Esophageal cancer related gene 4 (ECRG4) is a marker of articular chondrocyte differentiation and cartilage destruction

Gene 448 (2009) 7–15

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Gene j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / g e n e

Esophageal cancer related gene 4 (ECRG4) is a marker of articular chondrocyte differentiation and cartilage destruction Yun Hyun Huh a, Je-Hwang Ryu a, Sun Shin a, Dong-Uk Lee a, Siyoung Yang a, Kyung-Shin Oh a, Churl-Hong Chun b, Jeong-Keun Choi a, Woo Keun Song a, Jang-Soo Chun a,⁎ a b

Cell Dynamics Research Center and BioImaging Research Center, Department of Life Sciences, Gwangju Institute of Science and Technology, Buk-Gu, Gwangju, 500-712, Korea Department of Orthopaedic Surgery, Wonkwang University School of Medicine, Iksan 570-711, Korea

a r t i c l e

i n f o

Article history: Received 9 May 2009 Received in revised form 15 August 2009 Accepted 24 August 2009 Available online 6 September 2009 Received by A. Bernardi Keywords: Articular cartilage Chondrocytes Collagen II ECRG4 Osteoarthritis

a b s t r a c t With the aim of identifying novel genes regulating cartilage development and degeneration, we screened a cartilage-specific expressed sequence tag database. Esophageal cancer related gene 4 (ECRG4) was selected, based on the criteria of ‘chondrocyte-specific’ and ‘unknown function.’ ECRG4 expression was particularly abundant in chondrocytes and cartilage, compared to various other mouse tissues. ECRG4 is a secreted protein that undergoes cleavage after secretion. The protein is specifically expressed in chondrocytes in a manner dependent on differentiation status. The expression is very low in mesenchymal cells, and dramatically increased during chondrogenic differentiation. The ECRG4 level in differentiated chondrocytes is decreased during hypertrophic maturation, both in vitro and in vivo, and additionally in dedifferentiating chondrocytes induced by interleukin-1β or serial subculture, chondrocytes of human osteoarthritic cartilage and experimental mouse osteoarthritic cartilage. However, ectopic expression or exogenous ECRG4 treatment in a primary culture cell system does not affect chondrogenesis of mesenchymal cells, hypertrophic maturation of chondrocytes or dedifferentiation of differentiated chondrocytes. Additionally, cartilage development and organization of extracellular matrix are not affected in transgenic mice overexpressing ECRG4 in cartilage tissue. However, ectopic expression of ECRG4 reduced proliferation of primary culture chondrocytes. While the underlying mechanisms of ECRG4 expression and specific roles remain to be elucidated in more detail, our results support its function as a marker of differentiated articular chondrocytes and cartilage destruction. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes, which is triggered by precartilage condensation (DeLise et al., 2000; Goldring et al., 2006). Differentiated chondrocytes either remain as permanent chondrocytes in joint articular cartilage or mature into hypertrophic chondrocytes to serve as a template for long bone development during endochondral bone formation (Tickle, 2002; Mariani and Martin, 2003). These sequential events of cartilage and bone development are precisely modulated by various growth factors and regulatory genes released from cartilage elements and perichondrium (DeLise et al., 2000; Goldring et al., 2006). Articular chondrocytes Abbreviations: DMEM, Dulbecco's modified Eagle's medium; ECRG4, esophageal cancer related gene 4; ECM, extracellular matrix; EST, expressed sequence tag; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IGF, insulin-like growth factor; IL, interleukin; MMP, matrix metalloproteinase; OA, osteoarthritis; P, passage; qRT-PCR, quantitative real-time polymerase chain reaction; RT-PCR, reverse transcription– polymerase chain reaction; TG, transgenic; WT, wild-type. ⁎ Corresponding author. Tel.: +82 970 2479; fax: +82 62 970 2484. E-mail address: [email protected] (J.-S. Chun). 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.08.015

maintain cartilage homeostasis by synthesizing cartilage-specific extracellular matrix (ECM) molecules, such as collagen II and sulfated proteoglycans. However, differentiated phenotypes of chondrocytes are unstable and rapidly lose their characteristics (i.e., undergo dedifferentiation) in response to environmental changes, such as exposure to interleukin (IL)-1β and during serial monolayer culture (Gouttenoire et al., 2004). Dedifferentiation of chondrocytes appears to contribute to cartilage destruction in arthritic disease. Cartilage destruction is caused by imbalance of ECM catabolism and anabolism (Malemud, 1999). Cartilage ECM degradation is attributed to the induction and activation of matrix metalloproteinases (MMP) and/or inactivation of tissue inhibitors of MMP, and additionally, insufficient synthesis of ECM molecules as a result of apoptosis and dedifferentiation of chondrocytes. We analyzed the UniGene library with a view to identifying novel genes involved in cartilage development and degeneration. UniGene, a well organized gene-oriented cluster database, is automatically divided into a set of GeneBank sequences, including expressed sequence tags (ESTs) (Peale and Gerritsen, 2001). The EST database provides important information on novel genes displaying tissue-specific expression profiles (Hong et al., 2005).

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Y.H. Huh et al. / Gene 448 (2009) 7–15

