Title: Mechanisms for Asporin Function and Regulation in Articular Cartilage
Abstract: Osteoarthritis (OA), the most prevalent form of skeletal disease, represents a leading cause of disability following middle age. OA is characterized by the loss of articular cartilage; however, the details of its etiology and pathogenesis remain unclear. Recently, we demonstrated a genetic association between the cartilage extracellular matrix protein asporin and OA (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). Furthermore, we showed that asporin binds to transforming growth factor-β (TGF-β), a key cytokine in OA pathogenesis, and inhibits TGF-β-induced chondrogenesis. To date, functional data for asporin have come primarily from mouse cell culture models of developing cartilage rather than from human articular cartilage cells, in which OA occurs. Here, we describe mechanisms for asporin function and regulation in human articular cartilage. Asporin blocks chondrogenesis and inhibits TGF-β1-induced expression of matrix genes and the resulting chondrocyte phenotypes. Small interfering RNA-mediated knockdown of asporin increases the expression of cartilage marker genes and TGF-β1; in turn, TGF-β1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-β1 form a regulatory feedback loop. Asporin inhibits TGF-β/Smad signaling upstream of TGF-β type I receptor activation in vivo by co-localizing with TGF-β1 on the cell surface and blocking its interaction with the TGF-β type II receptor. Our results provide a basis for elucidating the role of asporin in the molecular pathogenesis of OA. Osteoarthritis (OA), the most prevalent form of skeletal disease, represents a leading cause of disability following middle age. OA is characterized by the loss of articular cartilage; however, the details of its etiology and pathogenesis remain unclear. Recently, we demonstrated a genetic association between the cartilage extracellular matrix protein asporin and OA (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). Furthermore, we showed that asporin binds to transforming growth factor-β (TGF-β), a key cytokine in OA pathogenesis, and inhibits TGF-β-induced chondrogenesis. To date, functional data for asporin have come primarily from mouse cell culture models of developing cartilage rather than from human articular cartilage cells, in which OA occurs. Here, we describe mechanisms for asporin function and regulation in human articular cartilage. Asporin blocks chondrogenesis and inhibits TGF-β1-induced expression of matrix genes and the resulting chondrocyte phenotypes. Small interfering RNA-mediated knockdown of asporin increases the expression of cartilage marker genes and TGF-β1; in turn, TGF-β1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-β1 form a regulatory feedback loop. Asporin inhibits TGF-β/Smad signaling upstream of TGF-β type I receptor activation in vivo by co-localizing with TGF-β1 on the cell surface and blocking its interaction with the TGF-β type II receptor. Our results provide a basis for elucidating the role of asporin in the molecular pathogenesis of OA. Osteoarthritis (OA 4The abbreviations used are: OA, osteoarthritis; ECM, extracellular matrix; SLRPs, small leucine-rich proteoglycans; TGF-β1, transforming growth factor-β1; siRNA, small interfering RNA; TβR, transforming growth factor-β receptor; NHAC, normal human articular chondrocyte; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.4The abbreviations used are: OA, osteoarthritis; ECM, extracellular matrix; SLRPs, small leucine-rich proteoglycans; TGF-β1, transforming growth factor-β1; siRNA, small interfering RNA; TβR, transforming growth factor-β receptor; NHAC, normal human articular chondrocyte; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum.; OMIM accession number 165720) is the most prevalent disease affecting bones and joints, and it represents a leading cause of disability in aging populations. OA is characterized by the loss of articular cartilage, which is composed of abundant extracellular matrix (ECM) proteins, including proteoglycan and type II collagen (1Muir H. BioEssays. 