Title: Proteolysis of Latent Transforming Growth Factor-β (TGF-β)-binding Protein-1 by Osteoclasts
Abstract: The binding of growth factors to the extracellular matrix (ECM) may be a key pathway for regulation of their activity. We have shown that a major mechanism for storage of transforming growth factor-β (TGF-β) in bone ECM is via its association with latent TGF-β-binding protein-1 (LTBP1). Although proteolytic cleavage of LTBP1 has been reported, it remains unclear whether this represents a physiological mechanism for release of matrix-bound TGF-β. Here we examined the role of LTBP1 in cell-mediated release of TGF-β from bone ECM. We first characterized the soluble and ECM-bound forms of latent TGF-β produced by primary osteoblasts. Next, we examined release of ECM-bound TGF-β by bone resorbing cells. Isolated avian osteoclasts and rabbit bone marrow-derived osteoclasts released bone matrix-bound TGF-β via LTBP1 cleavage. 1,25-Dihydroxyvitamin D3 enhanced LTBP1 cleavage, resulting in release of 90% of the ECM-bound LTBP1. In contrast, osteoblasts failed to cleave LTBP1 or release TGF-β from bone ECM. Cleavage of LTBP1 by avian osteoclasts was inhibited by serine protease and metalloproteinase (MMP) inhibitors. Studies using purified proteases showed that plasmin, elastase, MMP2, and MMP9 were able to cleave LTBP1 to produce 125–165-kDa fragments. These studies identify LTBP1 as a novel substrate for MMPs and provide the first demonstration that LTBP1 proteolysis may be a physiological mechanism for release of TGF-β from ECM-bound stores, potentially the first step in the pathway by which matrix-bound TGF-β is rendered active. The binding of growth factors to the extracellular matrix (ECM) may be a key pathway for regulation of their activity. We have shown that a major mechanism for storage of transforming growth factor-β (TGF-β) in bone ECM is via its association with latent TGF-β-binding protein-1 (LTBP1). Although proteolytic cleavage of LTBP1 has been reported, it remains unclear whether this represents a physiological mechanism for release of matrix-bound TGF-β. Here we examined the role of LTBP1 in cell-mediated release of TGF-β from bone ECM. We first characterized the soluble and ECM-bound forms of latent TGF-β produced by primary osteoblasts. Next, we examined release of ECM-bound TGF-β by bone resorbing cells. Isolated avian osteoclasts and rabbit bone marrow-derived osteoclasts released bone matrix-bound TGF-β via LTBP1 cleavage. 1,25-Dihydroxyvitamin D3 enhanced LTBP1 cleavage, resulting in release of 90% of the ECM-bound LTBP1. In contrast, osteoblasts failed to cleave LTBP1 or release TGF-β from bone ECM. Cleavage of LTBP1 by avian osteoclasts was inhibited by serine protease and metalloproteinase (MMP) inhibitors. Studies using purified proteases showed that plasmin, elastase, MMP2, and MMP9 were able to cleave LTBP1 to produce 125–165-kDa fragments. These studies identify LTBP1 as a novel substrate for MMPs and provide the first demonstration that LTBP1 proteolysis may be a physiological mechanism for release of TGF-β from ECM-bound stores, potentially the first step in the pathway by which matrix-bound TGF-β is rendered active. extracellular matrix fetal rat calvarial cells latent transforming growth factor-β-binding protein-1 matrix metalloproteinase parathyroid hormone-related peptide 25-D3, 1,25-dihydroxyvitamin D3 β-glycerophosphate phosphate-buffered saline fetal bovine serum α-modified minimal essential medium penicillin/streptomycin antibody fast pressure liquid chroma- tography latency-associated peptide l-glutamine aprotinin tartrate-resistant acid phosphatase plasmin tissue inhibitor of the matrix