TSG-6 Regulates Bone Remodeling through Inhibition of Osteoblastogenesis and Osteoclast Activation
2008; Elsevier BV; Volume: 283; Issue: 38 Linguagem: Inglês
10.1074/jbc.m802138200
ISSN1083-351X
AutoresDavid J. Mahoney, Katalin Mikecz, Tariq Ali, Guillaume Mabilleau, Dafna Benayahu, Anna Plaas, Caroline M. Milner, Anthony J. Day, A Sabokbar,
Tópico(s)Bone health and treatments
ResumoTSG-6 is an inflammation-induced protein that is produced at pathological sites, including arthritic joints. In animal models of arthritis, TSG-6 protects against joint damage; this has been attributed to its inhibitory effects on neutrophil migration and plasmin activity. Here we investigated whether TSG-6 can directly influence bone erosion. Our data reveal that TSG-6 inhibits RANKL-induced osteoclast differentiation/activation from human and murine precursor cells, where elevated dentine erosion by osteoclasts derived from TSG-6-/- mice is consistent with the very severe arthritis seen in these animals. However, the long bones from unchallenged TSG-6-/- mice were found to have higher trabecular mass than controls, suggesting that in the absence of inflammation TSG-6 has a role in bone homeostasis; we have detected expression of the TSG-6 protein in the bone marrow of unchallenged wild type mice. Furthermore, we have observed that TSG-6 can inhibit bone morphogenetic protein-2 (BMP-2)-mediated osteoblast differentiation. Interaction analysis revealed that TSG-6 binds directly to RANKL and to BMP-2 (as well as other osteogenic BMPs but not BMP-3) via composite surfaces involving its Link and CUB modules. Consistent with this, the full-length protein is required for maximal inhibition of osteoblast differentiation and osteoclast activation, although the isolated Link module retains significant activity in the latter case. We hypothesize that TSG-6 has dual roles in bone remodeling; one protective, where it inhibits RANKL-induced bone erosion in inflammatory diseases such as arthritis, and the other homeostatic, where its interactions with BMP-2 and RANKL help to balance mineralization by osteoblasts and bone resorption by osteoclasts. TSG-6 is an inflammation-induced protein that is produced at pathological sites, including arthritic joints. In animal models of arthritis, TSG-6 protects against joint damage; this has been attributed to its inhibitory effects on neutrophil migration and plasmin activity. Here we investigated whether TSG-6 can directly influence bone erosion. Our data reveal that TSG-6 inhibits RANKL-induced osteoclast differentiation/activation from human and murine precursor cells, where elevated dentine erosion by osteoclasts derived from TSG-6-/- mice is consistent with the very severe arthritis seen in these animals. However, the long bones from unchallenged TSG-6-/- mice were found to have higher trabecular mass than controls, suggesting that in the absence of inflammation TSG-6 has a role in bone homeostasis; we have detected expression of the TSG-6 protein in the bone marrow of unchallenged wild type mice. Furthermore, we have observed that TSG-6 can inhibit bone morphogenetic protein-2 (BMP-2)-mediated osteoblast differentiation. Interaction analysis revealed that TSG-6 binds directly to RANKL and to BMP-2 (as well as other osteogenic BMPs but not BMP-3) via composite surfaces involving its Link and CUB modules. Consistent with this, the full-length protein