A New Lysyl Oxidase-related Gene in Cartilage Ito H. et al. 1

A New Lysyl Oxidase-related Gene in Cartilage Ito H. et al.

1

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

in Cartilage

Hiromu Ito, Haruhiko Akiyama , Hiroshi Iguchi , Ken-ichi Iyama , Masahiro Miyamoto,

Kunitaka Ohsawa, and Takashi Nakamura

Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University,

Sakyo, Kyoto 606-8507, Department of Hygiene, Hyogo College of Medicine,

Nishinomiya, Hyogo 663-8501, Department of Surgical Pathology, Kumamoto University

School of Medicine, Kumamoto 860-8556, Japan

Running title: A New Lysyl Oxidase-related Gene in Cartilage

The first two authors equally contributed to the work.

To whom correspondence should be addressed: Department of Orthopaedic Surgery,

Graduate School of Medicine, Kyoto University, Sakyo, Kyoto 606-8507, Japan. Tel: 81-

75-751-3652; Fax: 81-75-751-8409; E-mail: [email protected]

This work was supported by a grant from the Japan Orthopaedics and Traumatology

Foundation, Inc. (No. 0123).

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 5, 2001 as Manuscript M100861200 by guest on M

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SUMMARY

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, while 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 ß-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.

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INTRODUCTION

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 by a variety of growth and differentiation modulating

factors, including bone morphogenetic proteins (3, 4), fibroblast growth factors (5, 6),

parathyroid hormone-related peptide (7-9) 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 (11-14). 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


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converted to type X collagen-expressing hypertrophic chondrocytes, following the process

of mineralization. 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 isolated a novel cDNA clone encoding a lysyl oxidase-related

protein expressed in cartilage as well as ATDC5 cells.

EXPERIMENTAL PROCEDURES

Cells and Culture Conditions

ATDC5 cells were plated in six-multiwell plates at an initial cell density of 6 x104

cells/well and cultured as previously described (11-15). Briefly, cells were cultured for the

initial 21 day-period in DMEM/Ham's F12 hybrid medium (ICN Pharmaceuticals, Inc., Costa

Mesa, CA) containing 5% FBS (GIBCO BRL, Gaithersburg, MD), 10 µM human transferrin

(Roche Molecular Biochemicals, Tokyo, Japan), 30 nM sodium selenite (Sigma Chemical

Co., St. Louis, MO), and 10 µM bovine insulin (Wako Pure Chemical, Osaka, Japan) at

37 C in a humidified 5% CO2/95% air atmosphere. On day 21, the culture medium was

switched to α-MEM (ICN Pharmaceuticals, Inc.) containing 5 % FBS, 10 µM human

transferrin, 30 nM sodium selenite and 10 µM bovine insulin, and the CO2 concentration

was shifted to 3% for facilitating cellular hypertrophy and mineralization in culture. Clonal


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mouse embryonic fibroblast C3H10T1/2 cells, clonal mouse osteogenic MC3T3-E1 cells,

clonal mouse fibroblast NIH3T3 cells, and clonal mouse myoblastic C2C12 cells (RIKEN

Cell Bank, Tsukuba, Japan) were plated in six-multiwell plates at an initial cell density of 6

x104 cells/well and cultured as previously described (16).

RNA Extraction and Suppression Subtractive Hybridization

Poly (A)+ RNA was isolated from differentiated (day 21) and calcified (day 42)

ATDC5 cells by a single-step method as previously described (17) and analyzed by

suppression subtractive hybridization according to the manufacture's instruction (PCR-

Select cDNA Subtractions Kit, Clontech Laboratories, Inc., Palo Alto, CA). The cDNA

fragment of approximately 600-basepairs (bp) 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

Oligo (dT)-primed cDNA library from poly (A)+ RNA of calcified ATDC5 cells was

constructed in ZAP Express vector (Stratagene, La Jolla, CA), and 1x106 plaques were

screened with the cDNA fragment as a probe (12, 16). Plaques were transferred to the

membranes (137-mm nylon membrane, NEN Life Science Products, Inc., Boston, MA), the

600-bp fragment was [32P]-labeled (BcaBEST labeling kit, Takara, Otsu, Japan), and

hybridization was performed as previously described (12). The membranes were washed


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to a final stringency of 0.1 X SSPE (3 M NaCl, 197 mM NaH2PO4, 25 mM EDTA), and 0.1 %

SDS at 55 C . The nucleotide sequence was determined with ALFred DNA Sequencer

(Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, U.K.).