The translation product of clustered ESTs is effectively analyzed using an in silico approach, a useful tool to predict domains, functions, localization and tissue distribution of proteins. Analysis of the human normal cartilage UniGene library (Lib. 8940) EST database (Liu et al., 2000; Kim et al., 2008) facilitated selection of the Hs.43125 EST clone as a chondrocyte-specific gene with unknown functions in chondrocytes and cartilage. We cloned a mouse homolog of Hs.43125, which appeared identical to mouse esophageal cancer related gene 4 (ECRG4). Mouse ECRG4 displays 84% amino acid sequence identity to its human counterpart. ECRG4 is a 17 kDa protein composed of 148 amino acids. In this study, we characterize mouse ECRG4 and investigate its possible functions in chondrogenesis as well as cartilage development and destruction during osteoarthritis (OA). Based on our current results, we propose that ECRG4 is a marker of differentiated articular chondrocyte and cartilage destruction, with a capacity to inhibit proliferation of differentiated chondrocytes. 2. Materials and methods 2.1. In silico analysis The cDNA sequences of novel genes were searched using Lib. 8940 of the UniGene transcriptome library (www.ncbi.nlm.nih.gov/UniGene). Goblet (www.goblet.molgen.mpg.de) and AmiGO (www. godatabase.org/cgi-bin/go.cgi) were used for predicting gene ontology. SMART (www.smart.embl-heidelberg.de) was performed to predict the presence of various protein domains. SignalP (www.cbs. dtu.dk/services/SignalP) was applied to investigate the presence of signal peptides with cleavage sites at the N-terminus. 2.2. Tissue preparation from mouse and human Mouse limb buds were prepared from both forelimb and hindlimb buds of embryos at the indicated embryonic days. RNA was extracted from limb buds, and expression of collagen IIb and ECRG4 was determined by reverse transcription–polymerase chain reaction (RT-PCR). Tissues from forelimb buds of 14.5 dpc embryo were used for in situ hybridization to detect transcripts of collagen IIb and ECRG4 (Kim et al., 2008). Cartilage and all other tissues were obtained from newborn mice and used to detect ECRG4 expression by RT-PCR. Human osteoarthritic cartilage was obtained from patients (aged between 51 and 72 years) undergoing arthroplasty for osteoarthritic knee joints. Transfer of material was approved by the appropriate Human Subjects Committees. All cases satisfied the American College of Rheumatology classification criteria for OA of the knee (Altman et al., 1986). Cartilage tissues of each specimen were sampled down to the subchondral bone from tibial plateau within 60 min after operation. The undamaged part of cartilage was used as control tissue, and the severely damaged part was used as OA cartilage tissue. RNA was extracted from both normal and OA cartilage tissues, and collagen II expression was examined by RT-PCR. 2.3. Generation of ECRG4 transgenic (TG) mice and embryo analysis For generation of chondrocyte-specific ECRG4 TG mice, the ECRG4 gene was cloned into the NotI site of an expression vector containing the promoter and enhancer of mouse Col2a1 gene, as described by Ueta et al. (2001). ECRG4 TG mice were generated by Macrogen, Inc. (Seoul, Korea). Transgenic founders were mated with wild-type C57BL/6 mice to produce F1 heterozygotes. F1–F4 generations were genetically screened for the transgene at 3–4 weeks of age. The following two primers were used in amplification of tail genomic DNA to identify wild-type or heterozygous ECRG4 TG mice: 5′-TGGGTCCAGATGGCATAAGTGG-3′ and 5′-ATAGTTGACACTGGCCTCCATGCC-3′. To

determine the function of ECRG4 during the developmental stage, embryos (E14.5 and E16.5) were skinned, eviscerated and fixed in 95% ethanol for 4 days prior to staining. After 3 days in acetone, skeletons were stained with Alcian blue/Alizarin red staining solution (0.015% Alcian blue in 70% EtOH, 0.05% Alizarin red in 95% EtOH and 5% acetic acid in 70% EtOH) for 10 days. Embryos were placed in 1% KOH for 2 days to clear and stored in 100% glycerol. 2.4. Collagenase-induced cartilage destruction Experimental knee OA was induced in 10-week-old male C57BL/6 mice by intra-articular injection of 4 units of collagenase (Sigma, St. Louis, MO) on days 0 and 2. After 4 weeks, knee joints were isolated and processed for histologic assessment. Briefly, knee joints were fixed in 5% paraformaldehyde and treated with decalcification solution (Merck, Darmstadt, Germany) for 14 days. Paraffin-embedded sections were cut (8 μm thickness) and mounted onto Superfrost slides (Menzel-Glaser, Braunschweig, Germany). Sections were deparaffinized, hydrated and stained with hematoxylin for 10 min. After washing in running tap water for 10 min, slides were stained with Fast Green solution for 3 min and rinsed quickly with 1% acetic acid solution for no more than 10 s Finally, samples on slides were stained with 0.1% Safranin-O solution for 5 min and washed in running tap water for 3 min. Cartilage destruction was scored using the methods of Mankin et al. (1971). For biochemical analysis, normal and osteoarthritic mouse knee joint cartilage were collected using the method described by Blom et al. (2007) to determine the transcript levels of various molecules, as described below. 2.5. Cell culture Mesenchymal cells were derived from the distal tips of the limb buds of 11.5 dpc mouse embryos and maintained as micromass culture to induce chondrogenesis, as described previously (Kim et al., 2008). Briefly, cells were suspended at a density of 4.0 × 107/ml in Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Gaithersburg, MD) containing 10% (v/v) fetal bovine serum and spotted onto culture dishes. Cells were maintained in the absence or presence of various reagents, as indicated in each experiment. Chondrogenesis was determined by examining collagen II expression with RT-PCR, and accumulation of sulfated glycosaminoglycan with Alcian blue staining. Hypertrophic maturation of chondrocytes was induced by switching DMEM to DMEM/F-12 (2:3) medium supplemented with 50 μg/ml ascorbic acid and 5 mM β-glycerol phosphate at 6 day of micromass culture, and maintained up to 21 days (Mello and Tuan, 1999; Kim et al., 2008). Hypertrophic maturation was confirmed by examining the expression of collagen X and MMP-13 by RT-PCR or Alizarin red staining to detect chondrocyte mineralization, as described in a previous report (Kim et al., 2008). Micromass culture spots at day 5 were detached and embedded in paraffin wax for in situ hybridization of collagen II and ECRG4. Rabbit articular chondrocytes were released from cartilage slices of 2-week-old New Zealand White rabbits by enzymatic digestion, based on the procedure of Yoon et al. (2002). Cartilage slices were dissociated enzymatically for 4 h in 0.2% collagenase type II (381 units/mg solid; Sigma) in DMEM. Rib chondrocytes were obtained from 3-day-old newborn mice, as described previously (Kim et al., 2008). Cartilaginous rib cages were preincubated for 45 min in 0.2% collagenase type II. Cells isolated from articular cartilage and rib were resuspended in DMEM supplemented with 10% (v/v) fetal bovine serum, 50 μg/ml streptomycin and 50 units/ ml penicillin, and plated on culture dishes at a density of 5 × 104 cells/cm2. The culture medium was changed every 1.5 days after seeding, and cells reached confluence by days 4–5. Confluent primary cultures, designated passage (P) 0, were subcultured up to P3 by plating at a density of 5 × 104 cells/cm2 (Yoon et al., 2002).

