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Fibronectin Regulates Latent Transforming Growth Factor-β (TGFβ) by Controlling Matrix Assembly of Latent TGFβ-binding Protein-1

2005; Elsevier BV; Volume: 280; Issue: 19 Linguagem: Inglês

10.1074/jbc.m410762200

ISSN

1083-351X

Autores

Sarah L. Dallas, Pitchumani Sivakumar, Carolyn Jones, Qian Chen, Donna M. Peters, Deane F. Mosher, Martin J. Humphries, Cay M. Kielty,

Tópico(s)

Connective tissue disorders research

Resumo

Latent transforming growth factor-β-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in the storage of latent TGFβ in the ECM and regulate its availability. Here we show that fibronectin is critical for the incorporation of LTBP1 and transforming growth factor-β (TGFβ) into the ECM of osteoblasts and fibroblasts. Immunolocalization studies suggested that fibronectin provides an initial scaffold that precedes and patterns LTBP1 deposition but that LTBP1 and fibronectin are later localized in separate fibrillar networks, suggesting that the initial template is lost. Treatment of fetal rat calvarial osteoblasts with a 70-kDa N-terminal fibronectin fragment that inhibits fibronectin assembly impaired incorporation of LTBP1 and TGFβ into the ECM. Consistent with this, LTBP1 failed to assemble in embryonic fibroblasts that lack the gene for fibronectin. LTBP1 assembly was rescued by full-length fibronectin and superfibronectin, which are capable of assembly into fibronectin fibrils, but not by other fibronectin fragments, including a 160-kDa RGD-containing fragment that activates α5β1 integrins. This suggests that the critical event for LTBP1 assembly is the formation of a fibronectin fibrillar network and that integrin ligation by fibronectin molecules alone is not sufficient. Not only was fibronectin essential for the initial incorporation of LTBP1 into the ECM, but the continued presence of fibronectin was required for the continued assembly of LTBP1. These studies highlight a nonredundant role for fibronectin in LTBP1 assembly into the ECM and suggest a novel role for fibronectin in regulation of TGFβ via LTBP1 interactions. Latent transforming growth factor-β-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in the storage of latent TGFβ in the ECM and regulate its availability. Here we show that fibronectin is critical for the incorporation of LTBP1 and transforming growth factor-β (TGFβ) into the ECM of osteoblasts and fibroblasts. Immunolocalization studies suggested that fibronectin provides an initial scaffold that precedes and patterns LTBP1 deposition but that LTBP1 and fibronectin are later localized in separate fibrillar networks, suggesting that the initial template is lost. Treatment of fetal rat calvarial osteoblasts with a 70-kDa N-terminal fibronectin fragment that inhibits fibronectin assembly impaired incorporation of LTBP1 and TGFβ into the ECM. Consistent with this, LTBP1 failed to assemble in embryonic fibroblasts that lack the gene for fibronectin. LTBP1 assembly was rescued by full-length fibronectin and superfibronectin, which are capable of assembly into fibronectin fibrils, but not by other fibronectin fragments, including a 160-kDa RGD-containing fragment that activates α5β1 integrins. This suggests that the critical event for LTBP1 assembly is the formation of a fibronectin fibrillar network and that integrin ligation by fibronectin molecules alone is not sufficient. Not only was fibronectin essential for the initial incorporation of LTBP1 into the ECM, but the continued presence of fibronectin was required for the continued assembly of LTBP1. These studies highlight a nonredundant role for fibronectin in LTBP1 assembly into the ECM and suggest a novel role for fibronectin in regulation of TGFβ via LTBP1 interactions. Recent evidence suggests that the binding of growth factors to the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; FRC, fetal rat calvarial cells; FN, fibronectin; LTBP1, latent transforming growth factor-β binding protein-1; TGFβ, transforming growth factor-β; FPLC, fast protein liquid chromatography; ELISA, enzyme-linked