Northern Analysis

ATDC5 cells, C3H10T1/2 cells, MC3T3-E1 cells, NIH3T3 cells, and C2C12 cells

were plated in six-multiwell plastic plates and cultured as described above. Total RNA from

various cell lines cultured in vitro as well as poly (A)+ RNA from rib cartilage of 3-week-old

ICR mice were isolated, and analyzed by northern hybridization as previously described

(12, 17). Briefly, total RNA (20 µg) and poly (A)+ RNA (2 µg) were denatured, separated by

1 % agarose gel electrophoresis, and transferred on Nytran membranes (Schleicher &

Schuell, Dassel, Germany). A 1.3-kilobasepairs (kb) cDNA fragment of LOXC, a 0.55-kb

cDNA fragment 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, Rochester, NY) at -80 C with Cronex lightening plus intensifying

screens (DuPont, Boston, MA).


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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 embedding in paraffin. Sections (6 µm thick) were then processed for in situ

hybridization as previously described (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(+) (Stratagene) were linealized with appropriate restriction enzymes to transcribe

either sense or antisense [35S]-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 system (TnT Coupled Transcription/Translation Systems, Promega Co.,

Madison, WI) in the presence of [35S]-methionine (Amersham Pharmacis Biotech, Cat.#

AG1094) according to the manufacturer's instructions. The 5.4-kb mouse LOXC cDNA


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cloned into pcDNA3.1 vector (Invitrogen) was used as a template (pCMV/mLOXC). The

translation product was electrophoresed on a 10-20 % polyacrylamide gel and detected by

autoradiography with the X-Omat films (Eastman Kodak) (8 h exposure at room

temperature). Prestained rainbow marker (Bio-Rad Laboratories, Hercules, CA) was

loaded in the adjacent lane to estimate a molecular size.

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 previously

described (19).

DNA Transfection of COS-7 Cells

COS-7 cells were cultured in DMEM medium (ICN Pharmaceuticals, Inc.) containing

10 % FBS. For the assay of lysyl oxidase activity, COS-7 cells were transiently transfected

with pCMV/mLOXC, or pcDNA3.1 by using DEAE-dextran (Sigma Chemical Co.), and the

conditioned media were prepared seven days after transfection. For western blot analysis,

COS-7 cells were transfected with pCMV/mLOXC, or pcDNA3.1 by using FuGene6 (Roche

Molecular Biochemicals, Tokyo, Japan) according to the manufacturer's instructions, and

total cellular proteins were prepared two days after transfection.


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Western Blot Analysis

Total cellular proteins were prepared from ATDC5 cells and COS-7 cells transfected

with pCMV/mLOXC, or pcDNA3.1 as previously described (17). Forty micrograms of

protein was separated by a 10-20 % polyacrylamide gel and transferred to nitrocellulose

filters. The filters were blocked in 3% gelatin in Tris-buffered saline containing 0.1% Tween

20 and then incubated with the anti-LOXC antibody. Prestained rainbow marker (Bio-Rad

Laboratories) 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 previously described (20). In brief, twenty 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 (GIBCO BRL). The suspension was supplemented with L-

[4,5-3H]lysine (200 µCi, NEN Life Science Products, Inc., Cat. # NET-376) and ß-

aminopropionitrile (ßAPN)-fumarate (50 µg/ml as ßAPN, Sigma Chemical Co.), 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 g for 20 min. Labeled collagens in the

supernatant fraction were precipitated by salting with 20 % NaCl and collected by

centrifugation at 30,000 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


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the same buffer for 24 h.