Y.H. Huh et al. / Gene 448 (2009) 7–15

The differentiation status of chondrocytes was determined by examining collagen II expression. Proliferation of primary culture rabbit chondrocytes was examined by Ez-cytox enhanced cell viability assay kit (Itsbio, Inc., Seoul, Korea). 2.6. Construction of ECRG4 expression vector and lentivirus Mouse ECRG4 (Mm.50109) cDNA was prepared by RT-PCR from mouse rib chondrocytes with specific primers (Table 1) designed to introduce BamHI and XbaI sites at the 5′ and 3′ ends, respectively. The resulting cDNA was cloned into pcDNA3.1/myc-His vector (Invitrogen Corp., Carlsbad, CA). pCDNA-ECRG4-myc/his was transfected into primary chondrocytes using Metafectene (Biontex, Martinsried, Germany), according to the manufacturer's protocol. Transfected chondrocytes were cultured for an additional 36 h and employed for further experiments. Lentiviruses expressing ECRG4 were constructed by the Macrogen LentiVector Institute (Seoul, Korea). Briefly, the pcDNA3.1-ECRG4-myc/his vector was digested and inserted into a lentiviral vector (Lenti-mCMV-IRES-puro). The expression vector was subsequently transfected into 293T cells using Lipofectamine Plus (Invitrogen), and culture supernatant containing viral particles was harvested at 48 h after transfection. Titers were determined using the human immunodeficiency virus type I p24 enzyme-linked immunosorbent assay. Mesenchymal cells and rabbit primary chondrocytes were infected with mock or ECRG4 lentivirus and cultured for the indicated times.

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2.7. Preparation of ECRG4-conditioned medium Rabbit primary chondrocytes were transfected with the pCDNAECRG4 vector. Following growth to 80% confluency, control and ECRG4-expressing chondrocytes were washed and maintained in serum-free DMEM for 48 h. Conditioned medium was clarified by filtration (0.2 μm pore size) and concentrated 30 times by ultrafiltration in Amicon stirred cells (Millipore, Billerica, MA) using a YM membrane with 5 kDa molecular mass cutoff. Mesenchymal cells were maintained as micromass culture in the presence of control conditioned medium or conditioned medium containing ECRG4, and cultured for up to 5 or 13 days. 2.8. RT-PCR analysis and quantitative real-time PCR (qRT-PCR) Total RNA was isolated using TRI reagent (MRC, Inc., Cincinnati, OH), according to the manufacturer's protocol, and reverse-transcribed with ImProm-II™ reverse transcriptase (Promega, Madison, WI). The cDNA generated was amplified by PCR with Taq polymerase (Intron, Gyeonggido, Korea). The PCR primers and conditions are summarized in Table 1. qRT-PCR was performed using a chromo 4 cycler (Bio-Rad, Hercules, CA) and SYBR Premix Ex Taq™ (TaKaRa Bio, Inc., Shiga, Japan). All qRT-PCR reactions were performed in duplicate, and the amplification signal from the target gene was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signal in the same reaction.

Table 1 Oligonucleotide primers and PCR conditions. Gene ECRG4 ECRG4 Collagen II Collagen IIa/b Collagen IIb Collagen II Chondromodulin-1 Aggrecan GAPDH GAPDH GAPDH MMP-2 MMP-3 MMP-9 MMP-12 MMP-13 MMP-13 MMP-14 COX-2

S As S As S As S As S As S As S As S As S As S As S As S As S As S As S As S As S As S As S As

Primer sequences

Size (bp)

AT (°C)

Origina

Detectionb

5′-TGGGTCCAGATGGCATAAGTGG-3′ 5′-ATAGTTGACACTGGCTCCATGCC-3′ 5′-CGGGATCCCGATGAGCACCTCGTCTGCGCG-3′ 5′-GCTCTAGAGCATAGTCATCATAGTTGACACTGGC-3′ 5′-GACCCCATGCAGTACATGCG-3′ 5′-AGCCGCCATTGATGGTCTCC-3′ 5′-GGGTCTCCTGCCTCCTCCTGCTC-3′ 5′-CTCCATCTCTGCCACCGGGGT-3′ 5′-GGGTCTCCTGCCTCCTCCTGCTC-3′ 5′-TCCTTTCTGCCCCTTTGGCCCTAATTTTCGGG-3′ 5′-CAGTTGGGAGTAATGCAAG-3′ 5′-GCCTGGATAACCTCTGTG-3′ 5′-ACAGTGACCAAGCAGAGCATC-3′ 5′-GCGGCATCCTTGGTAATTG-3′ 5′-GAAGACGACATCACCATCCAG-3′ 5′-CTGTCTTTGTCACCCACACATG-3′ 5′-TCACCATCTTCCAGGAGCGA-3′ 5′-CACAATGCCGAAGTGGTCGT-3′ 5′-TCACTGCCACCCAGAAGAC-3′ 5′-TGTAGGCCATGAGGTCCAC-3′ 5′-CGTCTTCACCACCATGGAGA-3′ 5′-CGGCCATCACGCCACAGTTT-3′ 5′-CCGTGTGAAGTATGGCAATGC-3′ 5′-GCGGTCATCGTCGTAGTTGG-3′ 5′-TGTACCCAGTCTACAACGCC-3′ 5′-TCCAGGGACTCTCTCTTCTC-3′ 5′-CTCCTCGTGCTGGGCTGTTG-3′ 5′-TACACGCGGGTGAAGGTGAG-3′ 5′-CCCAGAGGTCAAGATGGATG-3′ 5′-GGCTCCATAGAGGGACTGAA-3′ 5′-CCTACACCGGCAAGAGTCAC-3′ 5′-TCTTGGGAATCCCAGTTCAG-3′ 5′-TGATGGACCTTCTGGTCTTCTGG-3′ 5′-CATCCACATGGTTGGGAAGTTCT-3′ 5′-GCGTACGAGAGGAAGGATGG-3′ 5′-CCAGCACCAGGAGTAGCAGC-3′ 5′-TCAGCCACGCAGCAAATCCT-3′ 5′-GTCATCTGGATGTCAGCACG-3′

370

62

Mouse

444

60

Mouse

RT-PCR In situ Cloning

370

62

Rabbit

RT-PCR

584 (a) 380 (b)

55

Mouse

RT-PCR

Abbreviations: AT, annealing temperature; S, sense primer; As, antisense primer. a Species from which primers were designed. b Experimental purpose using primers.