is required for maximal inhibition of osteoblast differentiation and osteoclast activation, although the isolated Link module retains significant activity in the latter case. We hypothesize that TSG-6 has dual roles in bone remodeling; one protective, where it inhibits RANKL-induced bone erosion in inflammatory diseases such as arthritis, and the other homeostatic, where its interactions with BMP-2 and RANKL help to balance mineralization by osteoblasts and bone resorption by osteoclasts. TSG-6, 4The abbreviations used are: TSG-6tumor necrosis factor (TNF)-stimulated gene 6rhTSG-6recombinant human TSG-6SABstandard assay bufferALPalkaline phosphataseBMPbone morphogenetic proteinCUBcomplement C1r/C1s,Uegf, Bmp1CUB_C_TSG6recombinant CUB_C domain of TSG-6HAhyaluronanLink_TSG6recombinant Link module of TSG-6M-CSFmacrophage colony-stimulating factorOPGosteoprotegrinPBMCperipheral blood mononuclear cellRArheumatoid arthritis(s)RANKL(soluble) receptor activator of NF-κB ligandTGF-βtransforming growth factor βWTwild typeILinterleukinαMEMα-minimum essential mediumPBSphosphate-buffered salineCTmicro-computerized tomographyELISAenzyme-linked immunosorbent assayOPGosteoprotegerin. the ∼35-kDa secreted product of TNF-stimulated gene-6 (1Lee T.H. Wisniewski H.G. Vilcek J. J. Cell Biol. 1992; 116: 545-557Crossref PubMed Scopus (261) Google Scholar), is expressed in response to various inflammatory mediators and growth factors (2Milner C.M. Day A.J. J. Cell Sci. 2003; 116: 1863-1873Crossref PubMed Scopus (309) Google Scholar). It is comprised almost entirely of contiguous Link and CUB modules and binds to a diversity of protein and glycosaminoglycan ligands, including pentraxin-3, thrombospondin-1, thrombospondin-2, aggrecan, versican, inter-α-inhibitor, bikunin, bone morphogenetic protein-2 (BMP-2), fibronectin, hyaluronan (HA), heparin, heparan sulfate, chondroitin 4-sulfate, and dermatan sulfate (1Lee T.H. Wisniewski H.G. Vilcek J. J. Cell Biol. 1992; 116: 545-557Crossref PubMed Scopus (261) Google Scholar, 3Salustri A. Garanda C. Hirsch E. De Acetis M. Maccagno A. Bottazzi B. Doni A. Bastone A. Mantovani G. Beck Peccoz P. Salvatori G. Mahoney D.J. Day A.J. Siracusa G. Romani L. Mantovani A. Development. 2004; 131: 1577-1586Crossref PubMed Scopus (369) Google Scholar, 4Kuznetsova S.A. Day A.J. Mahoney D.J. Mosher D.F. Roberts D.D. J. Biol. 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Biol. 2007; 371: 669-684Crossref PubMed Scopus (22) Google Scholar), where the ligand binding sites for HA, bikunin, and heparin have been mapped onto this domain (8Mahoney D.J. Mulloy B. Forster M.J. Blundell C.D. Fries E. Milner C.M. Day A.J. J. Biol. Chem. 2005; 280: 27044-27055Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 19Mahoney D.J. Blundell C.D. Day A.J. J. Biol. Chem. 2001; 276: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 20Blundell C.D. Almond A. Mahoney D.J. DeAngelis P.L. Campbell I.D. Day A.J. J. Biol. Chem. 2005; 280: 18189-18201Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In addition, we have recently expressed the CUB_C domain from human TSG-6 (termed CUB_C_TSG6 (12Kuznetsova S. Mahoney D.J. Mosher D.F. Nentwich H.A. Ali T. Day A.J. Roberts D.D. Matrix Biol. 2008; 27: 201-210Crossref PubMed Scopus (35) Google Scholar)), which comprises the CUB module and C-terminal region of the protein, and have used this material to obtain an x-ray structure for the TSG-6 CUB module. 