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Potential Role for Heparan Sulfate Proteoglycans in Regulation of Transforming Growth Factor-β (TGF-β) by Modulating Assembly of Latent TGF-β-binding Protein-1

2007; Elsevier BV; Volume: 282; Issue: 36 Linguagem: Inglês

10.1074/jbc.m703341200

ISSN

1083-351X

Autores

Qian Chen, Pitchumani Sivakumar, Craig Barley, Donna M. Peters, Ronald R. Gomes, Mary C. Farach‐Carson, Sarah L. Dallas,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Latent transforming growth factor-β-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in storage of latent TGF-β in the ECM and regulate its availability. We have previously identified fibronectin as a key molecule for incorporation of LTBP1 and TGF-β into the ECM of osteoblasts and fibroblasts. Here we provide evidence that heparan sulfate proteoglycans may mediate binding between LTBP1 and fibronectin. We have localized critical domains in the N terminus of LTBP1 that are required for co-localization with fibronectin in osteoblast cultures and have identified heparin binding sites in the N terminus of LTBP1 between residues 345 and 487. Solid-phase binding assays suggest that LTBP1 does not bind directly to fibronectin but that the binding is indirect. Heparin coupled to bovine serum albumin (heparin-BSA) was able to mediate binding between fibronectin and LTBP1. Treatment of primary osteoblast cultures with heparin or heparin-BSA but not with chondroitin sulfate impaired LTBP1 deposition onto fibronectin without inhibiting expression of LTBP1. Inhibition of LTBP1 incorporation was accompanied by reduced incorporation of latent TGF-β into the ECM, with increased amounts of soluble latent TGF-β. Inhibition of attachment of glycosaminoglycans to the core proteins of proteoglycans by β-d-xylosides also reduced incorporation of LTBP1 into the ECM. These studies suggest that heparan sulfate proteoglycans may play a critical role in regulating TGF-β availability by controlling the deposition of LTBP1 into the ECM in association with fibronectin. Latent transforming growth factor-β-binding proteins (LTBPs) are extracellular matrix (ECM) glycoproteins that play a major role in storage of latent TGF-β in the ECM and regulate its availability. We have previously identified fibronectin as a key molecule for incorporation of LTBP1 and TGF-β into the ECM of osteoblasts and fibroblasts. Here we provide evidence that heparan sulfate proteoglycans may mediate binding between LTBP1 and fibronectin. We have localized critical domains in the N terminus of LTBP1 that are required for co-localization with fibronectin in osteoblast cultures and have identified heparin binding sites in the N terminus of LTBP1 between residues 345 and 487. Solid-phase binding assays suggest that LTBP1 does not bind directly to fibronectin but that the binding is indirect. Heparin coupled to bovine serum albumin (heparin-BSA) was able to mediate binding between fibronectin and LTBP1. Treatment of primary osteoblast cultures with heparin or heparin-BSA but not with chondroitin sulfate impaired LTBP1 deposition onto fibronectin without inhibiting expression of LTBP1. Inhibition of LTBP1 incorporation was accompanied by reduced incorporation of latent TGF-β into the ECM, with increased amounts of soluble latent TGF-β. Inhibition of attachment of glycosaminoglycans to the core proteins of proteoglycans by β-d-xylosides also reduced incorporation of LTBP1 into the ECM. These studies suggest that heparan sulfate proteoglycans may play a critical role in regulating TGF-β availability by controlling the deposition of LTBP1 into the ECM in association with fibronectin. Interaction between growth factors and extracellular matrix (ECM) 2The abbreviations used are: ECMextracellular matrixFRCfetal rat calvarial cellsLTBP1latent transforming growth factor-β-binding protein-1TGF-βtransforming growth factor-βEGFepidermal growth factorHSPGheparan sulfate proteoglycanaaamino