54

Mouse

204 300

58

Human

RT-PCR In situ RT-PCR

504

62

Mouse

RT-PCR

581

56

Mouse

RT-PCR

299

62

Rabbit

RT-PCR

450

62

Mouse

RT-PCR

300

62

Human

RT-PCR

493

55

Rabbit

RT-PCR

550

55

Rabbit

RT-PCR

449

58

Rabbit

RT-PCR

482

60

Mouse

RT-PCR

460

55

Rabbit

RT-PCR

473

55

Mouse

RT-PCR

396

55

Rabbit

RT-PCR

279

55

Rabbit

RT-PCR

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Y.H. Huh et al. / Gene 448 (2009) 7–15

2.9. Northern and Western blotting Northern and Western blotting were performed as described previously (Huh et al., 2007; Kim et al., 2008). Briefly, total RNA was fractionated on formaldehyde/agarose gels and transferred to S and S Nytran N nylon membranes. Following prehybridization and hybridization, the ECRG4 transcript was probed with partial cDNA generated by RT-PCR. Highly specific activity random primed probes were prepared from the PCR product using the T7 QuickPrime kit (Amersham Biosciences, Inc., Piscataway, NJ), as specified by the supplier. Filters were washed three times with 0.2× SSC/0.1% SDS and exposed to Kodak X-OMAT film with intensifying screens at −70 °C. For Western blot analysis, whole cell lysates were sizefractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Anti-myc antibody from Cell Signaling Technology (Beverly, MA) was used to detect ECRG4 protein. Blots were developed using a peroxidaseconjugated secondary antibody and the enhanced chemiluminescence system. 2.10. In situ hybridization Digoxigenin-conjugated riboprobes for collagen IIb and ECRG4 were synthesized using the digoxigenin RNA labeling mix (Roche Diagnostics Corp., Indianapolis, IN) (Ryu and Chun, 2006). Briefly, the cDNA fragments of mouse collagen II and ECRG4 were amplified by PCR using specific primers designed to introduce HindIII (sense) and EcoRI (antisense) restriction sites at the 5′ end (Table 1). Collagen IIb or ECRG4 cDNA was inserted into the pSPT-18 vector, linearized and transcribed with SP6 or T7 RNA polymerase to generate antisense and sense probes, respectively. For hybridization, forelimb buds of 14.5 dpc mouse embryos and spots of micromass culture were fixed in 4% paraformaldehyde for 18 h at 4 °C, dehydrated with graded ethanol, embedded in paraffin wax and cut into 4 μm sections. Dewaxed paraffin sections were treated with 0.2 N HCl for 10 min and permeabilized for 10 min at 37 °C with 20 μg/ml proteinase K. After acetylation for 10 min with 0.25% acetic anhydride, sections were incubated with hybridization buffer (40% formamide, 10% dextran sulfate, 1× Denhardt's solution, 4× SSC, 10 μM DTT, 1 mg/ml yeast tRNA and 1 mg/ml salmon sperm DNA) containing denatured sense or antisense digoxigenin-labeled riboprobes. Sections were treated for 30 min at 37 °C with 10 mg/ml RNase A, and processed using an antidigoxigenin detection assay kit (Roche Diagnostics Corp.). Hybridiza-

tion signals were visualized with a solution of 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-iodolyl-phosphate. 2.11. Transmission electron microscopy of mouse cartilage tissue For transmission electron microscopy, cartilage from 1-year-old mouse was dissected and fixed with 2% paraformaldehyde and 2% glutaraldehyde at 4 °C for 12 h. After postfixation with 1% osmium tetroxide for 2 h, samples were dehydrated in grade ethanol and embedded in LR white resin (London Resin Co. Ltd., London, England). Sections (0.07 μm thickness) were cut with diamond knives, stained with uranyl acetate and Raynold's lead citrate and observed under a Tecnai transmission electron microscope. 3. Results 3.1. ECRG4 is a secretory protein highly expressed in chondrocytes To identify genes that were expressed specifically or abundantly in cartilage, we analyzed the human normal cartilage library (Lib. 8940) deposited in the UniGene database at NCBI. Hs.43125 was isolated as a novel cartilage-specific gene from the library on the basis of the criteria ‘unknown’ and ‘cartilage-specific’ by serial analysis of gene expression (SAGE)/cDNA virtual Northern (Kim et al., 2008). The full sequence of mouse Hs.43125 is identical to that of mouse ECRG4 (Mm.50109). We initially examined the expression patterns of ECRG4 in various mouse tissues by in silico analysis to confirm cartilage-specific expression. RTPCR and qRT-PCR data revealed elevated expression of ECRG4 in cartilage, compared with that in other tissues (Fig. 1A). A single transcript of ECRG4 was detected at ∼1 kb in mouse rib chondrocytes (Fig. 1B). Sequence analysis disclosed that ECRG4 is composed of 4 exons and 3 introns, and its open reading frame encodes 148 amino acids with a signal peptide at the N-terminus. Ectopically expressed ECRG4 from chondrocyte lysates was detected at an apparent molecular weight of ∼17 kDa. A smaller cleaved form of ECRG4 (approximately ∼14 kDa) was additionally detected in culture medium (Fig. 1C), suggestive of post-translational cleavage after secretion. 3.2. ECRG4 is highly expressed in chondrocytes of developing limb buds To establish the in vivo expression pattern of ECRG4 during cartilage development, RT-PCR and qRT-PCR analyses were performed using the buds of fore and hindlimbs isolated from embryos at 11.5–15.5 dpc in