The substrate solution containing [3H]-type I or type II collagen equivalent to 2.5 X

105 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 -20 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 ßAPN (50 µg/ml), a specific inhibitor of

lysyl oxidase, after pre-incubation with [3H]-labeled collagen substrates for 1 h prior to the

addition of the conditioned media.

Statistical Analysis

Statistical significance was assessed by one-way 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 orderly in a synchronous manner as

evidenced by the expression of phenotypic marker genes with cartilage-characteristics, as


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previously reported (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 which began on day 35. To find genes upregulated

during a process of cellular hypertrophy and calcification of chondrocytes, we performed

suppression subtractive hybridization using the poly (A)+ RNA extracted from differentiated

and calcified ATDC5 cells, and obtained a 600-bp cDNA fragment corresponding to the 3

-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 X 106 independent plaques. Eight

out of twenty clones contained a cDNA of about 5.4-kb. Figure 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-daltons (Da) and an isoelectric

point of 7.8. The 3'-end of the sequence contains a poly (A) stretch, preceded by putative

polyadenilation 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


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homology with that of mouse lysyl oxidase (LO) and of mouse lysyl oxidase-related protein

2 (Lor2) (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 [35S] 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.

While no signal was observed in the mock-transfected cells, a specific band of

approximately 82-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 cultured cell

lines: ATDC5, C3H10T1/2, MC3T3-E1, NIH3T3, and C2C12. A major hybridization band of


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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. 6A 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


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western blot analysis. Significant activities were detected in the conditioned media of

COS-7 cells transfected with LOXC cDNA, but not with the control vector (Fig. 8). Moreover,

these enzyme activities were inhibited by ßAPN, 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, copper-dependent enzyme that initiates covalent cross-

linking between and within the molecular units of collagens by catalyzing the oxidative

deamination of peptidyl lysyne in these proteins to peptidyl α-aminoadipic-δ-semialdehyde

(22). Lysyl oxidase is synthesized as a preproprotein, secreted as a 50-kDa proenzyme,

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 a

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


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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: 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 [3H]-type I and type II

collagen substrates. Moreover, these enzyme activities were inhibited by ßAPN.

Endochondral bone formation includes a cascade of cellular events such as cellular

condensation of chondroprogenitor cells, proliferation, maturation, hypertrophic conversion

and 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


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elucidated, our in vitro and in vivo data that, LOXC is expressed in condensed

chondroprogenitor cells and hypertrophic and calcified chondrocytes as well as 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.


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FIGURE LEGENDS

FIG.1.

Structure of mouse LOXC cDNA. Nucleotide sequence of mouse LOXC and the deduced

amino acid sequence. The GenBank accession number for mouse LOXC is AF338440.

FIG. 2.

Comparison of the amino acid sequences of LOXC, lysyl oxidase-related protein 2 (Lor2),

and lysyl oxidase (LO). Identical residues are shown in gray. Conserved cysteine residues

are shown in black. The putative cleavage site is indicated by an arrow. The four scavenger

receptor cysteine-rich domains are overlined and numbered in parenthesis. The putative

copper binding site and the GHK site are boxed. The Lys and Tyr residues which form lysyl

tyrosylquinone by cross-linking are indicated by the arrow heads. The amino acid

sequences of mouse LO and mouse Lor2 are deduced from the nucleotide sequences of

the GenBank accession numbers, NM010728 and AF053368, respectively.

FIG. 3.

Translation of LOXC gene in vitro. A coupled transcription/translation reaction (TnT

Reticulocytes, Promega) was performed using mouse LOXC cDNA cloned into pcDNA3.1

vectors as a template. The translation products were separated by electrophoresis on a

gradient polyacrylamide gel (10-20 %) and detected by autoradiography. Three


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independent experiments were performed and gave similar results.