1995; 17: 1039-1048Crossref PubMed Scopus (343) Google Scholar). Genetic factors have been implicated in the onset and development of OA (2Stecher R.M. Am. J. Med. Sci. 1941; 201: 801-809Crossref Google Scholar, 3Kellgren J.H. Kim J.S. Bier F. Ann. Rheum. Dis. 1963; 22: 237-255Crossref PubMed Scopus (234) Google Scholar, 4Spector T.D. Kim F. Baker J. Loughlin J. Hart D. Br. Med. J. 1996; 312: 940-943Crossref PubMed Scopus (540) Google Scholar), and several susceptibility genes have been reported (5Spector T.D. Kim A.J. Osteoarthritis Cartilage. 2004; 12: S39-S44Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 6Peaches C.A. Kim A.J. Loughlin J. Trends Mol. Med. 2005; 11: 186-191Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar); however, the etiology and pathogenesis of OA remain unclear. Recently, we demonstrated the genetic association of a functional polymorphism in the gene encoding asporin (ASPN) with knee and hip OA in the Japanese population (7Kizawa H. Kim I. Iida A. Sudo A. Miyamoto Y. Fukuda A. Mabuchi A. Kotani A. Kawakami A. Yamamoto S. Uchida A. Nakamura K. Notoya K. Nakamura Y. Ikegawa S. Nat. Genet. 2005; 37: 138-144Crossref PubMed Scopus (369) Google Scholar). This association is replicated in knee OA in the Han Chinese population (8Jiang Q. Kim D. Yi L. Ikegawa S. Wang Y. Qiao D. Liu C. Dai J. J. Hum. Genet. 2006; 51: 1068-1072Crossref PubMed Scopus (80) Google Scholar). Asporin is an ECM protein that is abundantly expressed in OA articular cartilage, and its expression increases with progressive cartilage degeneration (7Kizawa H. Kim I. Iida A. Sudo A. Miyamoto Y. Fukuda A. Mabuchi A. Kotani A. Kawakami A. Yamamoto S. Uchida A. Nakamura K. Notoya K. Nakamura Y. Ikegawa S. Nat. Genet. 2005; 37: 138-144Crossref PubMed Scopus (369) Google Scholar, 9Lorenzo P. Kim A. Onnerfjord P. Bayliss M.T. Neame P.J. Heinegard D. J. Biol. Chem. 2001; 276: 12201-12211Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Asporin belongs to a family of small leucine-rich proteoglycans (SLRPs), which compose a major non-collagen component of the ECM (10Iozzo R.V. Kim A.D. FASEB J. 1996; 10: 598-614Crossref PubMed Scopus (550) Google Scholar). Although SLRPs play known roles in biological processes, such as skeletal growth, craniofacial structure, and collagen fibrillogenesis (11Iozzo R.V. J. Biol. Chem. 1999; 274: 18843-18846Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar), the exact role of asporin is not known. Previously, we showed that asporin binds directly to transforming growth factor-β1 (TGF-β1) in vitro (7Kizawa H. Kim I. Iida A. Sudo A. Miyamoto Y. Fukuda A. Mabuchi A. Kotani A. Kawakami A. Yamamoto S. Uchida A. Nakamura K. Notoya K. Nakamura Y. Ikegawa S. Nat. Genet. 2005; 37: 138-144Crossref PubMed Scopus (369) Google Scholar). Abundant in cartilage ECM, TGF-β1 regulates proliferation, differentiation, and matrix production of chondrocytes and their progenitor cells (12Denker A.E. Kim S.B. Tuan R.S. Differentiation. 1995; 59: 25-34Crossref PubMed Scopus (178) Google Scholar, 13Johnstone B. Kim T.M. Caplan A.I. Goldberg V.M. Yoo J.U. Exp. Cell Res. 1998; 238: 265-272Crossref PubMed Scopus (2026) Google Scholar). We have also demonstrated that asporin inhibits TGF-β1-induced expression of cartilage matrix genes in ATDC5 cells (7Kizawa H. Kim I. Iida A. Sudo A. Miyamoto Y. Fukuda A. Mabuchi A. Kotani A. Kawakami A. Yamamoto S. Uchida A. Nakamura K. Notoya K. Nakamura Y. Ikegawa S. Nat. Genet. 2005; 37: 138-144Crossref PubMed Scopus (369) Google Scholar), an in vitro mouse model for chondrogenesis (14Shukunami C. Kim C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (343) Google Scholar, 15Shukunami C. Kim K. Atsumi T. Ohta Y. Suzuki F. Hiraki Y. J. Bone Miner. Res. 1997; 12: 1174-1188Crossref PubMed Scopus (251) Google Scholar). These results strongly suggest that asporin acts as a negative regulator of TGF-β in cartilage, playing a critical role in the etiology and pathogenesis of OA. However, functional data for asporin have been obtained primarily from mouse cartilage cells rather than from human articular cartilage cells, in which OA occurs. In addition, the mechanism by which asporin