metalloproteinases no cells control control Recent evidence suggests that the binding of growth factors to the extracellular matrix (ECM)1 may be a major mechanism for regulation of growth factor activity (for review, see Ref. 1Taipale J. Keski-Oja J. FASEB J. 1997; 11: 51-59Crossref PubMed Scopus (769) Google Scholar). It has long been thought that growth factors released from bone matrix play important roles in the coupling of bone resorption to bone formation and in repair processes such as fracture healing. However, at present little is known about the mechanisms by which growth factors are stored in the ECM, the ECM proteins with which they interact, or the molecular mechanisms by which they are released. Bone ECM is the major storage site in the body for TGF-β (2Hauschka P.V. Mavrakos A.E. Lafrati S.E. Doleman S.E. Klagsbrun M. J. Biol. Chem. 1986; 261: 12665-12674Abstract Full Text PDF PubMed Google Scholar, 3Seyedin S.M. Thomas T.C. Thompson A.Y. Rosen D.M. Piez K.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2267-2271Crossref PubMed Scopus (406) Google Scholar). This ECM-bound TGF-β, which is predominantly the TGF-β1 isoform, is stored in a latent form and can be released and activated by resorbing osteoclasts (4Pfeilschifter J. Mundy G.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2024-2028Crossref PubMed Scopus (362) Google Scholar). Once released from the matrix and activated, TGF-β can influence many of the steps in the remodeling pathway, including inhibition of osteoclast formation and activity, stimulation of recruitment and proliferation of osteoblast precursors, and stimulation of mature osteoblasts to produce bone matrix proteins (reviewed in Ref.5Bonewald L.F. Crit. Rev. Eukaryotic Gene Expr. 1999; 9: 33-44Crossref PubMed Google Scholar). TGF-β has therefore been implicated as a coupling factor that coordinates the processes of bone resorption and subsequent bone formation. Although it is known that bone matrix-bound TGF-β can be released by osteoclasts (4Pfeilschifter J. Mundy G.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2024-2028Crossref PubMed Scopus (362) Google Scholar) and that osteoclasts are capable of activating both matrix-derived and exogenously added latent TGF-β (6Oreffo R.O.C. Mundy G.R. Seyedin S. Bonewald L.F. Biochem. Biophys. Res. Commun. 1989; 158: 817-823Crossref PubMed Scopus (243) Google Scholar, 7Oursler M.J. J. Bone Miner. Res. 1994; 9: 443-452Crossref PubMed Scopus (143) Google Scholar, 8Bonewald L.F. Oreffo R.O.C. Lee C.H. Park-Snyder S. Twardzik D. Mundy G.R. Endocrinology. 1997; 138: 657-666Crossref PubMed Scopus (23) Google Scholar), the molecular mechanism(s) by which TGF-β is released from ECM-bound stores remain unclear. We have shown previously (9Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (237) Google Scholar, 10Dallas S.L. Keene D.R. Bruder S.P. Saharinen J. Sakai L.Y. Mundy G.R. Bonewald L.F. J. Bone Miner. Res. 2000; 15: 68-81Crossref PubMed Scopus (131) Google Scholar) that a major mechanism for storage of latent TGF-β in bone matrix is via its association with the latent transforming growth factor-β-binding protein-1 (LTBP1). LTBP1 is a member of an emerging superfamily of ECM proteins, which includes fibrillins 1 and 2 and LTBPs 1–4 (reviewed in Refs. 11Sinha S. Nevett C. Shuttleworth C.A. Kielty C.M. Matrix Biol. 1998; 17: 529-545Crossref PubMed Scopus (85) Google Scholar and 12Saharinen J. Hyytiainen M. Taipale J. Keski-Oja J. Cytokine Growth Factor Rev. 1999; 10: 99-117Crossref PubMed Scopus (251) Google Scholar). LTBP1 was originally identified as a component of the large latent TGF-β1 complex (13Kanzaki T. Olofsson A. Morén A. Wernstedt