Fig. 1. ECRG4 is a secretory protein highly expressed in chondrocytes. ECRG4 expression was examined with RT-PCR and quantified using qRT-PCR in mouse tissues. The relative levels of ECRG4 mRNA were normalized against GAPDH as an internal control (A). Endogenous ECRG4 mRNA was detected as a single transcript in mouse rib chondrocytes by Northern blotting (B). Mouse rib chondrocytes were transfected with ectopic myc-tagged ECRG4 (1 μg) and incubated for 48 h in serum-free medium. ECRG4 was detected with an anti-myc antibody in both culture medium and cell lysates (C). The data in (A) to (C) represent typical results of more than four independent experiments.

Y.H. Huh et al. / Gene 448 (2009) 7–15

which cartilage development occurs (Fukumoto et al., 2003; Mariani and Martin, 2003). Peak expression of collagen IIb was detected in 14.5 and 15.5 dpc limb buds in which longitudinal growth of cartilage occurs gradually and individual digits are formed (Figs. 2A and B). Strong expression of ECRG4 was observed at 14.5 and 15.5 dpc, similar to the collagen IIb pattern (Figs. 2A and B). In situ hybridization indicated specific ECRG4 transcription in developing cartilage at the region where collagen IIb was detected (Fig. 2C). Further analysis of the distal phalange revealed ECRG4 expression in proliferating chondrocytes but not hypertrophic chondrocytes in the developing cartilage (Fig. 2D). 3.3. ECRG4 is expressed in proliferating but not hypertrophic chondrocytes Chondrocyte-specific ECRG4 expression was further examined during in vitro chondrogenesis and hypertrophic maturation of chondrocytes induced by micromass culture of mesenchymal cells. Mesenchymal cells were maintained as micromass culture to induce chondrogenesis up to day 6, and hypertrophic maturation was stimulated by switching chondrogenic medium to hypertrophic medium up to day 15. The chondrocyte-specific markers, collagen IIb and aggrecan, were expressed at day 3, reached peak levels at day 6 and decreased during hypertrophic maturation (Figs. 3A and B). Similar to the collagen II expression pattern, ECRG4 expression was very low in undifferentiated mesenchymal cells, dramatically increased during chondrogenesis and decreased during hypertrophic maturation (Figs. 3A and B). Collagen X and MMP-13, markers of hypertrophic chondrocytes, were detected on day 12 (Figs. 3A and B). In addition, in situ hybridization with sections of day 6 micromass culture spot revealed the presence of the ECRG4 transcript only in cartilage nodules composed of differentiated chondrocytes (Fig. 3C). 3.4. ECRG4 does not affect chondrogenesis or hypertrophic maturation of chondrocytes The chondrocyte-specific expression of ECRG4 during in vivo cartilage development and in vitro chondrogenesis suggests a possible

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role in chondrogenesis and/or hypertrophic maturation. Accordingly, we examined its possible function in chondrogenesis of mesenchymal cells and hypertrophic maturation of differentiated chondrocytes. The chondrogenic effects of ECRG4 were examined by ectopic expression with lentivirus-bearing mouse ECRG4 in the absence of serum. While insulin-like growth factor-I induced chondrogenesis as a positive control (Oh and Chun, 2003), ECRG4 did not exert chondrogenic effects (Fig. 4A). Additionally, ectopic expression of ECRG4 did not affect chondrogenesis of mesenchymal cells in the presence of serum (data not shown). To determine whether secreted ECRG4 plays a role, we prepared conditioned medium containing ECRG4 from primary culture rabbit articular chondrocytes transfected with the ECRG4 expression vector and treated with micromass culture. ECRG4conditioned medium did not modulate chondrogenesis in the absence (Fig. 4B) or presence (data not shown) of serum. Moreover, lentivirusmediated overexpression of ECRG4 and ECRG4-conditioned medium did not affect hypertrophic maturation of chondrocytes, as determined from Alizarin red staining or detection of the hypertrophic chondrocyte markers, collagen X and MMP-13 (Fig. 4C). 3.5. ECRG4 TG mice show no defects in cartilage and bone development To determine the in vivo function of ECRG4, we generated TG mice specifically overexpressing ECRG in chondrocytes using a collagen II promoter and enhancer (Fig. 5A). As shown in Fig. 5B, high levels of ECRG4 are expressed in the cartilage and chondrocytes of TG mice. However, cartilage development, determined from Alcian blue staining of 14.5 dpc embryos, and skeletal development, established based on Alizarin red/Alcian blue staining of 16.5 dpc embryos, were comparable between wild-type and TG mice (Fig. 5C), suggesting that cartilage-specific overexpression of ECRG4 does not affect cartilage and bone development. A possibility that ECRG4 regulates ECM organization was examined by transmission electron microscopy of cartilage tissue. As shown in Fig. 5D, ultrastructure of cartilage ECM and chondrocytes was not significantly different between wild-type and ECRG4 TG mice.

Fig. 2. Expression pattern of ECRG4 in developing limb buds. Limb buds were prepared from 11.5–15.5 dpc mouse embryos. Expression levels of ECRG4 and collagen (Coll)-IIb were determined with RT-PCR from forelimb and hindlimb (A) and quantified by qRT-PCR from forelimbs (B). Transcripts of ECRG4 and collagen IIb were detected in forelimb buds of 14.5 dpc mouse embryos by in situ hybridization (C). Sense riboprobes were used as a negative control. Phalange sections displaying positive signals with antisense probes of collagen IIb and ECRG4 were magnified using 20× and 40× objective lens of the microscope (D). No significant signal was observed with the sense probe (not shown). PC, proliferating chondrocytes; HC, hypertrophic chondrocytes. The data represent a typical result or mean values with standard deviation of five independent experiments.