inhibits TGF-β function, as well as the regulation of asporin in articular cartilage, is still undetermined. In this study, we investigated the function of asporin in articular cartilage and its mechanism for TGF-β inhibition during chondrogenesis. Asporin blocks chondrogenesis at both the early and late stages, inhibiting TGF-β1-induced gene expression and the resulting chondrocyte phenotypes. Knockdown of asporin by small interfering RNA (siRNA) increases the expression of cartilage marker genes, confirming its physiological role as a negative regulator of chondrogenesis. Asporin inhibits TGF-β/Smad signaling by co-localizing with TGF-β1 on the cell surface and inhibiting its binding to the TGF-β type II receptor (TβRII). TGF-β1 stimulates asporin expression, suggesting that asporin and TGF-β1 form a regulatory feedback loop. Human Articular Cartilage Samples—Articular cartilage samples were obtained during surgery with written informed consent: knee OA cartilage was obtained from total knee arthroplasties, and normal cartilage was obtained from knee and hip joints. None of the control subjects had a clinical history of joint diseases or showed radiographic signs of OA. For RNA extraction, samples were frozen in liquid nitrogen immediately after resection and stored at -80 °C. For immunohistochemistry, the samples were fixed in 10% buffered formaldehyde and embedded in paraffin. Cell Culture—We purchased normal human articular chondrocyte (NHAC) cells from Cambrex. To obtain a monolayer culture, NHAC cells were maintained in a standard chondrocyte growth medium (Cambrex) at 37 °C under 5% CO2.To redifferentiate chondrocytes and to maintain the differentiated phenotype, we carried out three-dimensional culture using alginate beads (Cambrex) according to the manufacturer's instructions. Unless noted otherwise, we used primary or secondary cells after redifferentiation in alginate bead culture for experiments. To examine the effects of asporin on TGF-β1-induced expression of various genes, NHAC cells were plated and cultured in a 12-well plate at a density of 5 × 104 cells/well in Dulbecco's modified Eagle's medium (DMEM)/nutrient mixture F-12 containing 10% fetal bovine serum (FBS) until they reached confluence. At this point, the medium was replaced with DMEM/nutrient mixture F-12 containing 0.2% FBS. After 12 h, cells were treated with recombinant mouse asporin (7Kizawa H. Kim I. Iida A. Sudo A. Miyamoto Y. Fukuda A. Mabuchi A. Kotani A. Kawakami A. Yamamoto S. Uchida A. Nakamura K. Notoya K. Nakamura Y. Ikegawa S. Nat. Genet. 2005; 37: 138-144Crossref PubMed Scopus (369) Google Scholar). After 12 h, cells were treated with TGF-β1 (40 ng/ml) for 12 h. To examine induction of asporin mRNA by TGF-β1, NHAC cells were cultured in a 12-well plate at a density of 1 × 105 cells/well in DMEM/nutrient mixture F-12 containing 10% FBS until they reached confluence. The medium was then replaced with DMEM/nutrient mixture F-12 containing 0.2% FBS. After 24 h, cells were treated with TGF-β1 (10 ng/ml) for 12 h. Chondrogenic ATDC5 cells were obtained from the RIKEN Cell Bank. ATDC5 cells were cultured in DMEM/F-12 containing 5% FBS at 37 °C under 5% CO2 as described (14Shukunami C. Kim C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (343) Google Scholar, 15Shukunami C. Kim K. Atsumi T. Ohta Y. Suzuki F. Hiraki Y. J. Bone Miner. Res. 1997; 12: 1174-1188Crossref PubMed Scopus (251) Google Scholar). For induction of chondrogenesis, confluent cells were cultured in medium containing 10 μg/ml insulin, 10 μg/ml transferrin, and 3 × 10-8 m selenium (ITS; Sigma), and the medium was changed every 2 or 3 days. Real-time Quantitative PCR Assays—We extracted total RNA from cells using Isogen (Wako Pure Chemical Industries, Ltd.) and purified them using the SV total RNA isolation system (Promega Corp.). We synthesized random-primed cDNA using MultiScribe reverse transcriptase (Applied Biosystems). Real-time PCR was carried out on a ABI PRISM 7700 sequence detection system (Applied Biosystems) using a QuantiTect SYBR Green PCR kit (Qiagen Inc.) in accordance with the manufacturers' instructions. High Density Oligonucleotide Microarray—We carried out high density oligonucleotide microarray analysis as described previously (7Kizawa H. Kim I. Iida A. Sudo A. Miyamoto Y. Fukuda A. Mabuchi A. Kotani A. Kawakami A. Yamamoto S. Uchida A. Nakamura K. Notoya K. Nakamura Y. Ikegawa S. Nat. Genet. 