C. Hellman U. Miyazono K. Claesson-Welsh L. Heldin C.H. Cell. 1990; 61: 1051-1061Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 14Tsuji T. Okada F. Yamaguchi K. Nakamura T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8835-8839Crossref PubMed Scopus (127) Google Scholar). This complex consists of the 25-kDa mature TGF-β homodimer, which is cleaved from but remains non-covalently associated with a 75-kDa portion of the propeptide homodimer (also known as latency-associated peptide or LAP). The propeptide is then disulfide-linked to the 190-kDa LTBP1 (13Kanzaki T. Olofsson A. Morén A. Wernstedt C. Hellman U. Miyazono K. Claesson-Welsh L. Heldin C.H. Cell. 1990; 61: 1051-1061Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 15Saharinen J. Keski-Oja J. Mol. Biol. Cell. 2000; 11: 2691-2704Crossref PubMed Scopus (219) Google Scholar). Although most cells produce latent TGF-β in the large latent complex, a small latent complex that lacks LTBP1 and consists of TGF-β non-covalently associated with its propeptide has also been reported as a naturally occurring form in bone and kidney (5Bonewald L.F. Crit. Rev. Eukaryotic Gene Expr. 1999; 9: 33-44Crossref PubMed Google Scholar, 16Bonewald L.F. Wakefield L. Oreffo R.O.C. Escobedo A. Twardzik D.R. Mundy G.R. Mol. Endocrinol. 1991; 5: 741-751Crossref PubMed Scopus (111) Google Scholar). LTBP1 facilitates secretion of latent TGF-β (17Miyazono K. Olofsson A. Colosetti P. Heldin C.H. EMBO J. 1991; 10: 1091-1101Crossref PubMed Scopus (423) Google Scholar) and may also modulate activation of latent TGF-β (18Flaumenhaft R. Abe M. Sato Y. Miyazono K. Heldin C.H. Rifkin D.B. J. Cell Biol. 1993; 120: 995-1002Crossref PubMed Scopus (226) Google Scholar). More recently it has been described as a stable component of the extracellular matrix that is important in storage of latent TGF-β in the matrix and may be a structural component of connective tissue microfibrils (9Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (237) Google Scholar, 10Dallas S.L. Keene D.R. Bruder S.P. Saharinen J. Sakai L.Y. Mundy G.R. Bonewald L.F. J. Bone Miner. Res. 2000; 15: 68-81Crossref PubMed Scopus (131) Google Scholar, 19Taipale J. Miyazono K. Heldin C.H. Keski-Oja J. J. Cell Biol. 1994; 124: 171-181Crossref PubMed Scopus (374) Google Scholar, 20Taipale J. Saharinen J. Hedman K. Keski-Oja J. J. Histochem. Cytochem. 1996; 44: 875-889Crossref PubMed Scopus (193) Google Scholar). Several studies have suggested that in addition to its role in storage of latent TGF-β in the ECM, LTBP1 may function as a vehicle for release of latent TGF-β from matrix-bound stores. Thus, purified proteases, such as plasmin and elastase, have been shown to release TGF-β from the matrix of fibroblasts and osteoblasts via cleavage of LTBP1 (9Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (237) Google Scholar, 21Taipale J. Lohi J. Saharinen J. Kovanen P.T. Keski-Oja J. J. Biol. Chem. 1995; 270: 4689-4696Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). However, it is unclear whether this is a phenomenon associated with pathologically high concentrations of proteases or whether proteolytic cleavage of LTBP1 represents a true physiologic mechanism for release of ECM-bound TGF-β. In the present study, we characterized the forms of latent TGF-β produced by primary bone cells and stored in the ECM. Next, by using osteoclast cell culture systems we investigated the molecular mechanism(s) by which TGF-β is released from bone ECM-bound stores during bone resorption. In particular, we examined whether proteolytic cleavage of LTBP1 may be a potential cellular mechanism for the release of TGF-β bound to bone ECM, and we determined whether proteases known to be important in osteoclast function may be involved in this process. Plasmin, elastase, aprotinin, leupeptin, and pepstatin A were purchased from Roche Molecular Biochemicals. Phenylmethylsulfonyl fluoride and E64 were purchased from Sigma. Matrix metalloproteinases (active MMP2 and MMP9) and the tissue inhibitor of the matrix metalloproteinases (TIMP1) were purchased from Oncogene Research Products (Cambridge, MA). 