immunosorbent assay. is a major mechanism for regulation of growth factor activity and plays a fundamental role in tissue morphogenesis and repair (1Taipale J. Keski-Oja J. FASEB J. 1997; 11: 51-59Crossref PubMed Scopus (762) Google Scholar). The latent transforming growth factor β-binding proteins (LTBPs) are members of a family of ECM proteins that are key regulators of transforming growth factor-βs (TGFβs) (2Saharinen J. Hyytiainen M. Taipale J. Keski-Oja J. Cytokine Growth Factor Rev. 1999; 10: 99-117Crossref PubMed Scopus (251) Google Scholar, 3Sinha S. Nevett C. Shuttleworth C.A. Kielty C.M. Matrix Biol. 1998; 17: 529-545Crossref PubMed Scopus (85) Google Scholar, 4Hyytiainen M. Penttinen C. Keski-Oja J. Crit. Rev. Clin. Lab. Sci. 2004; 41: 233-264Crossref PubMed Scopus (280) Google Scholar). LTBP1, the prototype member of this family, regulates TGFβ at multiple levels. First, LTBP1 associates with latent TGFβ inside the cell and facilitates secretion of the latent complex (5Miyazono K. Olofsson A. Colosetti P. Heldin C.H. EMBO J. 1991; 10: 1091-1101Crossref PubMed Scopus (420) Google Scholar). LTBP1 then targets latent TGFβ to the ECM for storage (6Taipale J. Miyazono K. Heldin C.H. Keski-Oja J. J. Cell Biol. 1994; 124: 171-181Crossref PubMed Scopus (373) Google Scholar, 7Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (236) Google Scholar). LTBP1 may also provide a vehicle for release of the latent growth factor, following proteolytic cleavage of LTBP1 and release of C-terminal LTBP1 fragments, still bound to the latent TGFβ (7Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (236) Google Scholar, 8Dallas S.L. Rosser J.L. Mundy G.R. Bonewald L.F. J. Biol. Chem. 2002; 277: 21352-21360Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 9Taipale 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 (343) Google Scholar). Finally, there is also evidence that LTBP1 plays a role in activation of the latent TGFβ complex (10Annes J.P. Chen Y. Munger J.S. Rifkin D.B. J. Cell Biol. 2004; 165: 723-734Crossref PubMed Scopus (380) Google Scholar, 11Flaumenhaft R. Abe M. Sato Y. Miyazono K. Harpel J. Heldin C.H. Rifkin D.B. J. Cell Biol. 1993; 120: 995-1002Crossref PubMed Scopus (226) Google Scholar). LTBPs are members of a larger superfamily of matrix proteins that include fibrillins 1 and 2, the recently reported fibrillin 3 (12Corson G.M. Charbonneau N.L. Keene D.R. Sakai L.Y. Genomics. 2004; 83: 461-472Crossref PubMed Scopus (141) Google Scholar), and the latent TGFβ-binding proteins 1–4 (reviewed in Refs. 2Saharinen J. Hyytiainen M. Taipale J. Keski-Oja J. Cytokine Growth Factor Rev. 1999; 10: 99-117Crossref PubMed Scopus (251) Google Scholar, 3Sinha S. Nevett C. Shuttleworth C.A. Kielty C.M. Matrix Biol. 1998; 17: 529-545Crossref PubMed Scopus (85) Google Scholar, 13Koli K. Saharinen J. Hyytiainen M. Penttinen C. Keski-Oja J. Microsc. Res. Tech. 2001; 52: 354-362Crossref PubMed Scopus (226) Google Scholar, and 14Oklu R. Hesketh R. Biochem. J. 2000; 352: 601-610Crossref PubMed Scopus (154) Google Scholar). At least three of the LTBPs (LTBPs 1, 3, and 4) can form complexes with latent TGFβ1, whereas LTBP2 appears not to bind to latent TGFβ (15Saharinen J. Keski-Oja J. Mol. Biol. Cell. 2000; 11: 2691-2704Crossref PubMed Scopus (219) Google Scholar). LTBP1 is also secreted by many cell types in a free form that is not bound to TGFβ. The percentage of free LTBP1 varies from 10 to 90%, depending on the cell type and the differentiation stage examined (6Taipale J. Miyazono K. Heldin C.H. Keski-Oja J. J. Cell Biol. 1994; 124: 171-181Crossref PubMed Scopus (373) Google Scholar, 9Taipale 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 (343) Google Scholar, 16Dallas S.L. Park-Snyder S. Miyazono K. Twardzik D. Mundy G.R. Bonewald L.F. J. Biol. Chem. 1994; 269: 6815-6821Abstract Full Text PDF PubMed Google Scholar). These observations raise the possibility that LTBP1 may also have important functions that are independent of TGFβ and may be related to its properties as an ECM protein. At present, little is known about how LTBPs function as matrix