2005; 37: 138-144Crossref PubMed Scopus (369) Google Scholar). Preparation of Recombinant Mouse Asporin—Recombinant mouse asporin expressed in Escherichia coli was purified as described previously (7Kizawa H. Kim I. Iida A. Sudo A. Miyamoto Y. Fukuda A. Mabuchi A. Kotani A. Kawakami A. Yamamoto S. Uchida A. Nakamura K. Notoya K. Nakamura Y. Ikegawa S. Nat. Genet. 2005; 37: 138-144Crossref PubMed Scopus (369) Google Scholar). The coding sequence for the mature mouse asporin protein was cloned into the pTriEx4 vector (Novagen). Recombinant mouse asporin was also expressed in COS-7 cells and purified from cell lysates using S-protein-agarose (Novagen) according to the manufacturer's instructions. Pulldown Assay—Pulldown assays to demonstrate direct interactions between asporin and TGF-β proteins were performed as described previously (7Kizawa H. Kim I. Iida A. Sudo A. Miyamoto Y. Fukuda A. Mabuchi A. Kotani A. Kawakami A. Yamamoto S. Uchida A. Nakamura K. Notoya K. Nakamura Y. Ikegawa S. Nat. Genet. 2005; 37: 138-144Crossref PubMed Scopus (369) Google Scholar). Briefly, purified recombinant mouse asporin (5 μg) was incubated with 0.1 μg of recombinant TGF-β proteins (R&D Systems) for 1 h at 4 °C in 0.3 ml of binding buffer (20 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 1% Triton X-100). We added 12.5 μl of S-protein-agarose to the reactions and incubated them for 30 min at 25 °C. The precipitates were washed three times with binding buffer and subjected to SDS-PAGE. The proteins were blotted and visualized with the corresponding antibody. Each blot was then treated with horseradish peroxidase-conjugated S-protein (Novagen) to confirm the precipitation of recombinant mouse asporin. Safranin O Staining—After redifferentiation in alginate bead culture, cells were plated on a Lab-Tek II chamber slide (Nunc) and cultured in DMEM/nutrient mixture F-12 containing 5% FBS and ITS in the absence or presence of TGF-β1 (40 ng/ml) with or without asporin (20 μg/ml) for 7 days. Cells were washed twice with phosphate-buffered saline, fixed with methanol, and stained with 0.1% safranin O for 5 min at room temperature. Sections of articular cartilage from individuals with OA were stained with safranin O/fast green. Safranin O-positive and -negative areas were defined as normal and degenerated areas, respectively. Alcian Blue Staining and Measurement of Staining Intensity—At day 21 of culture, cells were washed with phosphate-buffered saline, fixed with 4% paraformaldehyde for 10 min, and stained with Alcian blue solution (pH 1.0) (Sigma) overnight at 25 °C. The stained culture plates were rinsed with distilled water and extracted with 6 m guanidine HCl for 2 h at 25 °C. The optical density of the extracted dye was measured at 650 nm with a spectrophotometer. siRNA—We selected three siRNAs (Si1, Si9, and Si16) directed against human asporin using the siRNA design support system available on the Takara Bio web site. These siRNAs were synthesized and obtained from Takara Bio Inc. Among them, Si9 (target sequences 5′-UCCCUUCAGGAUUACCAGATT-3′ and 5′-UCUGGUAAUCCUGAAGGGATT-3′) showed the strongest knockdown effect in NHAC cells. As a control, we used a scrambled siRNA against Si1. We transfected siRNAs into NHAC cells using TransIT-TKO (Mirus Bio Corp.) and cultured the cells for 24 h in DMEM/nutrient mixture F-12 containing 0.2% FBS and 10 ng/ml TGF-β1 in the presence or absence of TGF-β-neutralizing antibodies (MAB240, R&D Systems). Western Blotting—Cells were plated at 5 × 104 cells/well in 12-well plastic tissue culture plates in medium containing ITS. After 24 h, the medium was changed to DMEM/F-12 containing 0.2% FBS and ITS. 16 h later, cells were treated with 10 ng/ml TGF-β1 for 5 min at 37 °C. Recombinant mouse asporin was added 1 h prior to TGF-β1 treatment. After incubation, the cells were lysed using the M-PER protein