1,25-Dihydroxyvitamin D3(1,25-D3) was purchased from Biomol (Plymouth Meeting, PA). Parathyroid hormone-related peptide (PTHrP) was purchased from Bachem (Torrance, CA). Two different rabbit polyclonal antibodies against LTBP1 were used: Ab39 that recognizes mouse, rat, and human LTBP1 (kindly supplied by K. Miyazono, Japanese Foundation for Cancer Research, Tokyo, Japan), and a second antibody, termed the “LTBP1 hinge antibody,” raised against a synthetic peptide corresponding to residues 721–744 of the rat LTBP1 sequence (GenBankTMaccession number M55431) that recognizes mouse and rat LTBP1. The specificity of these antibodies has been described elsewhere (10Dallas S.L. Keene D.R. Bruder S.P. Saharinen J. Sakai L.Y. Mundy G.R. Bonewald L.F. J. Bone Miner. Res. 2000; 15: 68-81Crossref PubMed Scopus (131) Google Scholar, 13Kanzaki T. Olofsson A. Morén A. Wernstedt C. Hellman U. Miyazono K. Claesson-Welsh L. Heldin C.H. Cell. 1990; 61: 1051-1061Abstract Full Text PDF PubMed Scopus (370) Google Scholar,20Taipale J. Saharinen J. Hedman K. Keski-Oja J. J. Histochem. Cytochem. 1996; 44: 875-889Crossref PubMed Scopus (193) Google Scholar). A mouse monoclonal antibody against human LTBP1, as well as neutralizing antibodies to TGF-β1 and -β2, a pan-specific TGF-β-neutralizing antibody, and a goat polyclonal antibody against latent TGF-β1 propeptide were purchased from R & D systems (Minneapolis, MN). The detection antibody for Western blotting was a peroxidase-conjugated donkey anti-rabbit (Amersham Biosciences). Tissue culture reagents were purchased from Invitrogen or JRH Biosciences (Lenexa, KS). Fetal rat calvarial osteoblasts (FRC) were isolated as described previously (9Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (237) Google Scholar, 10Dallas S.L. Keene D.R. Bruder S.P. Saharinen J. Sakai L.Y. Mundy G.R. Bonewald L.F. J. Bone Miner. Res. 2000; 15: 68-81Crossref PubMed Scopus (131) Google Scholar). The cells were stored for up to 1 year using standard cryopreservation procedures and used for experiments after one further passage. UMR-106 cells were a gift from T. J. Martin (St. Vincent Institute of Medical Research, Fitzroy, Victoria, Australia). TMLC-C32 cells were a gift from D. B. Rifkin (New York University, New York). Rabbit marrow cells were isolated using a modification of the method of Tezuka et al. (22Tezuka K. Sato T. Kamioka H. Nijweide P.J. Tanaka K. Matsuo T. Ohta M. Kurihara N. Hakeda Y. Kumegawa M. Biochem. Biophys. Res. Commun. 1992; 186: 911-917Crossref PubMed Scopus (202) Google Scholar). Femora, humeri, ulnae, and radii of 11-day-old New Zealand White rabbits were removed. Connective tissues were dissected away, and the bones were minced in α-modified minimal essential medium (αMEM). The bone pieces were vortexed to dissociate the cells, and bone particles were removed by sedimentation under gravity. The supernatant was centrifuged, and the cells were resuspended in αMEM supplemented with 10% fetal bovine serum (FBS), 2 mml-glutamine (LG), and 100 units/ml penicillin/streptomycin (P/S). The cells were then seeded into 12-well plates coated with 35S-labeled bone ECM (see below). Avian osteoclast precursors were isolated as described elsewhere from the medullary bone of egg-laying White Leghorn hens fed on a low calcium diet (23Alvarez I. Teitelbaum S.L. Blair H.C. Greenfield E.M. Athanasou N.A. Ross F.P. Endocrinology. 