5D. C. Briggs, T. Ali, D. J. Mahoney, C. M. Milner, and A. J. Day, unpublished information. tumor necrosis factor (TNF)-stimulated gene 6 recombinant human TSG-6 standard assay buffer alkaline phosphatase bone morphogenetic protein complement C1r/C1s,Uegf, Bmp1 recombinant CUB_C domain of TSG-6 hyaluronan recombinant Link module of TSG-6 macrophage colony-stimulating factor osteoprotegrin peripheral blood mononuclear cell rheumatoid arthritis (soluble) receptor activator of NF-κB ligand transforming growth factor β wild type interleukin α-minimum essential medium phosphate-buffered saline micro-computerized tomography enzyme-linked immunosorbent assay osteoprotegerin. Current data suggest that TSG-6 is not constitutively expressed in normal adult tissues, but rather that it is associated with inflammatory diseases (2Milner C.M. Day A.J. J. Cell Sci. 2003; 116: 1863-1873Crossref PubMed Scopus (309) Google Scholar, 21Wisniewski H.G. Vilcek J. Cytokine Growth Factor Rev. 2004; 15: 129-146Crossref PubMed Scopus (124) Google Scholar, 22Milner C.M. Higman V.A. Day A.J. Biochem. Soc. Trans. 2006; 34: 446-450Crossref PubMed Scopus (69) Google Scholar) such as asthma (23Forteza R. Casalino-Matsuda S. Monzon-Medina M.E. Rugg M.S. Milner C.M. Day A.J. Am. J. Respir. Cell Mol. Biol. 2007; 36: 20-31Crossref PubMed Scopus (62) Google Scholar) and arthritis (24Wisniewski H.G. Maier R. Lotz M. Lee S. Klampfer L. Lee T.H. Vilcek J. J. Immunol. 1993; 151: 6593-6601PubMed Google Scholar, 25Bayliss M.T. Howat S.L.T. Dudhia J. Murphy J.M. Barry F.P. Edwards J.C.W. Day A.J. Osteoarthritis Cartilage. 2001; 9: 42-48Abstract Full Text PDF PubMed Scopus (98) Google Scholar). However, TSG-6 is produced in ovulating ovarian follicles, where it has an essential physiological role in female fertility (26Ochsner S.A. Day A.J. Rugg M.S. Breyer R.M. Gomer R.H. Richards J.S. Endocrinology. 2003; 144: 4376-4384Crossref PubMed Scopus (130) Google Scholar, 27Fülöp C. Szántó S. Mukhopadhyay D. Bárdos T. Kamath R.V. Rugg M.S. Day A.J. Salustri A. Hascall V.C. Glant T.T. Mikecz K. Development. 2003; 130: 2253-2261Crossref PubMed Scopus (337) Google Scholar). TSG-6 has been most extensively studied in the context of articular joint disease; it has been detected in the synovial fluid, cartilage, and synovia of osteoarthritis and rheumatoid arthritis (RA) patients but not in the corresponding normal tissues (24Wisniewski H.G. Maier R. Lotz M. Lee S. Klampfer L. Lee T.H. Vilcek J. J. Immunol. 1993; 151: 6593-6601PubMed Google Scholar, 25Bayliss M.T. Howat S.L.T. Dudhia J. Murphy J.M. Barry F.P. Edwards J.C.W. Day A.J. Osteoarthritis Cartilage. 2001; 9: 42-48Abstract Full Text PDF PubMed Scopus (98) Google Scholar). It is likely that TSG-6 is produced locally at disease lesions in joints, as its expression can be induced in cultured human chondrocytes by TNF, IL-1, IL-6, TGF-β, and platelet-derived growth factor (28Maier R. Wisniewski H.G. Vilcek J. Lotz M. Arthritis Rheum. 1996; 39: 552-559Crossref PubMed Scopus (44) Google Scholar, 29Margerie D. Flechtenmacher J. Buttner F.H. Karbowski A. Puhl W. Schleyerbach R. Bartnik E. Osteoarthritis Cartilage. 1997; 5: 129-138Abstract Full Text PDF PubMed Scopus (31) Google Scholar), and it is constitutively expressed by synoviocytes from RA patients, where its production is further enhanced by treatment with IL-1, TNF (24Wisniewski H.G. Maier R. Lotz M. Lee S. Klampfer L. Lee T.H. Vilcek J. J. Immunol. 