acid(s)ELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineBSAbovine serum albuminCSchondroitin 6-sulfate.2The abbreviations used are: ECMextracellular matrixFRCfetal rat calvarial cellsLTBP1latent transforming growth factor-β-binding protein-1TGF-βtransforming growth factor-βEGFepidermal growth factorHSPGheparan sulfate proteoglycanaaamino acid(s)ELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineBSAbovine serum albuminCSchondroitin 6-sulfate. molecules may be a major mechanism for regulation of growth factor activity (1Taipale J. Saharinen J. Keski-Oja J. Adv. Cancer Res. 1998; 75: 87-134Crossref PubMed Google Scholar, 2Charbonneau N.L. Ono R.N. Corson G.M. Keene D.R. Sakai L.Y. Birth Defects Res. C Embryo Today. 2004; 72: 37-50Crossref PubMed Scopus (88) Google Scholar, 3Ramirez F. Sakai L.Y. Dietz H.C. Rifkin D.B. Physiol. Genomics. 2004; 19: 151-154Crossref PubMed Scopus (99) Google Scholar). The latent transforming growth factor-β-binding proteins (LTBPs) are a family of ECM glycoproteins that are key regulators of transforming growth factor-βs (TGF-βs) (4Hyytiainen M. Penttinen C. Keski-Oja J. Crit. Rev. Clin. Lab. Sci. 2004; 41: 233-264Crossref PubMed Scopus (273) Google Scholar, 5Annes J.P. Munger J.S. Rifkin D.B. J. Cell Sci. 2003; 116: 217-224Crossref PubMed Scopus (1309) Google Scholar, 6Saharinen J. Hyytiainen M. Taipale J. Keski-Oja J. Cytokine Growth Factor Rev. 1999; 10: 99-117Crossref PubMed Scopus (247) Google Scholar). These multifunctional growth factors have potent effects in multiple cell types and have been implicated in several human diseases, including cancer (reviewed in Ref. 7Blobe G.C. Schiemann W.P. Lodish H.F. N. Engl. J. Med. 2000; 342: 1350-1358Crossref PubMed Scopus (2169) Google Scholar). extracellular matrix fetal rat calvarial cells latent transforming growth factor-β-binding protein-1 transforming growth factor-β epidermal growth factor heparan sulfate proteoglycan amino acid(s) enzyme-linked immunosorbent assay phosphate-buffered saline bovine serum albumin chondroitin 6-sulfate. extracellular matrix fetal rat calvarial cells latent transforming growth factor-β-binding protein-1 transforming growth factor-β epidermal growth factor heparan sulfate proteoglycan amino acid(s) enzyme-linked immunosorbent assay phosphate-buffered saline bovine serum albumin chondroitin 6-sulfate. TGF-βs are produced by virtually all cells as one or more latent complexes, which must be activated in order for TGF-β to exert its biological activities (reviewed in Refs. 5Annes J.P. Munger J.S. Rifkin D.B. J. Cell Sci. 2003; 116: 217-224Crossref PubMed Scopus (1309) Google Scholar and 8Miyazono K. Heldin C.H. Ciba Found. Symp. 1991; 157: 81-89PubMed Google Scholar). The major secreted forms of latent TGF-β are known as the small and large latent TGF-β complexes (6Saharinen J. Hyytiainen M. Taipale J. Keski-Oja J. Cytokine Growth Factor Rev. 1999; 10: 99-117Crossref PubMed Scopus (247) Google Scholar, 9Kanzaki 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 (368) Google Scholar, 10Miyazono K. Olofsson A. Colosetti P. Heldin C.H. EMBO J. 1991; 10: 1091-1101Crossref PubMed Scopus (420) Google Scholar). The small latent complex consists of mature TGF-β, noncovalently bound to its latency-associated peptide. In the large latent TGF-β complexes, TGF-β and its latency-associated peptide are associated with a third protein from the family of LTBPs, of which four members (LTBPs 1–4) have been identified (reviewed in Refs. 4Hyytiainen M. Penttinen C. Keski-Oja J. Crit. Rev. Clin. Lab. Sci. 2004; 41: 233-264Crossref PubMed Scopus (273) Google Scholar and 6Saharinen J. Hyytiainen M. Taipale J. Keski-Oja J. Cytokine Growth Factor Rev. 1999; 10: 99-117Crossref PubMed Scopus (247) Google Scholar). Several studies have shown that LTBPs are major regulators of TGF-β. LTBP1 has been shown to facilitate secretion of latent TGF-β (10Miyazono K. Olofsson A. Colosetti P. Heldin C.H. EMBO J. 1991; 10: 1091-1101Crossref PubMed Scopus (420) Google Scholar), to target latent TGF-β to ECM for storage (11Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (233) Google Scholar, 12Taipale J. Miyazono K. Heldin C.H. Keski-Oja J. J. Cell Biol. 1994; 124: 171-181Crossref PubMed Scopus (373) Google Scholar), and LTBP1 cleavage may provide a mechanism for release of the latent TGF-β from ECM (13Taipale 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 (342) Google Scholar, 14Dallas 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 (310) Google Scholar). LTBPs may also be critical players in the activation of TGF-β by specific cell types (15Flaumenhaft R. Abe M. Sato Y. Miyazono K. Harpel J. Heldin C.H. Rifkin D.B. J. Cell Biol. 1993; 120: 995-1002Crossref PubMed Scopus (224) Google Scholar, 16Annes J.P. Chen Y. Munger J.S. Rifkin D.B. J. Cell Biol. 2004; 165: 723-734Crossref PubMed Scopus (372) Google Scholar). Interestingly, LTBP3-null mice showed craniofacial malformations and developed osteosclerosis and osteoarthritis (17Dabovic B. Chen Y. Colarossi C. Obata H. Zambuto L. Perle M.A. Rifkin D.B. J. Cell Biol. 2002; 156: 227-232Crossref PubMed Scopus (126) Google Scholar). The LTBP4-null mice showed pulmonary emphysema, cardiomyopathy, and colorectal cancer (18Sterner-Kock A. Thorey I.S. Koli K. Wempe F. Otte J. Bangsow T. Kuhlmeier K. Kirchner T. Jin S. Keski-Oja J. von Melchner H. Genes Dev. 2002; 16: 2264-2273Crossref PubMed Scopus (213) Google Scholar). In both cases, the phenotypes appear to be due to misregulation of TGF-β activity (17Dabovic B. Chen Y. Colarossi C. Obata H. Zambuto L. Perle M.A. Rifkin D.B. J. Cell Biol. 2002; 156: 227-232Crossref PubMed Scopus (126) Google Scholar, 18Sterner-Kock A. Thorey I.S. Koli K. Wempe F. Otte J. Bangsow T. Kuhlmeier K. Kirchner T. Jin S. Keski-Oja J. von Melchner H. Genes Dev. 2002; 16: 2264-2273Crossref PubMed Scopus (213) Google Scholar). LTBPs share homology with fibrillins, and it is now clear that the LTBPs 1–4 and fibrillins 1–3 constitute a superfamily of ECM proteins with overlapping and interacting functions (reviewed in Ref. 4Hyytiainen M. Penttinen C. Keski-Oja J. Crit. Rev. Clin. Lab. Sci. 2004; 41: 233-264Crossref PubMed Scopus (273) Google Scholar). Similar to the fibrillins, 60–70% of the structure of LTBPs consists mainly of two types of cysteine-rich repeats. These include epidermal growth factor (EGF)-like six-cysteine repeats, similar to motifs found in the EGF precursor, and eight-cysteine repeats that are unique to the LTBPs and fibrillins. The LTBPs contain 16–18 of the EGF-like repeats and 3–4 copies of the 8-cysteine repeats. The third 8-cysteine repeat of LTBP1 is critical for interaction with latent TGF-β (19Saharinen J. Taipale J. Keski-Oja J. EMBO J. 1996; 15: 245-253Crossref PubMed Scopus (186) Google Scholar, 20Gleizes P.E. Beavis R.C. Mazzieri R. Shen B. Rifkin D.B. J. Biol. Chem. 1996; 271: 29891-29896Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). A heparin binding consensus sequence (HRRRPIHHHVGK) is found in the "hinge" region of human LTBP1 at amino acids 414–425 (21Oklu R. Metcalfe J.C. Hesketh T.R. Kemp P.R. FEBS Lett. 1998; 425: 281-285Crossref PubMed Scopus (27) Google Scholar). At present, little is known concerning the mechanisms by which LTBP1 assembles into the ECM. Several studies suggest that the N terminus of LTBP1 contains a critical region for matrix binding (19Saharinen J. Taipale J. Keski-Oja J. EMBO J. 1996; 15: 245-253Crossref PubMed Scopus (186) Google Scholar, 22Dallas 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 (129) Google Scholar, 23Unsold C. Hyytiainen M. Bruckner-Tuderman L. Keski-Oja J. J. Cell Sci. 2001; 114: 187-197Crossref PubMed Google Scholar, 24Nunes I. Gleizes P.E. Metz C.N. Rifkin D.B. J. Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (346) Google Scholar). A binding region in the C terminus was also identified (23Unsold C. Hyytiainen M. Bruckner-Tuderman L. Keski-Oja J. J. Cell Sci. 2001; 114: 187-197Crossref PubMed Google Scholar). LTBP1 and fibrillin-1 co-localize in the matrix of early and late osteoblast cell cultures (22Dallas 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 (129) Google Scholar), and LTBP1 has been immunolocalized to fibrillin-containing microfibrils in the skin (25Raghunath M. Unsold C. Kubitscheck U. Bruckner-Tuderman L. Peters R. Meuli M. J. Invest. Dermatol. 1998; 111: 559-564Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and bone (11Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (233) Google Scholar, 22Dallas 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 (129) Google Scholar) as well as to microfibrillar structures in the heart (26Nakajima Y. Miyazono K. Kato M. Takase M. Yamagishi T. Nakamura H. J. Cell Biol. 1997; 136: 193-204Crossref PubMed Scopus (106) Google Scholar). LTBP1 also shows a time-dependent co-localization with fibronectin in osteoblast culture models, where co-localization is seen in early postconfluent cultures (1–3 days) but not in late cultures (14–21 days) (27Dallas S.L. Sivakumar P. Jones C.J. Chen Q. Peters D.M. Mosher D.F. Humphries M.J. Kielty C.M. J. Biol. Chem. 2005; 280: 18871-18880Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Binding studies have demonstrated direct interactions between LTBP1 and fibrillins (28Isogai Z. Ono R.N. Ushiro S. Keene D.R. Chen Y. Mazzieri R. Charbonneau N.L. Reinhardt D.P. Rifkin D.B. Sakai L.Y. J. Biol. Chem. 2003; 278: 2750-2757Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). However, by overexpressing LTBP1 in UMR-106 cells that lack endogenous LTBP1 or fibrillin-1, we previously showed that fibrillin-1 was not required for LTBP1 deposition into the ECM in association with fibronectin (27Dallas S.L. Sivakumar P. Jones C.J. Chen Q. Peters D.M. Mosher D.F. Humphries M.J. Kielty C.M. J. Biol. Chem. 2005; 280: 18871-18880Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Fibronectin has been shown to be required for assembly of several matrix proteins, including type I collagen, thrombospondin, fibulin-1, and fibrinogen (29Sottile J. Hocking D.C. Mol. Biol. Cell. 2002; 13: 3546-3559Crossref PubMed Scopus (435) Google Scholar, 30Godyna S. Mann D.M. Argraves W.S. Matrix Biol. 1995; 14: 467-477Crossref PubMed Scopus (61) Google Scholar, 31Pereira 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). Our recent studies have shown that fibronectin also plays a critical role for LTBP1 assembly into the ECM (27Dallas S.L. Sivakumar P. Jones C.J. Chen Q. Peters D.M. Mosher D.F. Humphries M.J. Kielty C.M. J. Biol. Chem. 2005; 280: 18871-18880Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). However, the mechanism by which fibronectin regulates LTBP1 assembly is still unclear. In these studies, we sought to determine whether LTBP1 binds directly to fibronectin and to localize the critical domains in LTBP1 that are required for LTBP1 deposition in association with fibronectin. Solid-phase binding assays suggested that fibronectin interacted with LTBP1 through an indirect mechanism. Because fibronectin contains heparin binding sites (32Dalton B.A. McFarland C.D. Underwood P.A. Steele J.G. J. Cell Sci. 1995; 108: 2083-2092Crossref PubMed Google Scholar) and LTBP1 contains a consensus heparin binding site, and because fibrillins have been shown to be dependent on heparan sulfate proteoglycans (HSPGs) for their incorporation (33Tiedemann K. Batge B. Muller P.K. Reinhardt D.P. J. Biol. Chem. 2001; 276: 36035-36042Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 34Ritty T.M. Broekelmann T.J. Werneck C.C. Mecham R.P. Biochem. J. 2003; 375: 425-432Crossref PubMed Scopus (67) Google Scholar), we examined the potential role of HSPGs in mediating interactions between LTBP1 and fibronectin. Antibodies and Purified Proteins—Antibodies for LTBP1 included an affinity-purified rabbit polyclonal against rat LTBP1 generated in our laboratory (22Dallas 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 (129) Google Scholar) (for