1991; 128: 2324-2335Crossref PubMed Scopus (72) Google Scholar). The cells were plated in 150-mm Petri dishes as described elsewhere (23Alvarez I. Teitelbaum S.L. Blair H.C. Greenfield E.M. Athanasou N.A. Ross F.P. Endocrinology. 1991; 128: 2324-2335Crossref PubMed Scopus (72) Google Scholar), and after overnight incubation, adherent cells were harvested by treatment with 2 mm EDTA in PBS for 5 min at 37 °C. The cells were then replated into 12-well plates coated with 35S-labeled bone ECM (see below) in αMEM containing 2.5% FBS, 2.5% chicken serum, 2 mm LG, 100 units/ml P/S, and 6 μg/ml arabinose-β-d-cytosine furanoside. These cells have been shown to fuse and become multinucleated in the presence of 1,25-D3 (23Alvarez I. Teitelbaum S.L. Blair H.C. Greenfield E.M. Athanasou N.A. Ross F.P. Endocrinology. 1991; 128: 2324-2335Crossref PubMed Scopus (72) Google Scholar) (see also Fig. 5, e and f). The cells were cultured for up to 6 days, with or without 1,25-D3 and/or protease inhibitors as described below. Culture media were changed every 2 days. FPLC fractionation was performed as described previously (24Dallas S.L. Park-Snyder S. Miyazono K. Twardzik D. Mundy G.R. Bonewald L.F. J. Biol. Chem. 1994; 269: 6815-6822Abstract Full Text PDF PubMed Google Scholar). FRC cells were grown in 150-cm2 flasks in αMEM supplemented with 10% FBS, 2 mm LG, 100 units/ml P/S, and 30 μg/ml gentamycin. At confluence the media were changed to αMEM, supplemented as above but with the addition of 50 μg/ml ascorbic acid and 3 mm β-glycerophosphate (β-GP) and the reduction of the serum to 5%. Thereafter the media were changed every 3 days. Conditioned medium was harvested from four time points representing sequential stages in the in vitro differentiation of FRC cells to form mineralized bone-like nodules. The “pre-confluent” stage was from proliferating cultures at 90% confluence; the “early post-confluent” stage was from 2-day post-confluent cultures; the “nodule-forming” stage was from 6-day post-confluent cultures in which multilayered cellular nodules had formed, but were not yet mineralized; and the “mineralization” stage was from 14-day post-confluent cultures in which mineralized bone nodules were present (see Fig. 1, inset micrographs). For collection of conditioned media, phenol red-free Dulbecco's modified Eagle's medium was used containing 2 mm LG, 100 units/ml P/S, 0.1% bovine serum albumin, 50 μg/ml ascorbic acid. 25 ml of conditioned medium was collected per flask over a 48-h culture period. A total of 150 ml of conditioned medium per time point was concentrated 10-fold over a 50-kDa cut-off membrane using a minisette concentrator (Millipore, Bedford, MA), and the samples were lyophilized and reconstituted with 1–2 ml of distilled water, pH 7.2. They were then dialyzed against 20 mm tris buffer, pH 7.2, and applied to an analytical Mono-Q anion exchange column (Amersham Biosciences). The column was eluted with a linear gradient of 0–0.5 m NaCl/20 mmtris buffer. Fractions were tested for TGF-β activity using the alkaline phosphatase microassay as described below. TGF-β was measured as described previously by using either the ROS 17/2.8 microassay (24Dallas S.L. Park-Snyder S. Miyazono K. Twardzik D. Mundy G.R. Bonewald L.F. J. Biol. Chem. 1994; 269: 6815-6822Abstract Full Text PDF PubMed Google Scholar), which measures stimulation of alkaline phosphatase activity by TGF-β in ROS 17/2.8 osteosarcoma cells, or by using the mink lung epithelial cell luciferase bioassay (25Abe M. Harpel J.G. Metz C.N. Nunes I. Loskutoff D.J. Rifkin D.B. Anal. Biochem. 