1993; 151: 6593-6601PubMed Google Scholar), and IL-17 (30Kehlen A. Pachnio A. Thiele K. Langner J. Arthritis Res. Ther. 2003; 5: 186-192Crossref PubMed Google Scholar). A number of studies have revealed that TSG-6 has a protective role in experimental models of arthritis (31Mindrescu C. Thorbecke G.J. Klein M.J. Vilcek J. Wisniewski H.G. Arthritis Rheum. 2000; 43: 2668-2677Crossref PubMed Scopus (61) Google Scholar, 32Mindrescu C. Dias A.A.M. Olszewski R.J. Klein M.J. Reis L.F.L. Wisniewski H.G. Arthritis Rheum. 2002; 46: 2453-2464Crossref PubMed Scopus (61) Google Scholar, 33Bárdos T. Kamath R.V. Mikecz K. Glant T.T. Am. J. Pathol. 2001; 159: 1711-1721Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 34Glant T.T. Kamath R.V. Bárdos T. Gál I. Szántó S. Murad Y.M. Sandy J.D. Mort J.S. Roughley P.J. Mikecz K. Arthritis Rheum. 2002; 46: 2207-2218Crossref PubMed Scopus (72) Google Scholar, 35Szántó S. Bárdos T. Gál I. Glant T.T. Mikecz K. Arthritis Rheum. 2004; 50: 3012-3022Crossref PubMed Scopus (75) Google Scholar). For example, in collagen-induced arthritis (an autoimmune polyarthritis with a histopathology similar to human RA) there was delayed onset of symptoms and reduction of both disease incidence and joint inflammation/destruction in TSG-6 transgenic mice or wild type mice treated systemically with recombinant human TSG-6 (31Mindrescu C. Thorbecke G.J. Klein M.J. Vilcek J. Wisniewski H.G. Arthritis Rheum. 2000; 43: 2668-2677Crossref PubMed Scopus (61) Google Scholar, 32Mindrescu C. Dias A.A.M. Olszewski R.J. Klein M.J. Reis L.F.L. Wisniewski H.G. Arthritis Rheum. 2002; 46: 2453-2464Crossref PubMed Scopus (61) Google Scholar), where the TSG-6 transgene mediated an effect comparable with anti-TNF antibody treatment in mice (32Mindrescu C. Dias A.A.M. Olszewski R.J. Klein M.J. Reis L.F.L. Wisniewski H.G. Arthritis Rheum. 2002; 46: 2453-2464Crossref PubMed Scopus (61) Google Scholar). In cartilage-specific TSG-6 transgenic mice, the instigation of antigen-induced arthritis (a model of monoarticular arthritis) resulted in delayed cartilage damage compared with controls, with reduced degradation of aggrecan by matrix metalloproteinases and aggrecanases (34Glant T.T. Kamath R.V. Bárdos T. Gál I. Szántó S. Murad Y.M. Sandy J.D. Mort J.S. Roughley P.J. Mikecz K. Arthritis Rheum. 2002; 46: 2207-2218Crossref PubMed Scopus (72) Google Scholar). Furthermore, there was evidence of cartilage regeneration 4–5 weeks after the onset of disease in these animals. Similar chondroprotective effects were seen in wild type mice where recombinant murine TSG-6 was injected directly into the affected joint in antigen-induced arthritis or intravenously in proteoglycan-induced arthritis (a model of human RA) (33Bárdos T. Kamath R.V. Mikecz K. Glant T.T. Am. J. Pathol. 2001; 159: 1711-1721Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The anti-inflammatory and chondroprotective effects of TSG-6 observed in these studies are likely to be due to more than one mechanism (22Milner C.M. Higman V.A. Day A.J. Biochem. Soc. Trans. 2006; 34: 446-450Crossref PubMed Scopus (69) Google Scholar). For example, TSG-6 is a potent inhibitor of neutrophil extravasation in vivo (36Wisniewski H.G. Hua J.C. Poppers D.M. Naime D. Vilcek J. Cronstein B.N. J. Immunol. 