g) were denatured, separated by 1% agarose gel electrophoresis, and transferred on Nytran membranes (Schleicher & Schuell). A 1.3-kilobase pair (kb) cDNA fragment of LOXC, a 0.55-kb cDNA frag-ment of mouse type II collagen, and a 0.65-kb cDNA fragment of mouse type X collagen were used for hybridization as probes. In analysis of tissue distribution in adult mice, a labeled cDNA was hybridized to a mouse multiple tissue Northern blot (CLONTECH Laboratories, Inc.). After hybridization, the membranes were exposed to X-Omat films (Eastman Kodak Co.) at Ϫ80°C with Cronex Lightning Plus intensifying screen (PerkinElmer Life Sciences).
In Situ Hybridization-Tibiae of male neonate C57BL/6J mice were collected and fixed in 4% paraformaldehyde in 10 mM phosphate-buffered saline, pH 7.4, overnight at 4°C. Tibiae were decalcified for 4 days in 10% EDTA. They were dehydrated in a graded series of ethanol and embedded in paraffin. Sections (6 m thick) were then processed for in situ hybridization as described previously (18). Subclones of the 1.3-kb mouse LOXC cDNA, the 0.4-kb mouse type II collagen cDNA, and the 0.55-kb mouse type X collagen cDNA into pBSII-KS(ϩ) (Stratagene) were linearized with appropriate restriction enzymes to transcribe either sense or antisense 35 S-labeled riboprobes. After hybridization, the slides were washed under conditions of high stringency, and the dried tissue sections were dipped into Kodak NTB-2 emulsion and exposed at 4°C. The sections were counterstained with hematoxylin. As a negative control, the slide was hybridized with the sense probe RNA (data not shown). In addition, the sections pretreated with RNase before in situ hybridization with the riboprobes showed no autographic signals, indicating that the hybridization signals were dependent on the presence of RNA (data not shown).
In Vitro Transcription/Translation-A coupled transcription/translation reaction was performed using the rabbit reticulocyte lysate sys- Polyclonal Antibody-Antisera were raised in rabbits (Takara, Otsu, Japan) using keyhole limpet hemocyanin conjugated to a peptide corresponding to a sequence in the C-terminal of mouse LOXC (AELSLEQEQRLRNNL). The specific antibody was purified as described previously (19).
DNA Transfection of COS-7 Cells-COS-7 cells were cultured in Dulbecco's modified Eagle's medium (ICN Pharmaceuticals, Inc.) containing 10% fetal bovine serum. For the assay of lysyl oxidase activity, COS-7 cells were transiently transfected with pCMV/mLOXC or pcDNA3.1 by using DEAE-dextran (Sigma), and the conditioned media were prepared 7 days after transfection. For Western blot analysis, COS-7 cells were transfected with pCMV/mLOXC or pcDNA3.1 by using FuGene6 (Roche Molecular Biochemicals) according to the manufacturer's instructions, and total cellular proteins were prepared 2 days after transfection.
Western Blot Analysis-Total cellular proteins were prepared from ATDC5 cells and COS-7 cells transfected with pCMV/mLOXC or pcDNA3.1 as described previously (17). Forty micrograms of protein were separated by a 10 -20% polyacrylamide gel and transferred to nitrocellulose filters. The filters were blocked in 3% gelatin in Trisbuffered saline containing 0.1% Tween 20 and then incubated with the anti-LOXC antibody. Prestained rainbow marker (Bio-Rad) was loaded in the adjacent lane to estimate molecular sizes.
Assay of Lysyl Oxidase Activity-Preparation of collagen substrates and assay of lysyl oxidase activity were performed as described previously (20). In brief, 20 pairs of calvariae or growth plates of proximal tibiae in 17-day-old chick embryos were suspended in 6 ml of Eagle's minimal essential medium without lysine (Life Technologies, Inc.). The suspension was supplemented with L-[4,5-3 H]lysine (200 Ci, PerkinElmer Life Sciences, catalog number NET-376) and ␤-aminopropionitrile (␤APN)-fumarate (50 g/ml as ␤APN, Sigma) and incubated at 37°C for 24 h. The calvariae or the growth plates were then homogenized in 0.05 M Tris-HCl buffer, pH 7.4, containing 1 M NaCl. The homogenates were stirred for 90 min at 4°C and then centrifuged at 20,000 ϫ g for 20 min. Labeled collagens in the supernatant fraction were precipitated by salting with 20% NaCl and collected by centrifugation at 30,000 ϫ g for 20 min. The precipitates were suspended in a minimum volume of 0.05 M Tris acetate buffer, pH 7.7, containing 0.15 M NaCl and dialyzed against the same buffer for 24 h.