1994; 216: 276-284Crossref PubMed Scopus (676) Google Scholar), which measures stimulation of activity of the plasminogen activator inhibitor-1 promoter by TGF-β. To determine the total (active + latent) TGF-β levels, the samples were acidified to pH 2 using 1 m HCl and then reneutralized using 1m NaOH immediately before addition to the assay plate. Latent TGF-β values were determined by subtracting active TGF-β measurements from total TGF-β. To examine further the secreted and matrix-bound forms of latent TGF-β produced by FRC cells, pulse-chase metabolic labeling and immunoprecipitation were performed. Cells were plated into 12-well multiwell plates at 10,000 cells/cm2 growth area. At 90% confluence, the cells were washed twice in PBS and incubated for 1 h in cysteine-free αMEM supplemented with 5% dialyzed FBS, 2 mm LG, 100 units/ml P/S, 50 μg/ml ascorbic acid. The cells were then labeled for 30 min using 100 μCi/well [35S]cysteine (Amersham Biosciences) in cysteine-free αMEM supplemented with 5% dialyzed FBS and additives as above. This was followed by a “cold chase” in complete αMEM (containing 0.1 mg/ml l-cysteine), supplemented with 5% FBS and additives as above for a time course of 15 and 30 min, and 2, 6, 24, and 48 h. Conditioned media were harvested and protease inhibitors added (1 mm phenylmethylsulfonyl fluoride, 50 μg/ml aprotinin, 1 μm pepstatin A, 10 μm leupeptin). The cells + matrix were washed twice in ice-cold PBS and then lysed in ice-cold radioimmunoprecipitation buffer (RIPA) (50 mm Tris, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate). The deoxycholate-insoluble material (essentially matrix) was washed two more times in ice-cold PBS, transferred to a 1.5-ml tube, and centrifuged for 5 min at 14,000 rpm. The pellet was then digested for 2 h at 37 °C on an end-over rotator in 500 μl of a solution consisting of 0.2 units/ml plasmin in plasmin digestion buffer (see under “Protease Digestions”). This digestion releases any LTBP1 that may still be bound to the ECM (9Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (237) Google Scholar, 21Taipale J. Lohi J. Saharinen J. Kovanen P.T. Keski-Oja J. J. Biol. Chem. 1995; 270: 4689-4696Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). LTBP1 and TGF-β in the supernatant and in the plasmin digest of the matrix were determined by immunoprecipitation followed by SDS-PAGE and autoradiography as described previously (9Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (237) Google Scholar, 24Dallas S.L. Park-Snyder S. Miyazono K. Twardzik D. Mundy G.R. Bonewald L.F. J. Biol. Chem. 1994; 269: 6815-6822Abstract Full Text PDF PubMed Google Scholar) using a rabbit polyclonal antibody specific for LTBP1 or using a goat polyclonal antibody specific for LAP. To prepare bone ECM for culturing with osteoclasts, FRC cells were plated into 12-well plates as described above. At confluence, the medium was changed to αMEM supplemented with 5% FBS, 2 mm LG, 100 units/ml P/S, 30 μg/ml gentamycin, 50 μg/ml ascorbic acid, and 3 mmβ-GP. Thereafter, the culture media were changed every 3 days. Cultures were maintained for 10–14 days, by which time mineralized bone nodules could be seen, and a thick ECM layer had formed. For preparation of ECM-coated plates, a modification of the method of Joneset al. (26Jones J.I. Gockerman A. Busby W.H. Camacho-Hubner C. Clemmons D.R. J. Cell Biol. 1993; 121: 679-687Crossref PubMed Scopus (482) Google Scholar) was used. The cells were washed twice in ice-cold PBS and lysed in PBS containing 0.5% Triton X-100 for 10 min at 4 °C. This leaves a membrane-like layer of bone ECM adhered