1996; 156: 1609-1615PubMed Google Scholar, 37Getting S.J. Mahoney D.J. Cao T. Rugg M.S. Fries E. Milner C.M. Perretti M. Day A.J. J. Biol. Chem. 2002; 277: 51068-51076Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 38Cao T. La M. Getting S.J. Day A.J. Perretti M. Microcirculation. 2004; 11: 615-624Crossref PubMed Scopus (49) Google Scholar) and has also been implicated in the inhibition of the protease network through its potentiation of the anti-plasmin activity of inter-α-inhibitor (8Mahoney D.J. Mulloy B. Forster M.J. Blundell C.D. Fries E. Milner C.M. Day A.J. J. Biol. Chem. 2005; 280: 27044-27055Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 36Wisniewski H.G. Hua J.C. Poppers D.M. Naime D. Vilcek J. Cronstein B.N. J. Immunol. 1996; 156: 1609-1615PubMed Google Scholar, 37Getting S.J. Mahoney D.J. Cao T. Rugg M.S. Fries E. Milner C.M. Perretti M. Day A.J. J. Biol. Chem. 2002; 277: 51068-51076Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), where plasmin is a key regulator of proteolysis during inflammation, e.g. via its activation of matrix metalloproteinases. In this regard, TSG-6-/- mice develop an accelerated and much more severe form of proteoglycan-induced arthritis than controls, having extensive cartilage degradation and bone erosion, which was attributed to increased neutrophil infiltration and plasmin activity in the inflamed paw joints (35Szántó S. Bárdos T. Gál I. Glant T.T. Mikecz K. Arthritis Rheum. 2004; 50: 3012-3022Crossref PubMed Scopus (75) Google Scholar). The data presented here demonstrate that TSG-6 inhibits bone erosion by osteoclasts and that this is likely to be mediated via a mechanism that involves its direct interaction with RANKL (the receptor activator of NF-κB ligand), the major regulator of osteoclast activity and of joint destruction in arthritis. We have also shown that TSG-6 binds to osteogenic bone morphogenetic proteins (i.e. BMP-2, -4, -5, -6, -7, -13, and -14) and provide evidence that TSG-6 has a physiological role in bone homeostasis via the regulation of both bone formation and resorption. Preparation of Recombinant TSG-6 Proteins—Full-length recombinant human (rh)TSG-6 was expressed in Drosophila S2 cells and purified as described previously (39Nentwich H.A. Mustafa Z. Rugg M.S. Marsden B.D. Cordell M.R. Mahoney D.J. Jenkins S.C. Dowling B. Fries E. Milner C.M. Loughlin J. Day A.J. J. Biol. Chem. 2002; 277: 15354-15362Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Link_TSG6 (the Link module of human TSG-6, corresponding to residues 36–133 of the pre-protein (1Lee T.H. Wisniewski H.G. Vilcek J. J. Cell Biol. 1992; 116: 545-557Crossref PubMed Scopus (261) Google Scholar)) and CUB_C_TSG6 (the CUB domain and C-terminal 27 amino acids; i.e. residues 129–277 of the human pre-protein) were expressed in E. coli, purified, and characterized as described in Day et al. (17Day A.J. Aplin R.T. Willis A.C. Protein Expression Purif. 1996; 8: 9-16Crossref Scopus (47) Google Scholar), Kahmann et al. (40Kahmann J.D. Koruth R. Day A.J. Protein Expression Purif. 