proteins. Like the fibrillins, their primary structure consists predominantly of 6 cysteine (epidermal growth factor-like) repeats similar to the motifs found in the epidermal growth factor precursor and 8 cysteine repeats (termed "TB repeats") unique to the LTBPs and fibrillins. Recent studies have demonstrated that the third TB repeats in LTBPs 1, 3, and 4 are the sites for covalent binding of latent TGFβ (15Saharinen J. Keski-Oja J. Mol. Biol. Cell. 2000; 11: 2691-2704Crossref PubMed Scopus (219) Google Scholar, 17Chen Y. Ali T. Todorovic V. O'Leary J.M. Downing K.A. Rifkin D.B. J. Mol. Biol. 2005; 345: 175-186Crossref PubMed Scopus (47) Google Scholar). Immunolocalization studies have shown that LTBP1 co-localizes with fibrillin-1 in 10-nm microfibrils in the ECM of osteoblasts (18Dallas 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 (130) Google Scholar). LTBP1 has also been shown to co-localize with fibronectin (18Dallas 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 (130) Google Scholar, 19Taipale J. Saharinen J. Hedman K. Keski-Oja J. J. Histochem. Cytochem. 1996; 44: 875-889Crossref PubMed Scopus (192) Google Scholar, 20Unsold C. Hyytiainen M. Bruckner-Tuderman L. Keski-Oja J. J. Cell Sci. 2001; 114: 187-197Crossref PubMed Google Scholar), and a binding interaction between LTBP1 and fibronectin has been suggested by ligand blotting studies (19Taipale J. Saharinen J. Hedman K. Keski-Oja J. J. Histochem. Cytochem. 1996; 44: 875-889Crossref PubMed Scopus (192) Google Scholar). Fibronectin is required for the assembly of several ECM proteins, including type I collagen (21Velling T. Risteli J. Wennerberg K. Mosher D.F. Johansson S. J. Biol. Chem. 2002; 277: 37377-37381Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar), fibulin I (22Godyna S. Mann D.M. Argraves W.S. Matrix Biol. 1995; 14: 467-477Crossref PubMed Scopus (61) Google Scholar), thrombospondin (23Sottile J. Hocking D.C. Mol. Biol. Cell. 2002; 13: 3546-3559Crossref PubMed Scopus (449) Google Scholar), and fibrinogen (24Pereira M. Rybarczyk B.J. Odrljin T.M. Hocking D.C. Sottile J. Simpson-Haidaris P.J. J. Cell Sci. 2002; 115: 609-617Crossref PubMed Google Scholar). We therefore hypothesized that fibronectin may play a critical role in LTBP1 assembly into the ECM and may thereby play a role in regulation of TGFβ. Multiple approaches were used to disrupt fibronectin assembly, and the effect on LTBP1 and TGFβ incorporation was determined. These studies highlight a novel role for fibronectin in the regulation of TGFβ via LTBP1 interactions. Reagents—Human plasma fibronectin was purified as described previously (25Mosher D.F. Johnson R.B. Ann. N. Y. Acad. Sci. 1983; 408: 583-594Crossref PubMed Scopus (55) Google Scholar) or purchased from Invitrogen. The 70-kDa N-terminal and 160-kDa fibronectin fragments were prepared as described previously (26Peters D.M. Mosher D.F. Scanning Microsc. 1987; 1: 757-763PubMed Google Scholar). 30- and 40-kDa fibronectin fragments as well as superfibronectin were purchased from Sigma. The H120 recombinant fibronectin fragment was prepared as described previously (27Mould A.P. Askari J.A. Craig S.E. Garratt A.N. Clements J. Humphries M.J. J. Biol. Chem. 1994; 269: 27224-27230Abstract Full Text PDF PubMed Google Scholar). Fibronectin-stripped serum was prepared by passing the serum over a gelatin-Sepharose column as described elsewhere (28McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar). Antibodies—Antibodies against LTBP1 included a rabbit polyclonal antibody (Ab39; kindly supplied by K. Miyazono, Japanese Foundation for Cancer Research, Tokyo) (18Dallas 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 (130) Google Scholar, 29Kanzaki T. Olofsson A. Moren A. Wernstedt C. Hellman U. Miyazono K. Claesson-Welsh L. Heldin C.H. Cell. 1990; 61: 1051-1061Abstract Full Text PDF PubMed Scopus (369) Google Scholar). A rabbit polyclonal antibody against a peptide in rat LTBP1 was also used, which recognizes mouse and rat but not human LTBP1 (18Dallas 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 (130) Google Scholar). Fibronectin antibodies