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Fig. 3. Expression pattern of ECRG4 during chondrogenesis and hypertrophic maturation. Undifferentiated mesenchymal cells obtained from 11.5 dpc mouse embryos were maintained as micromass culture for 5 days to induce chondrogenesis. Hypertrophic maturation of chondrocytes was induced by switching to hypertrophic medium on day 6. Transcripts of ECRG4, chondrocyte markers [collagen (Coll) IIa and IIb and aggrecan] and hypertrophic markers (collagen X and MMP-13) were observed using RT-PCR (A) and quantified with qRT-PCR (B). Cells expressing ECRG4 and collagen IIb were probed by in situ hybridization from cross-sections of micromass culture spots at day 5 (C). Data represent a typical result (A and C) and mean values with standard deviation (B) of four independent experiments.

Fig. 4. ECRG4 does not affect chondrogenesis of mesenchymal cells or hypertrophic maturation of chondrocytes. Mesenchymal cells at micromass culture day 1 were left untreated (none) or infected with 400 or 800 MOI of mock virus or ECRG4 lentivirus. Cells were maintained in serum-free conditions for 72 h (A, upper panel) or 86 h (A, lower panel). Micromass culture spots were stained with Alcian blue to assess chondrogenesis at day 4 (A, upper panel). Expression of ECRG4 and chondrocyte marker genes (collagen IIb, aggrecan and chondromodulin) was determined by RT-PCR at day 5 of micromass culture (A, lower panel). Mesenchymal cells were maintained as micromass culture for 4 days in the absence (None) or presence of control conditioned medium (100 μl), ECRG4-conditioned medium (100 μl) or IGF-1 (100 ng/ml) under serum-free conditions. Cells were stained with Alcian blue to determine chondrogenesis at day 4 (B, upper panel), and expression of ECRG4 and chondrocyte marker genes was determined using RT-PCR (B, lower panel). Mesenchymal cells were left untreated (None), infected with mock (400 and 800 MOI) or ECRG4-containing lentivirus (400 and 800 MOI) on day 6 of micromass culture for 2 h and maintained in hypertrophic media up to 13 days. Alternatively, cells on day 6 were treated with 50 μl of control or ECRG4-conditioned medium for up to day 13 in hypertrophic medium with daily changes. Alizarin red staining was used to detect hypertrophic phenotype (C, upper panel), and RT-PCR analysis was employed to determine ECRG4 and hypertrophic markers (MMP-13 and collagen X) (C, lower panel). The data represent a typical result of more than five independent experiments.

Y.H. Huh et al. / Gene 448 (2009) 7–15

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Fig. 5. Analysis of cartilage-specific ECRG4 TG mice. Design of an ECRG4 vector construct with collagen II (CII) promoter and enhancer to generate cartilage-specific TG mouse (A). Expression levels of ECRG4 mRNA and collagen II were observed by RT-PCR in the indicated tissues of ECRG4 TG mice (B, right panel). Tail tips of mouse pups were analyzed for genotyping, and ECRG4 and collagen II transcripts were determined by RT-PCR analysis (B, left panel). Embryos of wild-type (WT) and ECRG4 TG mice at 14.5 dpc were stained with Alcian blue to detect cartilage tissue (C, lower panel). Embryos of WT and ECRG4 TG mice at 16.5 dpc were stained with Alizarin red to detect skeletal structure (C, upper panel). Ultrastructure of cartilage tissue of wild-type (WT) and ECRG4 TG mice was shown (D).

3.6. ECRG4 expression is decreased in dedifferentiating chondrocytes Differentiated chondrocytes lose their characteristics in response to IL-1β and during serial monolayer culture. As shown in Figs. 6A and B, exposure of chondrocytes to IL-1β or subculture led to loss of collagen II expression, indicating dedifferentiation. Similar to collagen II, ECRG4 expression was significantly decreased in IL-1β-treated cells (Fig. 6A) and subcultured chondrocytes up to P3 (Fig. 6B), clearly signifying a decrease in ECRG4 expression in dedifferentiating chondrocytes. To determine the function of ECRG4 in chondrocyte dedifferentiation, the protein was overexpressed using lentivirus in IL1β-treated chondrocytes. Ectopic expression of ECRG4 in IL-1β-

treated chondrocytes did not affect the expression of collagen II, cyclooxygenase-2, a typical inflammatory molecule and various MMP isoforms, which play major roles in cartilage destruction (Fig. 6C). In contrast to the effects on differentiation and dedifferentiation of chondrocytes, ectopic expression of ECRG4 by lentivirus infection caused slight but significant decrease of the proliferation of differentiated chondrocytes in primary culture (Fig. 6D). 3.7. ECRG4 expression is decreased in OA cartilage and chondrocytes We finally examined the expression patterns of ECRG4 in OA cartilage. Human OA cartilage was obtained from patients undergoing

Fig. 6. Decrease in ECRG4 expression in dedifferentiating chondrocytes. Rabbit articular chondrocytes were exposed to 5 ng/ml IL-1β for the indicated periods. Expression of ECRG4 and collagen II was detected by RT-PCR (A). Cells were subcultured serially up to P3. Expression of ECRG4 and collagen II was examined by RT-PCR (B). Rabbit articular chondrocytes were infected with mock or ECRG4 lentivirus (400 and 800 MOI) at day 2.5 for 2 h in the absence or presence of 5 ng/ml IL-1β for 48 h. Expression patterns of ECRG4, collagen II, cyclooxygenase (COX-2) and MMPs were determined using RT-PCR (C). Proliferation assay was performed in rabbit articular chondrocytes infected with mock (800 MOI) or ECRG4 lentivirus (200, 400 and 800 MOI) (n = 4) (D). Data represent a typical result of more than four independent experiments.