1997; 9: 315-318Crossref PubMed Scopus (26) Google Scholar), and in Kuznetsova et al. (12Kuznetsova S. Mahoney D.J. Mosher D.F. Nentwich H.A. Ali T. Day A.J. Roberts D.D. Matrix Biol. 2008; 27: 201-210Crossref PubMed Scopus (35) Google Scholar), respectively. Effect of TSG-6 on RANKL-induced Human Osteoclast Formation—To determine the effects of TSG-6 on RANKL-induced human osteoclast formation, human peripheral blood mononuclear cells (PBMCs) were isolated from healthy male volunteers (age range 25–35 years) as described in Sabokbar and Athanasou (41Sabokbar A. Athanasou N.A. Methods Mol. Med. 2003; 80: 101-111PubMed Google Scholar). Briefly, blood was collected in EDTA-treated tubes and diluted 1:1 in α-minimum essential medium (αMEM) (Invitrogen), layered over Histopaque (Sigma-Aldrich), then centrifuged (693 × g), washed, and resuspended in αMEM with 10% (v/v) heat-inactivated fetal calf serum (Invitrogen). PBMCs were counted after lysis of red cells with 5% (v/v) acetic acid and seeded (at 5 × 105 cells/well) into 96-well tissue culture plates containing either dentine slices or coverslips. After 2 h of incubation, dentine slices and coverslips were removed from the wells and washed vigorously in αMEM to remove non-adherent cells before transfer into 24-well tissue culture plates containing 1 ml/well αMEM supplemented with 10% (v/v) fetal calf serum, 10 mm l-glutamine, antibiotics (100 IU/ml penicillin and 10 μg/ml streptomycin), 25 ng/ml macrophage colony-stimulating factor (M-CSF; R&D Systems Europe), and 50 ng/ml soluble (s)RANKL (PeproTech). After the addition of rhTSG-6 (30.1 kDa (39Nentwich H.A. Mustafa Z. Rugg M.S. Marsden B.D. Cordell M.R. Mahoney D.J. Jenkins S.C. Dowling B. Fries E. Milner C.M. Loughlin J. Day A.J. J. Biol. Chem. 2002; 277: 15354-15362Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar)) at 0–50 ng/ml or equimolar concentrations of Link_TSG6 (10.9 kDa (17Day A.J. Aplin R.T. Willis A.C. Protein Expression Purif. 1996; 8: 9-16Crossref Scopus (47) Google Scholar)) or CUB_C_TSG6 (16.8 kDa (12Kuznetsova S. Mahoney D.J. Mosher D.F. Nentwich H.A. Ali T. Day A.J. Roberts D.D. Matrix Biol. 2008; 27: 201-210Crossref PubMed Scopus (35) Google Scholar)), cultures were maintained for up to 21 days, during which time the entire culture medium containing all factors was replenished every 2–3 days. Osteoclast formation was assessed cytochemically by determining the number of multinucleated tartrate-resistant acid phosphatase-positive cells after 14 days in culture. Cells on coverslips were fixed in 10% (v/v) formalin and stained using napthol AS-BI as a substrate in the presence of 1.0 m acetate-tartrate; the reaction was stopped with 0.5 m NaF. Bone resorptive activity, an indicator of osteoclast activation, was determined by the measurement of resorption lacunae on dentine slices. After 21 days in culture dentine slices were removed from the wells, rinsed in PBS, placed in 1.0 m NH4OH overnight, and then sonicated for 5–10 min; this resulted in complete removal of cells from the dentine slice, permitting examination of its surface. The slices were washed in distilled water, stained with 0.5% (w/v) aqueous toluidine blue, pH 5.0, and examined by light microscopy. Tartrate-resistant acid phosphatase staining was analyzed using ImageJ software (rsb.info.nih.gov/ij), whereas for lacunar resorption the data were expressed as the mean percentage area resorbed from four dentine slices per treatment. Each set of experiments was repeated at least three times. Extent of Osteoclast Formation in TSG-6-deficient Mice—TSG-6-/- mice were generated as described previously (27Fülöp C. Szántó S. Mukhopadhyay D. Bárdos T. Kamath R.V. Rugg M.S. Day A.J. Salustri A. Hascall V.C. Glant T.T. Mikecz K. Development. 