extraction kit (Pierce) containing protease inhibitor mixture (Roche Applied Science). Proteins in the cell lysate were separated by electrophoresis on SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Amersham Biosciences). The membranes were incubated in 5% nonfat dry milk in 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, and 0.05% Tween 20 to block nonspecific binding. Primary antibodies against phosphorylated Smad2 (Cell Signaling Technology, Inc.) and Smad2 (Invitrogen) were used at 1:1000 dilution. Membranes were then incubated in horseradish peroxidase-conjugated anti-rabbit IgG (Cell Signaling Technology, Inc.) at 1:3000 dilution. Construction of Plasmids—The SBE4-luciferase plasmid (where SBE is Smad-binding element) was constructed as described previously (16Zawel L. Kim J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar). We cloned a cDNA encoding human TβRI (ALK5 (activin receptor-like kinase-5)) into the pcDNA3.1 vector (Invitrogen), and a constitutively active TβRI mutant (T204D) was engineered by PCR using mutagenic primers as described previously (17Wieser R. Kim J.L. Massagué J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (597) Google Scholar). Each construct was verified by DNA sequencing (Model 3700, Applied Biosystems). Luciferase Assay—Cells were plated at a density of 2.5 × 104 cells/well in 24-well plastic tissue culture plates and cultured in DMEM/F-12 containing 5% FBS. Cells were transiently transfected with the SBE4-luciferase reporter plasmid and the pRL-TK vector (Promega Corp.) as an internal control using FuGENE 6 (Roche Applied Science) as a transfection reagent. After 24 h, cells were transferred to medium containing 0.2% FBS with or without recombinant mouse asporin and, after 1 h, treated with TGF-β1 for 24 h. To demonstrate the effect of asporin on ligand (extracellular TGF-β)-independent Smad activation, cells were transiently transfected with SBE4-luciferase and pcDNA3.1 containing the constitutively active TβRI (T204D) mutant construct (17Wieser R. Kim J.L. Massagué J. EMBO J. 1995; 14: 2199-2208Crossref PubMed Scopus (597) Google Scholar). After 24 h, cells were transferred to medium containing 0.2% FBS with or without recombinant mouse asporin and cultured for 24 h. Luciferase activity was measured using the PG-DUAL-SP reporter assay system (Toyo Ink Co.). Affinity Cross-linking—Recombinant human TGF-β1 was iodinated using the chloramine-T method as described (18ten Dijke P. Kim H. Ichijo H. Franzen P. Laiho M. Miyazono K. Heldin C.H. Science. 1994; 264: 101-104Crossref PubMed Scopus (511) Google Scholar). ATDC5 cells were incubated for 3 h at 4 °C with 10 ng/ml 125I-labeled TGF-β1 in binding buffer (phosphate-buffered saline containing 1 mm CaCl2, 0.5 mm MgCl2, and 1 mg/ml bovine serum albumin). After incubation, the cells were washed with binding buffer without bovine serum albumin, and cross-linking was performed in buffer containing 0.27 mm disuccinimidyl suberate (Pierce) for 15 min at 4 °C. The cells were then washed once with 10 mm Tris-HCl (pH 7.4) containing 1 mm EDTA, 10% glycerol, and protease inhibitor mixture. Cells were scraped, centrifuged, and resuspended in solubilization buffer (1% Triton X-100, 1% deoxycholate, 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, and protease inhibitor mixture), followed by incubation on ice for 30 min. Cell lysates were mixed with an equal amount of SDS sample buffer (100 mm Tris-HCl (pH 6.8), 0.01% bromphenol blue, 36% glycerol, and 4% SDS) containing 10 mm dithiothreitol and analyzed on 7% SDS-polyacrylamide gels. The gels were dried and subjected to analysis using a BAS-2500 imaging analyzer (Fujifilm Corp.). Immunohistochemistry—Immunohistochemistry was carried out as described previously (19Kobayashi T. Kim K. Naito T. Unno S. Nakamura A. Martel-Pelletier J. Pelletier J.P. Arthritis Rheum. 