included a mouse monoclonal antibody (IgM) against cellular fibronectin (Sigma), a mouse monoclonal antibody (IgG) against the human fibronectin ED-A domain (Harlan), a mouse monoclonal antibody IgG (39B6) directed against type II repeats 12–14 of human fibronectin (30Mostafavi-Pour Z. Askari J.A. Whittard J.D. Humphries M.J. Matrix Biol. 2001; 20: 63-73Crossref PubMed Scopus (66) Google Scholar), and a rabbit polyclonal antibody against fibronectin purified from human platelets (Neomarkers, Freemont, CA). A peroxidase-conjugated donkey anti-rabbit antibody (Amersham Biosciences) was used for Western blotting and ELISA. All secondary antibodies for immunofluorescent staining were purchased from Jackson ImmunoResearch (West Grove, PA). Various combinations of fluorochrome-conjugated secondary antibodies were used as appropriate for each combination of primary antibodies, as stated in the figure legends. In some experiments, biotinylated secondary antibodies were used in conjunction with fluorescein isothiocyanate-conjugated streptavidin (Vector Laboratories, Burlingame, CA). Cell Culture—Tissue culture reagents were purchased from Invitrogen. Primary fetal rat calvarial (FRC) osteoblasts were prepared as described previously (7Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (236) Google Scholar, 18Dallas 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 (130) Google Scholar). UMR-106 cells were a gift from T. J. Martin (St. Vincent Institute of Medical Research, Fitzroy, Victoria, Australia) and were maintained in Dulbecco's modified Eagle's medium supplemented with 2 mm l-glutamine, 100 units/ml penicillin/streptomycin, and 10% fetal bovine serum. FN-null (FN–/–) and heterozygous (FN+/–) embryonic fibroblasts were derived as described elsewhere (31Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2805-2810Crossref PubMed Scopus (336) Google Scholar). For experiments examining LTBP1 assembly in FN-null fibroblasts, the cells were cultured in the presence of FN-stripped serum with or without the addition of 10 μg/ml plasma fibronectin. For growth curve experiments, cells were plated in 96-well plates at 20,000 cells/cm2. Cells were trypsinized at the specified time points, and cell number was determined by using a Coulter counter (Beckman Coulter Inc.). Immunocytochemistry—For immunocytochemistry, cells were plated in Lab-Tek chamber slides at 20,000 cells/cm2 in Dulbecco's modified Eagle's medium supplemented with 10% FN-stripped fetal bovine serum, 2 mm l-glutamine, and 100 units/ml penicillin/streptomycin with or without 10 μg/ml plasma fibronectin. At confluence, the media were changed to Dulbecco's modified Eagle's medium supplemented with 5% FN-stripped fetal bovine serum, 50 μg/ml ascorbic acid, and other additives as above, with or without the addition of fibronectin or fibronectin fragments. The cells were cultured for the specified periods, with media changed every 3 days. Co-localization of LTBP1, fibronectin, and type I collagen was performed using double staining indirect immunofluorescence techniques as described previously (18Dallas 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 (130) Google Scholar). The specimens were viewed and photographed digitally using a Leica DMRXA microscope with epifluorescence illumination and a Photometric Cool-snap FX CCD camera. For double labeling experiments, multiple controls were performed, including using nonimmune serum or control IgG in place of primary antibody, testing each primary antibody individually using both secondary antibodies to exclude cross-species reactivity of the secondary antibodies, and preincubating the primary antibody with the immunizing peptide (where available). TGFβ ELISA and Modified ELISA for Detection of Relative Amounts of LTBP1—A commercial ELISA (TGFβ1 Emax; Promega Corp., Madison, WI) was used for measurement of TGFβ1 in conditioned media samples collected over 48 h or in guanidine HCl extracts of the ECM, prepared as described elsewhere (32Pfeilschifter J. Erdmann J. Storch S. Ziegler R. Weinreb M. Calcif. Tissue Int. 1999; 64: 78-82Crossref PubMed