2003; 130: 2253-2261Crossref PubMed Scopus (337) Google Scholar) and back-crossed into the BALB/c background (35Szántó S. Bárdos T. Gál I. Glant T.T. Mikecz K. Arthritis Rheum. 2004; 50: 3012-3022Crossref PubMed Scopus (75) Google Scholar). Age-matched pairs of female TSG-6-/- and wild type (WT) BALB/c mice (between 18 and 25 weeks of age) were used for experiments. All procedures carried out on animals were approved by the Institutional Animal Care and Use Committee (Rush University Medical Center, Chicago, IL). Bone marrow cells from the long bones of TSG-6-/- mice were isolated as previously described (42Wani M.R. Fuller K. Kim N.S. Choi Y. Chambers T. Endocrinology. 1999; 140: 1927-1935Crossref PubMed Google Scholar). Briefly, mice were killed by CO2 inhalation, and the femora and tibiae were aseptically removed and dissected free of adherent soft tissue. The bone ends were cut, and the marrow cavity was flushed out into a Petri dish by slowly injecting αMEM at one end of the bone using a sterile 21-gauge needle. The bone marrow suspension was carefully agitated with a plastic Pasteur pipette to obtain a homogeneous suspension of cells, and these were incubated for 2 h at a density of 5 × 104 cells/ml in 96-well plates containing dentine slices. Non-adherent cells were removed from the dentine slices (by vigorous washing in αMEM), which were then transferred to 24-well plates containing αMEM with 10% (v/v) heat-inactivated serum and antibiotics (as described above). Cultures on dentine slices were maintained in the presence of 25 ng/ml M-CSF (R&D Systems Europe) and 30 ng/ml murine sRANKL (PeproTech) for 10 days during which the culture media and factors were replenished every 2–3 days. The extent of lacunar resorption was then determined as described above. Micro-computerized Tomography (CT) Analysis of Long Bones from TSG-6-deficient and WT Mice—Micro-CT analysis of the long bones in TSG-6-deficient and WT mouse femurs was performed to determine any differences in trabecular bone; four pairs of age-matched male mice (24–28 weeks) were analyzed. Briefly, plastic-wrapped knees were mounted vertically on a Skyscan 1172 micro-CT scanner and scans (6.77 μm with a voxel resolution of 6.9 μm2) were performed using a 20–100-kV microfocus x-ray source with a 10-megapixel digital x-ray camera. The images obtained were subjected to three-dimensional angular resampling (using Skyscan 3D-creator software), and morphometric parameters were calculated for trabecular regions of interest using a Marching Cubes model (43Lorensen W.E. Cline H.E. ACM SIGGRAPH Comp. Graphics. 1987; 21: 163-169Crossref Scopus (7291) Google Scholar) as described previously (44Parfitt A.M. Drezner M.K. Glorieux F.H. Kanis J.A. Malluche H. Meunier P.J. Ott S.M. Recker R.R. J. Bone Miner. Res. 1987; 2: 595-610Crossref PubMed Scopus (4914) Google Scholar). Immunolocalization of TSG-6 in the Mouse Knee Joint—Mouse knee joints were prepared for confocal immunohistochemistry essentially as described in Plaas et al. (45Plaas A. Osborn B. Yoshihara Y. Bai Y. Bloom T. Nelson F. Mikecz K. Sandy J.D. Osteoarthritis Cartilage. 