to the bottom of the culture well. The matrix layer was then washed twice in ice-cold PBS and treated with ice-cold 50 mm ammonium acetate, pH 7.5, for 10 min at 4 °C (the pH was reduced from 9.0, as described in Jones et al. (26Jones J.I. Gockerman A. Busby W.H. Camacho-Hubner C. Clemmons D.R. J. Cell Biol. 1993; 121: 679-687Crossref PubMed Scopus (482) Google Scholar), to prevent activation of TGF-β by the alkaline pH). The matrix layers were then washed 4 times in PBS, air-dried, and stored at −70 °C. An immunoprecipitation-based assay was developed for examining release of bone matrix-bound LTBP1 and latent TGF-β by cells cultured on bone ECM (27Dallas S.L. Methods Mol. Biol. 2000; 139: 231-243PubMed Google Scholar). FRC cells were cultured in 12-well plates as described above until 10–14 days of culture. The cells were then labeled for 48 h using 100 μCi/well [35S]cysteine in αMEM containing one-fifth the normal cysteine content and supplemented with 5% dialyzed FBS, 50 μg/ml ascorbic acid, 3 mm β-GP, 100 units/ml P/S, 2 mm LG. The labeled bone ECM was prepared as described above. To examine release of LTBP1/TGF-β by osteoclasts, two sources of osteoclast precursors were used, rabbit marrow cells and avian osteoclasts. The cells were plated onto the radiolabeled ECM at 2.5 × 106 cells/well (rabbit marrow cells) or 5 × 106 cells/well (avian osteoclast precursors) in 2 ml of αMEM supplemented as described above. The cells were incubated with or without addition of 1,25-D3, PTHrP, or protease inhibitors as specified under “Results.” Release of LTBP1 and latent TGF-β was monitored by immunoprecipitating the labeled protein fragments released into the culture media using anti-LTBP1 antibodies (the LTBP1 antibodies will precipitate both free LTBP1 and LTBP1 complexed to latent TGF-β). Because the osteoclasts themselves were never exposed to [35S]cysteine, any radiolabeled proteins present in the culture supernatants were assumed to have been released from the pre-labeled matrix. The supernatant was transferred to a fresh tube and protease inhibitors added as described above. The deoxycholate-insoluble pellet was digested in 0.2 units/ml plasmin as described above. LTBP1 in the supernatant and in the plasmin digest of the matrix was then determined by immunoprecipitation with anti-LTBP1 antibodies, as described above. Plasmin and elastase digestions of ECM preparations were performed in a digestion buffer consisting of 0.1% 1-O-n-octyl-β-glucopyranoside, 3 mmMgCl2, 3 mm CaCl2, 150 mm NaCl, 10 mm Tris, pH 8. MMP2 and MMP9 digestions were performed in MMP digestion buffer (50 mmTris, 0.2 m NaCl, 5 mm CaCl2, 0.02% Brij 35, pH 7.6). Activity of the MMPs was confirmed in all experiments by gelatin zymography. Samples were separated on SDS-PAGE using 4–20% gradient gels, transferred onto nitrocellulose, and immunoblotted as described previously (9Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (237) Google Scholar, 10Dallas S.L. Keene D.R. Bruder S.P. Saharinen J. Sakai L.Y. Mundy G.R. Bonewald L.F. J. Bone Miner. Res. 2000; 15: 68-81Crossref PubMed Scopus (131) Google Scholar). The immunostained bands were visualized using the ECL detection system according to manufacturer's instructions (Amersham Biosciences). The immunostained Western blots were then exposed to X-Omat AR film (Eastman Kodak). For immunofluorescent staining, FRC cells were cultured on lab-tek 8-chamber slides at 10,000 cells/cm2 growth area and maintained for 10–14 days as described above. ECM was prepared as described above and incubated with or without proteases as described above. After incubation in proteases, the ECM layers were fixed in 95% ethanol and stained