Scopus (12) Google Scholar). Values were normalized to cell number. For measurement of total (active + latent) TGFβ, the samples were acidified using HCl and then reneutralized prior to measurement using NaOH according to the ELISA manufacturer's instructions. To quantify the relative amounts of LTBP1 in the ECM, a modified ELISA was used. Cells were grown in 96-well plates in media with or without fibronectin. The plates were fixed in 95% ethanol, then blocked with 5% bovine serum albumin + 1% milk, followed by incubation in primary antibodies against LTBP1. After washing, the amount of bound LTBP1 antibody was determined using the Vectorstain-Elite ABC immunodetection kit (Vector Laboratories, Burlingame, CA) in conjunction with O-phenylenediamine as a reaction substrate. Alternatively, the vector VIP substrate kit was used, followed by solubilization of the reaction product by incubating for 10 min at room temperature in 50 μl of 2 m KCl, then adding 50 μl of Me2SO and incubating for a further 10 min. The plates were read on an ELX 800 plate reader (Bio-Tek Instruments, Winooski, VT) at 450 nm, and background subtraction was performed using a blank control that had been incubated with nonspecific IgG in place of primary antibody. Values were normalized to total protein content of the cell lysate or to cell number as determined using a Coulter counter. Immunoelectron Microscopy—For immunoelectron microscopy, FN-null and heterozygous fibroblasts were cultured on Thermanox coverslips (Nalge Nunc International) in media with or without added fibronectin as described above. Media were changed every 3 days, and coverslips were harvested on days 6, 14, and 21. Immunogold staining was performed on unfixed specimens as described previously (18Dallas 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 (130) Google Scholar) using anti-LTBP1 and anti-fibronectin antibodies each diluted 1:10. For detection, a donkey anti-rabbit secondary antibody-6-nm gold conjugate and a donkey anti-mouse secondary antibody-18-nm gold conjugate were used (Jackson ImmunoResearch). The samples were fixed after immunogold staining and processed for transmission electron microscopy as described previously (18Dallas 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 (130) Google Scholar). They were then embedded in Taab epoxy resin (Taab Laboratories Equipment Ltd., Aldermaston, UK). After polymerization, the coverslips were removed to leave the cell layer exposed on the outer surface of the block. Ultrathin sections were cut at a slight angle to the growth substrate by using a diamond knife. Sections were examined with or without contrast in a Philips EM 301 transmission electron microscope at an accelerating voltage of 60 kV. LTBP1 Stably Expressing Cell Lines—To generate UMR-106 cell lines stably overexpressing LTBP1, a full-length human LTBP1 construct in the PSV7d vector (kindly provided by K. Miyazono, Japanese Foundation for Cancer Research, Tokyo) was co-transfected at a 10:1 ratio with an RSVneo selection vector, using calcium phosphate precipitation as described elsewhere (33Chen D. Harris M.A. Rossini G. Dunstan C.R. Dallas S.L. Feng J.Q. Mundy G.R. Harris S.E. Calcif. Tissue Int. 1997; 60: 283-290Crossref PubMed Scopus (204) Google Scholar). The transfected cells were selected with 400 μg/ml G418, and resistant single cell clones were screened for LTBP1 expression using an ELISA, as described previously (7Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (236) Google Scholar). Three high expressing clones (0.2–1.5 μg/ml) were selected for further study. Control clones transfected with RSVneo alone were used for comparison. FPLC Analysis—FPLC fractionation was performed as described previously (16Dallas S.L. Park-Snyder S. Miyazono K. Twardzik D. Mundy G.R. Bonewald L.F. J. Biol. Chem. 1994; 269: 6815-6821Abstract Full Text PDF PubMed Google Scholar) using 48-h serum-free, phenol red-free conditioned media harvested from 90–95% confluent cultures. 