FIG. 4.

Expression of LOXC protein. pCMV/mLOXC, or pcDNA3.1 vector as a control, was

transiently transfected into COS-7 cells, and LOXC protein was detected using anti-LOXC

antibody. Original magnification, x 100. Three independent experiments were performed

and gave similar results.

FIG. 5.

Expression of LOXC mRNA in various cell lines. A, 20 µg per lane of total RNA extracted

from each cells, C3H10T1/2, MC3T3-E1 (undifferentiated and calcified), NIH3T3, or C2C12,

was analyzed by northern blot hybridization using 1.3-kb LOXC cDNA. B and C, ATDC5

cells were cultured described in Experimental Procedures. 20 µg of total RNA and the

conditioned media were prepared at the time point indicated and analyzed by northern blot

using 1.3-kb LOXC cDNA, 0.55-kb type II collagen cDNA and 0.65-kb type X collagen

cDNA (B) and by western blot using anti-LOXC antibody (C). Three independent

experiments were performed and gave similar results.

FIG. 6.

Expression of LOXC mRNA in various adult mouse tissues. A, multiple tissue blot


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containing 2 µg of poly (A)+ RNA from various mouse tissues (Clontech) was hybridized

with the LOXC cDNA (top) and a ß-actin probe (bottom). The mRNA in each lane was

isolated from 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; and

8, testis. A DNA fragment of ß-actin was also hybridized to the same blot as a control. B, 2

µg of poly (A)+ RNA from rib cartilage was hybridized with the LOXC cDNA (top) and a ß-

actin probe (bottom). Three independent experiments were performed and gave similar

results.

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 µm thick) were processed for in situ hybridization as

described in 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.

FIG. 8.

Lysyl oxidase activity of LOXC. The lysyl oxidase activities of the conditioned media of


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COS-7 cells transfected with plasmids containing LOXC cDNA and control plasmids were

assayed as described in Experimental Procedures. Data are shown as the mean SD of

six separate determinations. The figure represents one of three independent experiments

with similar results. * denotes statistically significant differences from the corresponding

control and ßAPN groups at p<0.001.


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FIG.1

1 ATGATGTGGCCCCAACCACCCACCTTCTCCCTGTTCCTGCTACTGCTGCTAAGCCAAGCCCCTTCCAGTAGGCCACAGTCATCAGGCACCAAGAAGCTCAGGCTTGTGGGGCCAGCGGAC M M W P Q P P T F S L F L L L L L S Q A P S S R P Q S S G T K K L R L V G P A D 121 AGACCAGAGGAGGGCCGCTTGGAGGTGCTGCACCAGGGCCAGTGGGGCACGGTGTGTGATGATGATTTCGCTCTCCAGGAGGCTACTGTGGCCTGCCGACAGCTGGGCTTTGAGTCAGCC R P E E G R L E V L H Q G Q W G T V C D D D F A L Q E A T V A C R Q L G F E S A

241 TTGACCTGGGCACACAGTGCCAAGTATGGTCAAGGAGAGGGTCCCATCTGGCTGGACAATGTTCGTTGTTTGGGCACCGAGAAGACCCTAGATCAGTGTGGCTCTAACGGCTGGGGTATC L T W A H S A K Y G Q G E G P I W L D N V R C L G T E K T L D Q C G S N G W G I

361 AGTGACTGCAGACACTCAGAAGATGTTGGGGTGGTATGTCACCCACGGCGCCAGCACGGATATCACTCTGAGAAGGTCTCCAATGCCCTCGGGCCTCAGGGCCGGCGGCTAGAAGAGGTA S D C R H S E D V G V V C H P R R Q H G Y H S E K V S N A L G P Q G R R L E E V