2005; 52: 479-487Crossref PubMed Scopus (88) Google Scholar). Sections were stained with an anti-asporin antibody (2229-B01; 1 μg/ml) or an anti-TGF-β1 antibody (catalog number sc-146, Santa Cruz Biotechnology, Inc.; 1 μg/ml) at 4 °C for 18 h. Immunofluorescence Microscopy—Cells were fixed for 15 min in 4% paraformaldehyde at 25 °C and blocked with 10% normal goat serum (Invitrogen) in phosphate-buffered saline for 1 h at 25 °C. The fixed cells were incubated with the antiasporin antibody (2210-B02; 3 μg/ml) and subsequently incubated with Alexa Fluor 546-conjugated goat-anti rabbit IgG (H + L; Invitrogen) for 30 min at 25 °C. After staining, the cells were mounted in VECTASHIELD mounting medium (Vector Laboratories) with 4′,6-diamidino-2-phenylindole and examined with an Olympus IX70 FluoView confocal microscope. For double staining with TGF-β1, a human TGF-β1 biotinylated Fluorokine kit (R&D Systems) was used according to the manufacturer's protocol with a slight modification. Prior to fixation, cells were incubated with biotinylated TGF-β1 for 1 h at 4 °C. Biotinylated TGF-β1 bound to cells was visualized with fluorescein isothiocyanate-labeled avidin. TGF-β1 Is Predominant among TGF-β Isoforms in Human Articular Cartilage—Three TGF-β isoforms (β1, β2, and β3) with similar functions exist in mammals, and all are expressed in ATDC5 cells (20Kawai J. Kim H. Shigeno C. Ito H. Konishi J. Nakamura T. Eur. J. Cell Biol. 1999; 78: 707-714Crossref PubMed Scopus (29) Google Scholar). Upon finding that asporin bound to TGF-β2 and TGF-β3 as well as to TGF-β1 (Fig. 1A), we evaluated the significance of each isoform in OA. Using microarray and real-time PCR, we examined the expression of all isoforms in cartilage samples from normal subjects and OA patients as well as in NHAC cells. Among the isoforms, TGF-β1 was most abundantly expressed in OA and non-OA articular cartilage (Fig. 2, A and B) and in NHAC cells (Fig. 2C), and its expression levels were higher in OA cartilage than in non-OA samples (Fig. 2A). Increased expression in of TGF-β3 in the microarray analysis was not replicated in the real-time PCR analysis. These results indicate that TGF-β1 is the most important of the three isoforms in human articular cartilage and OA.FIGURE 2TGF-β1 mRNA is most abundantly expressed among TGF-β isoforms in human articular cartilage and chondrocytes and is increased in OA. A, microarray analysis of human OA and normal cartilage. B and C, real-time PCR analysis of TGF-β isoform expression in human OA articular cartilage and NHAC cells, respectively. OA-A and OA-B, OA articular cartilage obtained from two individuals with OA; Beads, cells cultured for 2 weeks in alginate beads and then seeded in 12-well plates for the experiment; P2 and P7, cells after two and seven passages, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of Asporin on Cartilage Differentiation and TGF-β1-induced Expression of Cartilage Matrix Genes in Articular Cartilage—We demonstrated previously that asporin overexpression inhibits early chondrogenic differentiation in ATDC5 cells (7Kizawa H. Kim I. Iida A. Sudo A. Miyamoto Y. Fukuda A. Mabuchi A. Kotani A. Kawakami A. Yamamoto S. Uchida A. Nakamura K. Notoya K. Nakamura Y. Ikegawa S. Nat. Genet. 2005; 37: 138-144Crossref PubMed Scopus (369) Google Scholar). To confirm the influence of asporin in this model, we measured the expression of chondrogenic differentiation marker genes (aggrecan and type II and X collagens) in the presence of recombinant mouse asporin. Asporin decreased the expression of marker genes in a dose-dependent manner (Fig. 3, A-C). Asporin also inhibited glycosaminoglycan accumulation measured at 21 days of culture (Fig. 3D). To verify that the TGF-β binding (Fig. 1A) and chondrogenesis (Fig. 3, A-D) inhibitory activities of recombinant asporin prepared from E. coli were comparable with those of native asporin, we examined these activities using recombinant asporin produced using a eukaryotic expression system (COS-7). As shown in Figs. 1 (B and C) and 3 (E and F), the activities were similar. ATDC5 is an excellent model for chondrogenesis; sequential steps of chondrocyte differentiation are reflected in ATDC5 cells by increased expression of the chondrogenic differentiation marker genes type II and X collagens in the presence of insulin (14Shukunami C. Kim C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (343) Google Scholar, 15Shukunami