150 ml of conditioned medium was concentrated 10-fold over a 50-kDa cut-off membrane using a minisette concentrator (Millipore, Bedford, MA). The samples were then lyophilized, reconstituted, and dialyzed against 20 mm Tris buffer, pH 7.2, prior to application over an analytical Mono-Q anion exchange column (Amersham Biosciences). The column was eluted with a linear gradient of 0–0.5 m NaCl, 20 mm Tris buffer. Fractions were tested for TGFβ activity using the alkaline phosphatase microassay as described previously (16Dallas S.L. Park-Snyder S. Miyazono K. Twardzik D. Mundy G.R. Bonewald L.F. J. Biol. Chem. 1994; 269: 6815-6821Abstract Full Text PDF PubMed Google Scholar). Metabolic Labeling and Immunoprecipitation—For metabolic labeling and immunoprecipitation, cells were plated into 12-well multiwell plates at 10,000 cells/cm2 growth area. At 90% confluence, the cells were metabolically labeled as described previously (8Dallas S.L. Rosser J.L. Mundy G.R. Bonewald L.F. J. Biol. Chem. 2002; 277: 21352-21360Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar) by using 100 μCi/well [35S]cysteine for 6 h. LTBP1 was measured in the culture supernatants as well as the ECM by immunoprecipitation followed by SDS-PAGE and autoradiography as described previously (8Dallas S.L. Rosser J.L. Mundy G.R. Bonewald L.F. J. Biol. Chem. 2002; 277: 21352-21360Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 16Dallas S.L. Park-Snyder S. Miyazono K. Twardzik D. Mundy G.R. Bonewald L.F. J. Biol. Chem. 1994; 269: 6815-6821Abstract Full Text PDF PubMed Google Scholar). A plasmin digestion was used to release the ECM-bound LTBP1 (7Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (236) Google Scholar, 19Taipale J. Saharinen J. Hedman K. Keski-Oja J. J. Histochem. Cytochem. 1996; 44: 875-889Crossref PubMed Scopus (192) Google Scholar). Western Blotting—Proteins in concentrated conditioned media samples or plasmin matrix digests were separated by SDS-PAGE using 4–20% gradient polyacrylamide mini gels, and immunoblotting was performed as described previously (7Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (236) Google Scholar). The immunostained bands were visualized using the Renaissance ECL detection system according to manufacturer's instructions (PerkinElmer Life Sciences). Samples were normalized to total protein content or to cell number prior to loading on the gels. Time Course of LTBP1 and Fibronectin Assembly in Primary Osteoblast Cultures—To determine the time course of LTBP1 and fibronectin assembly, immunolocalization studies were performed over a time course of 1–21 days (Fig. 1). Fibronectin initially appeared as short fibrillar structures on the surface of fetal rat calvarial cells after 24 h in culture (Fig. 1, d1). At this stage, no immunoreactive LTBP1 was present on the cell surface or in the extracellular matrix. By 2 days in culture (Fig. 1, d2), the fibronectin became organized into a fibrillar network in the ECM, and a small amount of LTBP1 was observed that co-localized with fibronectin. By 3 and 5 days of culture (Fig. 1, d3 and d5), LTBP1 was localized in a fibrillar network that co-distributed with fibronectin. The incorporation of LTBP1 therefore lagged 1–2 days behind that of fibronectin, and there was always a proportion of fibronectin-positive fibrils that did not stain for LTBP1. Between days 5 and 21, there was an increase in formation of LTBP1 fibrils, which became organized into long parallel fibrillar arrays. At the same time, there was a progression from distinct fibronectin fibrils to fibrils with a more diffuse appearance. The co-localization of LTBP1 and fibronectin started to diverge until day 21, when LTBP1 became localized in fibrils that were clearly distinct from fibronectin. Identical results were obtained by using the human MG63 osteosarcoma cell line as well as with human skin fibroblasts and using two independent antibodies for LTBP1 and fibronectin (data not shown). Double-labeled immunogold localization for LTBP1 and fibronectin demonstrated co-localization of these two molecules in fibrillar structures in 6-day fetal rat calvarial cell cultures (Fig. 2). In later (14-day) cultures, some fibrils could be found that were labeled with