481 CGGCTGAAACCCATCCTCGCCAGTGCCAAAAGGCACAGCCCAGTGACTGAAGGGGCTGTGGAAGTACGGTACGACGGCCACTGGAGGCAGGTGTGTGACCAGGGCTGGACCATGAACAAC R L K P I L A S A K R H S P V T E G A V E V R Y D G H W R Q V C D Q G W T M N N

601 AGCAGGGTTGTATGCGGGATGCTGGGCTTTCCCAGTCAGACATCTGTCAACAGCCACTACTACAGAAAAGTCTGGAATCTGAAGATGAAGGATCCCAAGTCTAGGCTCAACAGCCTGACA S R V V C G M L G F P S Q T S V N S H Y Y R K V W N L K M K D P K S R L N S L T

721 AAAAAGAATTCCTTCTGGATTCACCGGGTTGACTGTTTCGGGACAGAGCCCCACTTGGCCAAGTGCCAGGTACAGGTGGCTCCAGGAAGGGGCAAGCTTCGGCCAGCCTGTCCAGGCGGC K K N S F W I H R V D C F G T E P H L A K C Q V Q V A P G R G K L R P A C P G G

841 ATGCACGCTGTGGTCAGCTGTGTGGCAGGGCCCCACTTCCGCCGACAGAAGCCAAAGCCCACGCGCAAGGAGTCCCATGCAGAGGAGCTGAAAGTGCGCCTGCGCTCTGGGGCTCAGGTG M H A V V S C V A G P H F R R Q K P K P T R K E S H A E E L K V R L R S G A Q V

961 GGTGAGGGCCGTGTGGAAGTGCTCATGAACCGCCAGTGGGGCACAGTCTGTGACCACAGGTGGAACCTCATCTCAGCCAGCGTCGTGTGTCGCCAGCTTGGCTTTGGCTCTGCCCGGGAG G E G R V E V L M N R Q W G T V C D H R W N L I S A S V V C R Q L G F G S A R E

1081 GCCCTTTTTGGGGCCCAGTTGGGTCAAGGGCTAGGACCCATCCACCTGAGTGAGGTGCGCTGCCGGGGGTATGAGCGGACCCTGGGTGACTGCCTTGCCCTGGAAGGGTCCCAGAATGGT A L F G A Q L G Q G L G P I H L S E V R C R G Y E R T L G D C L A L E G S Q N G

1201 TGTCAACATGCAAACGACGCTGCTGTCAGGTGCAACATCCCAGACATGGGCTTCCAGAACAAGGTGCGCTTGGCTGGTGGGCGCAACTCCGAAGAGGGAGTGGTGGAGGTGCAGGTGGAG C Q H A N D A A V R C N I P D M G F Q N K V R L A G G R N S E E G V V E V Q V E

1321 GTGAATGGGGGTCCACGATGGGGGACTGTATGCAGTGACCACTGGGGGCTCACCGAAGCCATGGTGACCTGTCGGCAACTTGGTCTGGGATTTGCCAACTTTGCTCTCAAGGACACCTGG V N G G P R W G T V C S D H W G L T E A M V T C R Q L G L G F A N F A L K D T W

1441 TACTGGCAGGGGACACCAGAGGCCAAAGAAGTGGTGATGAGTGGAGTTCGCTGCTCCGGCACAGAAATGGCCCTGCAGCAGTGTCAGAGGCATGGGCCGGTGCACTGTTCCCACGGCCCA Y W Q G T P E A K E V V M S G V R C S G T E M A L Q Q C Q R H G P V H C S H G P

1561 GGGCGCTTCTCGGCTGGCGTTGCTTGTATGAACAGTGCTCCAGACCTCGTGATGAACGCCCAGCTGGTACAAGAGACGGCGTACTTGGAGGATCGTCCACTCAGCATGCTGTACTGTGCT G R F S A G V A C M N S A P D L V M N A Q L V Q E T A Y L E D R P L S M L Y C A