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arthroplasty for OA knee joints, and experimental OA in mouse knee induced by intra-articular injection of collagenase. Similar to collagen II, ECRG4 expression was significantly decreased in human OA cartilage, compared with the relatively undamaged part of cartilage (Fig. 7A). Collagenase injection caused cartilage destruction, as determined from Safranin-O staining of the cartilage section (Fig. 7B). Mankin scores of PBS and collagenase injected knee joints were 0.4 ± 0.9 and 6.6 ± 1.3, respectively. RT-PCR and qRT-PCR analyses revealed that cartilage destruction was accompanied by a significant decrease in expression of collagen II as well as ECRG4. Clearly, ECRG4 expression is reduced during chondrocyte dedifferentiation and cartilage destruction in vivo. 4. Discussion In this investigation, we sought to identify a novel chondrocytespecific gene involved in cartilage development and destruction using an in silico approach based on the EST database. Our results show that the expression pattern of ECRG4 is correlated with differentiation status, indicating that the protein is a novel specific marker of differentiated chondrocytes, similar to collagen II. Sequence analysis revealed that ECRG4 is a secretory protein. Moreover, ECRG4 was detected in both cellular and culture media, confirming this finding. Indeed, mutation of ECRG4 by deleting N-terminal signal peptide blocked its secretion (data not shown). Secretion of ECRG4 is accompanied by cleavage into a lower molecular weight protein. While we did not focus on the related mechanisms, it is possible that furin-type protease is involved in cleavage of ECRG4, since the consensus recognition sequence is ‘RxK’ or ‘RRxR’ (‘x’ represents a basic amino acid) and ECRG4 contains a RAKR sequence that is

conserved among known ECRG homologs, including human, mouse, bovine and Xenopus. The biological function of the post-translationally modified (cleaved) protein remains to be confirmed. Only few reports have mentioned the expression of ECRG4 in chondrocytes and/or cartilage, but none of those works delineated the function(s) of ECRG4. In view of the finding that ECRG4 is specifically expressed in chondrocytes and cartilage in a manner dependent on differentiation status, we initially hypothesized that ECRG4 functions in aspects of cartilage development, such as chondrogenesis, hypertrophic maturation and/or cartilage destruction. However, under our experimental conditions, ectopic expression of ECRG4 and conditioned medium from chondrocytes overexpressing the protein did not affect chondrogenesis or hypertrophic maturation in the primary cell culture system. Similarly, TG mice overexpressing ECRG4 in a cartilage-specific manner did not display phenotype changes in cartilage and bone development as well as ECM organization in cartilage. The reason for no phenotype changes in TG mice may due to the fact that endogenous expression of ECRG4 is high in differentiated chondrocytes. If endogenous ECRG4 is an excess in chondrocytes, overexpression of ECRG4 in chondrocytes of TG mice may not induce marked phenotypes in cartilage. It is possible that constitutive expression of ECRG4 in TG mice may show clear phenotypes. However, our primary purpose for the production of ECRG4 TG mice was to examine the phenotypes when ECRG4 is specifically overexpressed in chondrocytes. Additionally, we tried to establish ECRG4 null mice. However, we failed to target ES cell line from more than 2000 ES cell clones. Similarly, failure of gene targeting of ECRG4 was also reported from European Conditional Mouse Mutagenesis Program to get the ECRG4 null mice. We also found in this study that ECRG4 expression is decreased in dedifferentiating

Fig. 7. ECRG4 expression is decreased in osteoarthritic cartilage. A typical OA cartilage tissue showing clinical severity of arthritis is presented (A, upper panel). OA and normal (N) cartilage tissue from human patients were analyzed for expression of ECRG4 and collagen II using RT-PCR (A, middle panel) and qRT-PCR (A, lower panel). Experimental OA in mouse was induced by collagenase injection. PBS-injected mice were used as the control. Safranin-O staining was performed to determine induction of OA (B, upper panel). ECRG4 and collagen II mRNA levels were analyzed with RT-PCR (B, middle panel) and qRT-PCR (B, lower panel). The data represent a typical result of more than four independent experiments.

Y.H. Huh et al. / Gene 448 (2009) 7–15

chondrocytes and osteoarthritic cartilage although the underlying reason for decreased expression of ECRG4 remains to be clarified, since ectopic expression of ECRG4 in IL-1β-treated cells did not lead to recovery of collagen II expression or inhibition of IL-1β-induced expression of MMPs. ECRG4 was originally identified as a gene downregulated in esophageal cancer (Su et al., 1998). A recent study showed that expression of ECRG4 is modulated by hypermethylation of its own promoter (Vanaja et al., 2009) and overexpression of ECRG4 inhibits tumor cell proliferation (Li et al., 2009). Consistent with its function as tumor suppressor gene, ectopic expression of ECRG4 inhibited proliferation of primary culture chondrocytes. Because chondrocytes in vivo are not rapidly proliferating cells, the functional significance of this inhibition remains to be elucidated. The function of ECRG4 is unclear at present. Since ECRG4 does not modulate chondrogenesis and hypertrophy, one of its possible functions is cartilage ECM reorganization. Here, we focus on anabolic regulation, such as expression of chondrogenic or hypertrophic marker genes. It is possible that secreted ECRG4 plays a role in catabolic regulation by ECM reorganization or affects synovial fluids in joint cartilage or adjacent tissues, such as bone and synovial tissue. However, ultrastructure of cartilage ECM and chondrocytes was not significantly different between wild-type and ECRG4 TG mice as determined by transmission electron microscopy of cartilage tissue. To date, only one report on ECRG4 expression in cartilage is available in the literature (Steck et al., 2002). It was found that ECRG4 is expressed in other tissues, such as heart and kidney, but the expression levels were not compared with those in cartilage. Our experiments show that ECRG4 is most highly expressed in cartilage. Steck et al. (2002) reported comparable expression levels of ECRG4 in normal and human OA cartilage tissue. However, we showed significantly decreased expression of ECRG4 in human OA cartilage, which was confirmed in more than 10 human samples. This discrepancy in the ECRG4 expression patterns in human OA cartilage may be attributed to the source of normal cartilage. Steck and coworkers (2002) obtained normal cartilage from crime victims and patients undergoing amputation for tumor resection, whereas we directly compared ECRG4 levels in chondrocytes from undamaged normal part and damaged OA-affected part of human cartilage originating from the same individuals. This comparison is valuable because two parts show dramatic differences in collagen II expression as shown in Fig. 7A. Accumulation of collagen II and sulfated proteoglycans was also significantly reduced in OA-affected part of human cartilage compared with undamaged normal part (data not shown). In addition to the decrease of ECRG4 levels in human OA, ECRG4 expression was also decreased in an experimental mouse OA model, confirming its downregulation in OA cartilage and chondrocytes. Our results clearly indicate that ECRG4 is an effective marker of differentiated articular chondrocytes and cartilage destruction. Indeed, there is a need for good biomarkers to diagnose OA. Because decreased expression and fragmentation of collagen II in OA can not be easily detected in vitro, ECRG4 can be a good biomarker for in vitro diagnosis of OA if secreted intact or cleaved form of ECRG4 can be measured in serum or urine. Although it remains to be determined whether ECRG4 is secreted into serum or urine and whether OA causes decrease of this secretion of ECRG4, we tried to detect secreted ECRG4 in cultured media using anti-ECRG4 antibody generated against ECRG4 peptide. However, the sensitivity of our antibody, compared to that of anti-myc tagging antibody, was very low to detect ECRG4 (data not shown). Although the specific function of this protein in chondrocytes and cartilage requires further investigation, we