both antibodies; however, LTBP1 was also frequently found in bundles of 10-nm microfibrils that were not labeled with fibronectin antibodies (Fig. 2). Inhibition of LTBP1 and TGFβ1 Incorporation by a 70-kDa N-terminal Fibronectin Fragment—To determine whether fibronectin was required for assembly of LTBP1 into bone ECM, fetal rat calvarial cell cultures were treated for 6 days with a 70-kDa N-terminal fibronectin fragment (Fig. 3a). This fragment blocks assembly of both endogenous and exogenous fibronectin in fibroblasts by occupying binding sites that are required for fibronectin self-association (28McKeown-Longo P.J. Mosher D.F. J. Cell Biol. 1985; 100: 364-374Crossref PubMed Scopus (247) Google Scholar). Treatment with 70 μg/ml of the fibronectin fragment (Fig. 3a, 70K) resulted in a dramatic reduction in fibronectin incorporation compared with untreated controls (control) or control cultures treated with plasma fibronectin (pFN). This was paralleled by a reduction in LTBP1 incorporation into the ECM. Dose-response experiments showed a significant reduction in LTBP1 incorporation at doses between 10 and 70 μg/ml, as determined by immunofluorescence (data not shown) and also by quantitation of the relative amounts of incorporated LTBP1 using a modified ELISA (Fig. 3b). Time course experiments indicated that at early time points (days 1–3) there was a virtual absence of both fibronectin and LTBP1 in the ECM of cells treated with the fibronectin 70-kDa fragment. However, by day 5, fibronectin and LTBP1 incorporation began to partially recover. This recovery of LTBP1 incorporation appeared to be due to the fact that even the highest dose of the 70-kDa fibronectin fragment failed to completely block fibronectin incorporation. The above results were replicated by using an LTBP1 antibody that recognizes the proline-rich hinge region and using MG63 human osteosarcoma cells (data not shown). TGFβ 1 is the major TGFβ isoform produced in bone (7Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (236) Google Scholar, 34Seyedin 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). To determine whether inhibition of fibronectin assembly also affected incorporation of TGFβ into the ECM, the amount of TGFβ1 in the ECM was measured using a commercial ELISA (Fig. 3c). Treatment of fetal rat calvarial cells with the 70-kDa fibronectin fragment reduced the amount of latent TGFβ1 stored in the ECM by 80%. This was not because of reduced synthesis and secretion of TGFβ as increased amounts of latent TGFβ were detected in the conditioned media of the treated cultures (Fig. 3c). LTBP1 Assembly in Fibronectin-null Embryonic Fibroblasts—To define further the role of fibronectin in the assembly of LTBP1 into the ECM, we examined LTBP1 assembly in fibroblasts differentiated from mouse embryonic stem cells that were null for the fibronectin gene (FN-null) (Fig. 4). Both heterozygous (+/–) and FN-null (–/–) embryonic fibroblasts synthesized and secreted LTBP1 in amounts that were grossly equivalent to the amounts produced by fetal rat calvarial cells. 2S. L. Dallas, unpublished observations.Fig. 4a shows double-stained immunolocalization of LTBP1 and fibronectin in the ECM of 6-day cultures of FN-null fibroblasts (–/–), as compared with heterozygous controls (+/–). The cells were cultured in fibronectin-stripped serum to ensure that no exogenous fibronectin was present. In control cells, a network of fibronectin fibrils was present in the ECM, and LTBP1 was found to co-distribute with this network. In contrast, no fibronectin was detected in the ECM of FN-null cells, and this was associated with a failure in LTBP1 incorporation. Western analysis (Fig. 4b) indicated that this effect was not because of reduced expression of LTBP1 in the FN-null cells, as they secreted a large amount of LTBP1 into the conditioned media (Fig. 4b, black arrow) but failed to incorporate it into the ECM (Fig. 4b, gray arrow). In contrast, the heterozygous cells had lower amounts of LTBP1 in the conditioned media, with the major proportion in t

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