The substrate solution containing 3 H-labeled type I or type II collagen equivalent to 2.5 ϫ 10 5 cpm was added to each assay tube. To this solution was added 0.9 ml of the conditioned media of COS-7 cells described above. The reaction mixture was incubated at 37°C for 3 h, and the reaction was stopped by freezing the mixture at Ϫ20°C. Tritiated water formed was collected by vacuum distillation according to the method of Pinnell and Martin (21), and 0.8-ml portions of the distillate were counted in a liquid scintillation counter. Assays were also carried out in the presence of ␤APN (50 g/ml), a specific inhibitor of lysyl oxidase, after preincubation with 3 H-labeled collagen substrates for 1 h prior to the addition of the conditioned media. Statistical Analysis-Statistical significance was assessed by oneway analysis of variance and unpaired Student's t test.

RESULTS
Cloning and the Structure of Mouse LOXC cDNA-In the presence of insulin, ATDC5 cells reached confluence 5 days after plating and initiated chondrogenesis. The process proceeded in an orderly and synchronous manner as evidenced by the expression of phenotypic marker genes with cartilage characteristics, as reported previously (12,14,15). Cellular condensation occurred on day 7, and the formation and growth of cartilaginous nodules were observed from day 9 to day 21, followed by a calcification process that began on day 35. To find genes up-regulated during a process of cellular hypertrophy and calcification of chondrocytes, we performed suppression subtractive hybridization using the poly(A) ϩ RNA extracted from differentiated and calcified ATDC5 cells, and we obtained a 600-bp cDNA fragment corresponding to the 3Ј-untranslated sequence of a novel gene named LOXC. This cDNA fragment was then used as a probe to screen at high stringency a mouse cDNA library generated from calcified ATDC5 cells. Twenty cDNA clones were obtained from 1 ϫ 10 6 independent plaques. Eight out of twenty clones contained a cDNA of about 5.4-kb. Fig. 1 shows the nucleotide sequence of LOXC cDNA and the deduced amino acid sequence of the putative LOXC protein. LOXC cDNA contains a 2271-bp open reading frame starting an ATG codon. Therefore, LOXC protein is predicted to consist of 757 amino acid residues. The predicted LOXC protein has a calculated molecular mass of 84,705 Da and an isoelectric point of 7.8. The 3Ј-end of the sequence contains a poly(A) stretch, preceded by putative polyadenylation signals (AATAAA). The 25 amino acids, starting from the first ATG initiation codon, possessed features characteristic of signal peptide sequences. These data suggest that the LOXC cDNA is likely to encode an extracellular protein. Cleavage of the signal peptide would yield a protein of 732 amino acids residues, having a calculated molecular mass of 81,864 Da. The amino acid sequence of LOXC protein showed significant homology with that of mouse lysyl oxidase and of mouse lysyl oxidase-related protein 2 (50 and 52% amino acid identity at the amino acid level, respectively) (Fig. 2).
Expression of LOXC Protein by in Vitro Transcription/ Translation-To confirm that the cloned 5.4-kb LOXC cDNA contained a functional open reading frame, we performed an in vitro transcription/translation using the rabbit reticulocyte lysate system in the presence of [ 35 S]methionine. A single protein product of the expected size was detectable (Fig. 3). No protein product was detectable with the vector control (pcDNA3.1) (data not shown).
Expression of Recombinant LOXC in COS-7 Cells and Western Blot Analysis-A recombinant LOXC protein was expressed in COS-7 cells by transfection with pCMV/mLOXC and identified in cell lysate upon polyacrylamide gel by immunoblotting using anti-LOXC antibody (Fig. 4). A protein band of LOXC was observed in the cell lysate. Whereas no signal was observed in the mock-transfected cells, a specific band of ϳ82-kDa was in reasonable agreement with the calculated value of the molecular mass.
Expression of LOXC in Various Cell Lines-We assessed by Northern analysis the expression of LOXC mRNA in the following cultured cell lines: ATDC5, C3H10T1/2, MC3T3-E1, NIH3T3, and C2C12. A major hybridization band of about 5.4 kb was detected in C3H10T1/2 cells and MC3T3-E1 cells but not in NIH3T3 cells and C2C12 cells (Fig. 5A). Interestingly, the LOXC gene exhibited a distinct temporal pattern of expression along with the phenotypic transitions of ATDC5 cells. The LOXC mRNA level increased in parallel with the induction of type II collagen mRNA level in the culture and peaked on day 7 (Fig. 5B). The level declined until day 14 and then increased until cells became calcified on day 42. As shown in Fig. 5C, LOXC protein was not detected in undifferentiated ATDC5 by Western blot analysis, and the protein level in the culture increased during a process of chondrogenic differentiation in these cells.
Expression of LOXC mRNA in the Adult Mice Tissues and Growth Plates of Neonate Mice-Northern analysis showed that among the various adult mice tissues, LOXC mRNA was expressed in cartilage and not in heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis (Fig. 6, A and B). By in situ hybridization, LOXC mRNA was localized in hypertrophic and calcified chondrocytes of growth plates in neonate mice (Fig. 7).