2007; 15: 719-734Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Briefly, mouse legs were fixed in 10% (v/v) neutral-buffered formalin for 48 h, skin and muscle tissue were removed, and joints were decalcified in 5% (w/v) EDTA in PBS for 14 days. Specimens were then processed and embedded in paraffin. Thin sections (4 μm) were deparaffinized, rehydrated, and exposed to a rabbit anti-TSG-6 polyclonal antibody (prepared in collaboration with Dr. J. D. Sandy (Rush University, Chicago, IL) and Affinity Bioreagents (Golden, CO) against the peptide ASVTAGGFQIK, affinity-purified, and shown to recognize recombinant murine TSG-6 (R&D Systems) and rhTSG-6 (39Nentwich H.A. Mustafa Z. Rugg M.S. Marsden B.D. Cordell M.R. Mahoney D.J. Jenkins S.C. Dowling B. Fries E. Milner C.M. Loughlin J. Day A.J. J. Biol. Chem. 2002; 277: 15354-15362Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) by Western blotting; see supplemental Fig. 1) at 10 μg/ml (IgG) in 1.5% (v/v) goat serum in PBS for 30 min, washed in PBS for 5 min, and then incubated with AlexaFluor-568 goat anti-rabbit IgG (Molecular Probes; 1:250 in PBS) for 1 h at room temperature. Sections were co-stained for HA using 5 μg/ml biotinylated bovine cartilage HA-binding protein/link protein complexes (isolated in the A1A1D6 fraction after CsCl gradient centrifugation) followed by AlexaFluor-488-streptavidin (Molecular Probes) as described in Plaas et al. (45Plaas A. Osborn B. Yoshihara Y. Bai Y. Bloom T. Nelson F. Mikecz K. Sandy J.D. Osteoarthritis Cartilage. 2007; 15: 719-734Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Nuclei were stained with TOTO-3 (Molecular Probes; 1:500 in PBS), and sections were examined by confocal microscopy as detailed previously (45Plaas A. Osborn B. Yoshihara Y. Bai Y. Bloom T. Nelson F. Mikecz K. Sandy J.D. Osteoarthritis Cartilage. 2007; 15: 719-734Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Effect of TSG-6 on BMP-2-induced Osteoblast Differentiation—Murine MC3T3-E1 osteoblastic cells (European Collection of Cell Cultures) (46Sudo H. Kodama H.A. Amagai Y. Yamamoto S. Kasai S. J. Cell Biol. 1983; 96: 191-198Crossref PubMed Scopus (1498) Google Scholar) and MBA-15.4 pre-osteoblastic marrow-derived cells (47Benayahu D. Sela J. Calcif. Tissue Int. 1996; 59: 254-258Crossref PubMed Scopus (6) Google Scholar) were seeded on 24-well plates (1.25 × 104 cells/ml) in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Invitrogen), 10 mm l-glutamine, and antibiotics (100 IU/ml penicillin and 10 μg/ml streptomycin). Differentiation into osteoblasts was induced with 100 ng/ml BMP-2 (R&D Systems Europe), and the effects of adding rhTSG-6 (0–10000 ng/ml) or molar equivalents of Link_TSG6 or CUB_C_TSG6 proteins were determined. Cells were cultured for 7 days in a humidified atmosphere with 5% (v/v) CO2 at 37 °C, during which time the culture media (including all factors) was replaced every 72 h. Cells were washed in PBS and freeze-thawed 3 times, and alkaline phosphatase (ALP) was released by scraping cells into 50 μl of 0.2% (v/v) Nonidet P-40 (Fluka) and subsequent sonication (5 s at 6 watts). ALP activity (pmol/μl of material) was determined using the fluorescent substrate 4-methyl umbelliferyl phosphate (Sigma-Aldrich). Briefly, 10 μl of cell lysate was diluted in 50 μl of 0.16% (v/v) Nonidet P-40, 10 mm Tris, pH 8.0, in the wells of a Nunclon Delta (Nunc) plate, and 100 μl of 0.2 mm methyl umbelliferyl phosphate was added. Plates were incubated at 37 °C for 45 min, and the reaction was stopped by adding 100 μl of 0.6 m Na2CO3 to each well. Fluorescence was measured using an excitation wavelength of 360 nm and an emission wavelength of 450 nm (w
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