by immunofluorescence as described previously (10Dallas S.L. Keene D.R. Bruder S.P. Saharinen J. Sakai L.Y. Mundy G.R. Bonewald L.F. J. Bone Miner. Res. 2000; 15: 68-81Crossref PubMed Scopus (131) Google Scholar). As matrix laid down by primary osteoblast cultures was to be used to examine release of bone matrix-bound TGF-β by osteoclasts, it was first necessary to characterize the latent TGF-β complexes produced by the osteoblast cultures and determine what proportion of the latent TGF-β was complexed to LTBP1. Fig. 1 shows results from Mono-Q FPLC analysis of conditioned media samples from FRC cultures at the pre-confluent, early post-confluent, nodule-forming, and mineralizing stages (see “Materials and Methods”). The insets in Fig. 1 show the appearance of the cultures at each of these stages. Two major peaks of latent TGF-β activity were observed, one eluting at 0.22 m NaCl (peak II), which is the expected elution position for the 100-kDa small latent TGF-β complex (16Bonewald L.F. Wakefield L. Oreffo R.O.C. Escobedo A. Twardzik D.R. Mundy G.R. Mol. Endocrinol. 1991; 5: 741-751Crossref PubMed Scopus (111) Google Scholar, 24Dallas S.L. Park-Snyder S. Miyazono K. Twardzik D. Mundy G.R. Bonewald L.F. J. Biol. Chem. 1994; 269: 6815-6822Abstract Full Text PDF PubMed Google Scholar), and the other eluting at 0.3m NaCl (peak III), which corresponds to the 290-kDa large latent TGF-β complex, containing LTBP1 (16Bonewald L.F. Wakefield L. Oreffo R.O.C. Escobedo A. Twardzik D.R. Mundy G.R. Mol. Endocrinol. 1991; 5: 741-751Crossref PubMed Scopus (111) Google Scholar, 24Dallas S.L. Park-Snyder S. Miyazono K. Twardzik D. Mundy G.R. Bonewald L.F. J. Biol. Chem. 1994; 269: 6815-6822Abstract Full Text PDF PubMed Google Scholar). The pre-confluent and early post-confluent cultures produced predominantly the small latent TGF-β complex (peak II). However, with maturation in culture, the cells switched to producing larger amounts of the 290-kDa (LTBP1-containing) complex (peak III), which made up ∼40% of the total latent TGF-β secreted. In addition, a minor peak eluting at 0.05 m NaCl (peak I) was also observed in nodule-forming and mineralizing cultures. At present the nature of this peak is unknown. Antibody neutralization studies indicated that the latent TGF-β activity was predominantly TGF-β1 in all three peaks with smaller amounts of TGF-β2 (data not shown). In the pre-confluent and early post-confluent cultures, peak III showed ∼20–30% TGF-β2 activity, in contrast to the nodule-forming and mineralizing cultures, which showed no detectable TGF-β2 activity in this peak (data not shown). LTBP1 has been shown to be cross-linked in the ECM via the action of transglutaminase (28Nunes I. Gleizes P. Metz C.N. Rifkin D.B. J. Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (348) Google Scholar) and is highly insoluble. Previous studies have shown that proteases such as plasmin and elastase can release proteolytic fragments of LTBP1 from the ECM (9Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (237) Google Scholar, 21Taipale J. Lohi J. Saharinen J. Kovanen P.T. Keski-Oja J. J. Biol. Chem. 1995; 270: 4689-4696Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar) and that plasmin can activate latent TGF-β (29Lyons R.M. Gentry L.E. Purchio A.F. Moses H.L. J. Cell Biol. 1990; 110: 1361-1367Crossref PubMed Scopus (672) Google Scholar, 30Yee J.A. Yan L. Dominguez J.C. Allan E.H. Martin T.J. J. Cell. Physiol. 1993; 157: 528-534Crossref PubMed Scopus (108) Google Scholar). These serine proteases were therefore used to examine the ECM-bound forms of latent TGF-β in primary bone cell cultures (see Fig. 2). Treatment of 35S-labeled bone ECM with