1681 CACGAGGAAAACTGCCTCTCCAAGTCCGCTGATCACATGGACTGGCCCTACGGGTACCGGCGCTTGCTGCGCTTCTCCTCACAGATCTACAACCTTGGCCGGGCCGACTTCCGTCCCAAG H E E N C L S K S A D H M D W P Y G Y R R L L R F S S Q I Y N L G R A D F R P K

1801 GCTGGACGCCACAGCTGGATTTGGCACCAGTGCCACAGGCATAACCACAGCATCGAAGTCTTCACTCATTATGACCTGCTCACGCTCAATGGCTCCAAGGTGGCCGAGGGACACAAGGCC A G R H S W I W H Q C H R H N H S I E V F T H Y D L L T L N G S K V A E G H K A

1921 AGCTTCTGTCTAGAAGATACAAACTGCCCCTCAGGAGTGCAGCGGCGCTATGCATGTGCCAACTTTGGGGAACAGGGAGTGGCTGTAGGCTGCTGGGACACCTACCGGCATGACATCGAT S F C L E D T N C P S G V Q R R Y A C A N F G E Q G V A V G C W D T Y R H D I D

2041 TGCCAGTGGGTGGATATCACAGATGTGGGTCCAGGGGACTATATCTTCCAGGTGGTTGTGAACCCCACAAACGATGTTGCAGAGTCCGATTTCTCCAATAACATGATACGGTGCCGCTGC C Q W V D I T D V G P G D Y I F Q V V V N P T N D V A E S D F S N N M I R C R C

2161 AAGTATGATGGACAGCGAGTCTGGTTGCACAACTGCCACACAGGAGATTCCTACCGAGCCAATGCAGAGCTCTCCCTGGAGCAGGAACAGCGACTCAGGAACAACTTGATCTGA 2274 K Y D G Q R V W L H N C H T G D S Y R A N A E L S L E Q E Q R L R N N L I *


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1) LOXC 1 MMWPQPPTFSLFLLLLLSQAPSSRPQSSGTKKLRLVGPADRPEEGRLEVLHQGQWGTVCDDDFALLor2 1 MRAVSVWYCCPWGLLLLHCLCSFSVGSPSPSISPEKKVGSQGLRFRLAGFPRKPYEGRVEIQRAGEWGTICDDDFTL

LOXC 66 QEATVACRQLGFESALTWAHSAKYGQGEGPIWLDNVRCLGTEKTLDQCGSNGWGISDCRHSEDVGVVCHPRRQHGYHLor2 78 QAAHVLCRELGFTEATGWTHSAKYGPGTGRIWLDNLSCRGTEGSVTECASRGWGNSDCTHDEDAGVICKDQRLPGFS

2) LOXC 143 SEKVSNALGPQGRRLEEVRLKPILASAKRHSPVTEGAVEVRYDGHWRQVCDQGWTMNNSRVVCGMLGFPSQTSVNSHLor2 155 DSNVIE--VEHQLQVEEVRLRPAVEWGRRPLPVTEGLVEVRLPEGWSQVCDKGWSAHNSHVVCGMLGFPGEKRVNMA

LOXC 220 YYRKVWNLKMKDPKSRLNSLTKKNSFWIHRVDCFGTEPHLAKCQVQVAPGRGKLRPACPGGMHAVVSCVAGPHFRRQLor2 230 FY-------------RMLAQKKQHSFGLHSVACVGTEAHLSLCSLEFYRANDTTR--CSGGNPAVVSCVLGPLYATF

3)LOXC 297 KPKPTRKESHAE-ELKVRLRSGAQVGEGRVEVLMNRQWGTVCDHRWNLISASVVCRQLGFGSAREALFGAQLGQGLGLor2 292 TGQKKQQHSKPQGEARVRLKGGAHQGEGRVEVLKAGTWGTVCDRKWDLQAASVVCPELGFGTAREALSGARMGQGMG