Lysyl Oxidase Enzyme Activity of LOXC-The deduced amino acid sequence of LOXC showed homology with that of lysyl oxidase, suggesting that LOXC may possess the lysyl oxidase enzyme activity. We assessed such activity by a tritium release assay procedure using type I and type II collagen substrates prepared from chick embryos. LOXC protein was produced in the conditioned media of COS-7 cells transfected with LOXC cDNA and was detected by Western blot analysis. Significant activities were detected in the conditioned media of FIG. 7. Localization of LOXC, type II collagen, and type X collagen gene expression in growth plates in neonate mice. Growth plates of tibiae of neonate mice were fixed, dehydrated, and embedded in paraffin. Sections (6 m thick) were processed for in situ hybridization as described under "Experimental Procedures." Silver grains were accumulated in hypertrophic and calcified chondrocytes of growth plates of tibiae. Three independent experiments were performed and gave similar results. COS-7 cells transfected with LOXC cDNA but not with the control vector (Fig. 8). Moreover, these enzyme activities were inhibited by ␤APN, a mechanism-based, irreversible inhibitor of lysyl oxidase. DISCUSSION We have isolated by suppression subtractive hybridization a novel gene, LOXC, encoding a protein of 757 amino acids, which is expressed in cartilage in vivo. The amino acid sequence of mouse LOXC showed 50% identity with that of a mouse lysyl oxidase. Lysyl oxidase is an extracellular, copperdependent enzyme that initiates covalent cross-linking between and within the molecular units of collagens by catalyzing the oxidative deamination of peptidyl lysine in these proteins to peptidyl ␣-aminoadipic-␦-semialdehyde (22). Lysyl oxidase is synthesized as a preproprotein, secreted as a 50-kDa proen-zyme, and then proteolytically cleaved to the 32-kDa catalytically active, mature enzyme. There are three lines of evidence for the notion that LOXC is an extracellular protein secreted as an 82-kDa active, mature enzyme. First, LOXC protein has a putative signal peptide at its N-terminal (Fig. 2). Second, amino acid sequence alignment revealed that LOXC contains four repeats of the scavenger receptor cysteine-rich domains, found in diverse secreted and cell membrane-associated proteins (23). Finally, LOXC protein was recognized as a specific band of 82-kDa in the conditioned media of COS-7 cells transfected with LOXC cDNA on Western blotting with anti-LOXC antibody.
Lysyl oxidase contains one tightly bound copper (II) cofactor (22). Two sequence motifs important for the binding to copper have been determined, which are highly conserved among different lysyl oxidase proteins, WXWHXCHXHXH is involved in the copper binding coordination and includes the four histidines that supply the nitrogen ligands, and GHK, another putative collagen-related copper affinity site, is also conserved in LOXC (Fig. 2). Lysyl oxidase also contains a lysine tyrosylquinone as a carbonyl cofactor (24). The Tyr and Lys residues in the C-terminal of lysyl oxidase participate together in the formation of this cofactor, and these Lys (amino acid 639) and Tyr (amino acid 675) residues are conserved in LOXC (Fig.  2). These data raise the possibility that LOXC possesses the lysyl oxidase enzyme activity and is involved in cross-linking of extracellular matrix. Indeed, the conditioned media of COS-7 cells transfected with LOXC cDNA exhibited significant lysyl oxidase enzyme activity, using chick 3 H-labeled type I and type II collagen substrates. Moreover, these enzyme activities were inhibited by ␤APN.
Endochondral bone formation includes a cascade of cellular events such as cellular condensation of chondroprogenitor cells, proliferation, maturation, hypertrophic conversion, calcification of chondrocytes, and the cartilage replacement by bone. During these processes, collagenous components of the extracellular matrix are transitionally changed from type II collagen to type X collagen. Furthermore, condensed chondroprogenitors and the chondro-osseous mineralizing border exhibit the expression of type I collagen. Although the expression pattern of LOXC protein in the growth plate remains to be elucidated, our in vitro and in vivo data that LOXC is expressed in condensed chondroprogenitor cells and hypertrophic and calcified chondrocytes and possesses lysyl oxidase enzyme activity provide evidence for the hypothesis that LOXC may regulate the endochondral bone formation by modulating collagenous extracellular matrix.
In this study, we identified LOXC gene by suppression subtractive hybridization to screen genes highly expressed in hypertrophic and calcified ATDC5 cells. Our observations that LOXC is expressed in hypertrophic and calcified chondrocytes in vivo and certain cell lines in vitro and that LOXC has lysyl oxidase enzyme activity raise the possibility that LOXC may play a critical role in endochondral bone formation.