C. Kim K. Atsumi T. Ohta Y. Suzuki F. Hiraki Y. J. Bone Miner. Res. 1997; 12: 1174-1188Crossref PubMed Scopus (251) Google Scholar); however, it most precisely represents growing cartilage in the mouse (21Atsumi T. Kim Y. Kimata K. Ikawa Y. Cell Differ. Dev. 1990; 30: 109-116Crossref PubMed Scopus (336) Google Scholar) rather than human articular cartilage, in which OA occurs. To clarify asporin function in human articular cartilage, we examined its effect on TGF-β1-induced expression of type II collagen and aggrecan mRNAs in NHAC cells. Recombinant mouse asporin inhibited TGF-β1-induced expression of cartilage matrix genes in a dose-dependent manner (Fig. 4, A and B), supporting the observations made in the ATDC5 model. Asporin Inhibits Redifferentiation of NHAC Cells Induced by TGF-β1—Articular cartilage chondrocytes lose their characteristic phenotype (dedifferentiate) during passage. TGF-β induces the restoration of the cartilage phenotype (redifferentiation), as evidenced by increased proteoglycan synthesis and a change from a fibroblastic to a chondrocytic (polygonal or spherical) cell shape (22Inoue H. Kim Y. Iwamoto M. Hiraki Y. Sakuda M. Suzuki F. J. Cell. Physiol. 1989; 138: 329-337Crossref PubMed Scopus (83) Google Scholar, 23Yan W.Q. Kim K. Iwamoto M. Kato Y. J. Biol. Chem. 1990; 265: 10125-10131Abstract Full Text PDF PubMed Google Scholar, 24Lafeber F.P. Kim H.L. van der Kraan P.M. van den Berg W.B. Bijlsma J.W. J. Rheumatol. 1997; 24: 536-542PubMed Google Scholar). We examined the effect of asporin on TGF-β1-induced changes in the cartilage phenotype of NHAC cells. Most TGF-β1-stimulated cells exhibited a spherical shape typical of chondrocytic cells, with areas of clustered cells staining strongly with safranin O, a marker for proteoglycan synthesis (Fig. 4D). However, control (unstimulated) cells showed a fibroblastic morphology with no safranin O staining (Fig. 4C). Addition of asporin changed the morphology of TGF-β1-stimulated cells to a fibroblastic phenotype and reduced safranin O staining (Fig. 4E). Together, these results indicate that asporin reverses the TGF-β1-induced chondrocyte phenotype in human articular chondrocytes. Endogenous Asporin Suppresses Expression of Cartilage Matrix Genes—To clarify the physiological role of asporin in human articular chondrocytes, we decreased endogenous asporin levels in NHAC cells via siRNA and measured the expression of cartilage marker genes. Knockdown of asporin significantly increased type II collagen and aggrecan mRNA expression (Fig. 5, A-C) as well as TGF-β1 (Fig. 5D), indicating that asporin negatively regulates cartilage matrix gene expression in articular cartilage under physiological conditions. The siRNA-induced increase in type II collagen and aggrecan mRNA expression was inhibited by TGF-β-neutralizing antibodies (Fig. 5, B and C), suggesting that increased matrix gene expression is due to increased available TGF-β. Asporin Inhibits TGF-β1/Smad Signaling—We further investigated the influence of asporin on chondrogenesis by examining its effects on TGF-β1/Smad signaling. In ATDC5 cells, recombinant asporin inhibited TGF-β1-induced phosphorylation of Smad2 in a dose-dependent manner (Fig. 6A). Next, we measured the effect of asporin on TGF-β1-induced Smad3/4-specific reporter activity using a luciferase construct containing four tandem repeats of Smad-binding elements (SBE4-luciferase) (16Zawel L. Kim J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Mol. Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (890) Google Scholar). Asporin significantly reduced TGF-β1-induced SBE4-luciferase activity (Fig. 6B). Confirmation of these inhibitory effects in NHAC cells (Fig. 6, C and D) indicates that asporin negatively regulates TGF-β1/Smad signaling in articular cartilage. Smad signaling is modulated not only by TβRI kinase, but also by various molecules, such as inhibitory Smad proteins, and by the ERK (extracellular signal-regulated kinase) MAPK (mitogen-activated protein kinase) and phosphatidylinositol 3-kinase/Akt pathways (25Derynck R. Kim Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4270) Google Sc