4)LOXC 373 PIHLSEVRCRGYERTLGDCLALEGSQNGCQHANDAAVRCNIPDMGFQNKVRLAGGRNSEEGVVEVQVEVNGGPRWGTLor2 369 AIHLSEVRCSGQEPSLWRCPSKNITAEDCSHSQDAGVRCNLPYTGVETKIRLSGGRSRYEGRVEVQIGIPGHLRWGL

LOXC 450 VCSDHWGLTEAMVTCRQLGLGFANFALKDTWYWQGTPEAKEVVMSGVRCSGTEMALQQCQRHGP-VHCSHGPGRFSALor2 446 ICGDDWGTLEAMVACRQLGLGYANHGLQETWYWDSG-NVTEVVMSGVRCTGSELSLNQCAHHSSHITCKKTGTRFTALO 1 DDPYNPYKYSDDNPYYNYYDTYER-PRPGSRNRPGYG

LOXC 526 GVACMNSAPDLVMNAQLVQETAYLEDRPLSMLYCAHEENCLSKSADHMD-WPYGYRRLLRFSSQIYNLGRADFRPKALor2 522 GVICSETASDLLLHSALVQETAYIEDRPLHMLYCAAEENCLASSARSAN-WPYGHRRLLRFSSQIHNLGRADFRPKALO 37 TGYFQYGLPDLVPDPYYIQASTYVQKMSMYNLRCAAEENCLASSAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSR

LOXC 602 GRHSWIWHQCHRHNHSIEVFTHYDLLTLNG-SKVAEGHKASFCLEDTNCPSGVQRRYACANFGEQGVAVGCWDTYRHLor2 598 GRHSWVWHECHGHYHSMDIFTHYDILTPNG-TKVAEGHKASFCLEDTECQEDVSKRYECANFGEQGITVGCWDLYRHLO 114 PRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTA-HTQGLSPGCYDTYAA

LOXC 678 DIDCQWVDITDVGPGDYIFQVVVNPTNDVAESDFSNNMIRCRCKYDGQRVWLHNCHTGDSYRANAELSLEQEQRLRNLor2 674 DIDCQWIDITDVKPGNYILQVVINPNFEVAESDFTNNAMKCNCKYDGHRIWVHNCHIGDAFSEEANRRFERYPGQTSLO 190 DIDCQWIDITDVQPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHHAYASGCTISPY

LOXC 755 NLILor2 751 NQIV

FIG.2


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~ 84 kDa

( kDa )

34.3

50

28.8

77103

20.7

FIG.3


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LOXC-tr

ansf

ecte

d

moc

k-tra

nsfe

cted

( kDa )

50

~ 82 kDa77

103

FIG.4


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LOXC

C3H10

T1/2

NIH3T

3

MC3T3-

E1 undiff

.

MC3T3-

E1 calc

if.

C2C12

FIG.5A


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α1(II)

LOXC

α1(X)

3 5 7 10 14 21 42 (day)FIG.5B


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( kDa )

34.3

50

28.8

~ 82 kDa77103

20.7

3 21 42 (day)FIG.5C


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1 2 3 4 5 6 7 8

kb

7.5

4.4

2.4

1.35

A

ß-actin

5.4kb

FIG.6A


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cart

ilageB

FIG.6B

ß-actin

18S

28S5.4kb


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500

400

300

200

100LOXCcontrol LOXC APN

600

500

400

300

200

100control LOXC LOXC APN

type I collagen

type II collagen

FIG.8

*

*

cpm

cpm


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Kunitaka Ohsawa and Takashi NakamuraHiromu Ito, Haruhiko Akiyama, Hiroshi Iguchi, Ken-ichi Iyama, Masahiro Miyamoto,

expressed in cartilageMolecular cloning and biological activity of a novel lysyl oxidase-related gene

published online April 5, 2001J. Biol. Chem.

10.1074/jbc.M100861200Access the most updated version of this article at doi:

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Documents

A New Lysyl Oxidase-related Gene in Cartilage Ito H. et al. 1