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propose that ECRG4 is a marker of differentiated articular chondrocyte and cartilage destruction, with a capacity to inhibit proliferation of differentiated chondrocytes. Acknowledgments This work was supported by grants from Cell Dynamics Research Center, Korea Science and Engineering Foundation (KOSEF R11-2007007-01001-0) and Korea Research Foundation (KRF-2006-312C00611). References Altman, R., et al., 1986. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum. 29, 1039–1049. Blom, A.B., et al., 2007. Crucial role of macrophages in matrix metalloproteinasemediated cartilage destruction during experimental osteoarthritis: involvement of matrix metalloproteinase 3. Arthritis Rheum. 56, 147–157. DeLise, A.M., Fischer, L., Tuan, R.S., 2000. Cellular interactions and signaling in cartilage development. Osteoarthritis Cartilage 8, 309–334. Fukumoto, T., et al., 2003. Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 on periosteal mesenchymal cells during chondrogenesis in vitro. Osteoarthritis Cartilage 11, 55–64. Goldring, M.B., Tsuchimochi, K., Ijiri, K., 2006. The control of chondrogenesis. J. Cell. Biochem. 97, 33–44. Gouttenoire, J., Valcourt, U., Ronzière, M.C., Aubert-Foucher, E., Mallein-Gerin, F., Herbage, D., 2004. Modulation of collagen synthesis in normal and osteoarthritic cartilage. Biorheology 41, 535–542. Hong, S., et al., 2005. Identification and integrative analysis of 28 novel genes specifically expressed and developmentally regulated in murine spermatogenic cells. J. Biol. Chem. 280, 7685–7693. Huh, Y.H., Ryu, J.H., Chun, J.S., 2007. Regulation of type II collagen expression by histone deacetylase in articular chondrocytes. J. Biol. Chem. 282, 17123–17131. Kim, J.S., Ryoo, Z.Y., Chun, J.S., 2008. Cytokine-like 1 (CYTL1) regulates the chondrogenesis of mesenchymal cells. J. Biol. Chem. 282, 29359–29367. Li, LW, et al., 2009. Expression of esophageal cancer related gene 4 (ECRG4), a novel tumor suppressor gene, in esophageal cancer and its inhibitory effect on the tumor growth in vitro. Int. J. cancer. 125, 1505–1513. Liu, X., Rapp, N., Deans, R., Cheng, L., 2000. Molecular cloning and chromosomal mapping of a candidate cytokine gene selectively expressed in human CD34+ cells. Genomics 65, 283–292. Malemud, C.J., 1999. Fundamental pathways in osteoarthritis: overview. Frontiers Biosci. 4, d659–d661. Mankin, H.J., Dorfman, H., Lippiello, L., Zarins, A., 1971. Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J. Bone Jt. Surg. Am. 53, 523–537. Mariani, F.V., Martin, G.R., 2003. Deciphering skeletal patterning: clues from the limb. Nature 423, 319–325. Mello, M.A., Tuan, R.S., 1999. High density micromass cultures of embryonic limb bud mesenchymal cells: an in vitro model of endochondral skeletal development. In Vitro Cell Dev. Biol. Anim. 35, 262–269. Oh, C.D., Chun, J.S., 2003. Signaling mechanisms leading to the regulation of differentiation and apoptosis of articular chondrocytes by insulin-like growth factor-1. J. Biol. Chem. 278, 36563–36571. Peale Jr, F.V., Gerritsen, M.E., 2001. Gene profiling techniques and their application in angiogenesis and vascular development. J. Pathol. 195, 7–19. Ryu, J.H., Chun, J.S., 2006. Opposing roles of WNT-5A and WNT-11 in interleukin-1beta regulation of type II collagen expression in articular chondrocytes. J. Biol. Chem. 281, 22039–22047. Steck, E., Breit, S., Breusch, S.J., Axtm, M., Richter, W., 2002. Enhanced expression of the human chitinase 3-like 2 gene (YKL-39) but not chitinase 3-like 1 gene (YKL-40) in osteoarthritic cartilage. Biochem. Biophys. Res. Commun. 299, 109–115. Su, T., Liu, H., Lu, S., 1998. Cloning and identification of cDNA fragments related to human esophageal cancer. Zhonghua Zhong Liu Za Zhi 20, 254–257. Tickle, C., 2002. Molecular basis of vertebrate limb patterning. Am. J. Med. Genet. 112, 250–255. Ueta, C., et al., 2001. Skeletal malformations caused by overexpression of Cbfa1 or its dominant negative form in chondrocytes. J. Cell Biol. 153, 87–100. Vanaja, D.K., et al., 2009. Hypermethylation of genes for diagnosis and risk stratification of prostate cancer. Cancer Invest 27, 549–560. Yoon, Y.M., et al., 2002. Maintenance of differentiated phenotype of articular chondrocytes by protein kinase C and extracellular signal-regulated protein kinase. J. Biol. Chem. 277, 8412–8420.