immunostaining this antibody recognizes rat and mouse LTBP1 but not human) and a rabbit polyclonal against human LTBP1 (Ab39) that cross-reacts with multiple species (a kind gift of Dr. C. H. Heldin, Ludwig Institute for Cancer Research, Uppsala, Sweden) (10Miyazono K. Olofsson A. Colosetti P. Heldin C.H. EMBO J. 1991; 10: 1091-1101Crossref PubMed Scopus (420) Google Scholar). A monoclonal antibody against human LTBP1 was also used (R&D Systems, Minneapolis, MN). Our laboratory has also recently developed a rabbit polyclonal antibody (Ab-K) against a recombinant human LTBP1 fragment (aa 526–1014) (Fig. 1A). This antibody recognizes LTBP1 in human, mouse, and rat cells. Antibodies for fibronectin included a commercially available mouse monoclonal against the ED-A domain (Sigma) that cross-reacts with human, mouse, and rat fibronectin and a rabbit polyclonal antibody that also recognizes human, mouse, and rat fibronectin (Sigma). For detection of His-tagged recombinant LTBP1 fragments, an anti-His monoclonal antibody was used (EMD Biosciences, Madison, WI). Human plasma fibronectin and N-terminal 70-kDa proteolytic fragment were obtained from commercial sources (Invitrogen or Sigma). The 160-kDa fibronectin fragment was prepared as described previously (35Peters D.M. Mosher D.F. J. Cell Biol. 1987; 104: 121-130Crossref PubMed Scopus (63) Google Scholar). The H120 fibronectin fragment, consisting of type III repeats 12–15 (36Mould 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), was provided by Dr. Martin Humphries (University of Manchester, United Kingdom) (Fig. 4C shows a schematic diagram of these fibronectin fragments). HSPG (Perlecan) was purchased from Sigma, and purified perlecan domain I was produced as described elsewhere (37Yang W.D. Jr. Gomes R.R. Alicknavitch M. Farach-Carson M.C. Carson D.D. Tissue Eng. 2005; 11: 76-89Crossref PubMed Scopus (51) Google Scholar). Cell Culture—Tissue culture reagents were purchased from Invitrogen or Mediatech Inc. (Herndon, VA). 293-EBNA cells were purchased from the American Type Tissue Culture Collection (Manassas, VA) and were routinely maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 2 mm l-glutamine, 100 units/ml penicillin/streptomycin, 250 μg/ml G418. 2T3 cells were a kind gift of Dr. S. E. Harris (University of Texas Health Science Center at San Antonio, TX). These cells were maintained as described previously (38Ghosh-Choudhury N. Windle J.J. Koop B.A. Harris M.A. Guerrero D.L. Wozney J.M. Mundy G.R. Harris S.E. Endocrinology. 1996; 137: 331-339Crossref PubMed Scopus (82) Google Scholar). Primary cultures of fetal rat calvarial osteoblasts (FRCs) were isolated and maintained as described previously (11Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (233) Google Scholar). TMLC-C32 cells were a kind gift of Dr. D. B. Rifkin (New York University, New York, NY) and were cultured as described previously (11Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (233) Google Scholar, 39Abe M. Oda N. Sato Y. J. Cell. Physiol. 1998; 174: 186-193Crossref PubMed Scopus (66) Google Scholar). Expression and Purification of Recombinant LTBP1 Peptides—For expression and purification of recombinant LTBP1 peptides, cDNA sequences corresponding to the desired recombinant LTBP1 fragments were generated by PCR amplification using Vent DNA polymerase, a high fidelity DNA polymerase, according to the manufacturer's instructions (New England Biolabs, Beverly, MA). The template was a human LTBP1 cDNA in the vector pSV7d (10Miyazono K. Olofsson A. Colosetti P. Heldin C.H. EMBO J. 1991; 10: 1091-1101Crossref PubMed Scopus (420) Google Scholar) (kindly donated by Dr. K. Miyazono, University of Tokyo, Tokyo, Japan). A 6- or 10-histidine epitope tag was engineered into the primers at the C terminus of the recombinant LTBP1 fragments, followed by a stop codon and XhoI restriction site. An NheI restriction site was engineered at the N terminus. Schematic diagrams of the LTBP1 constructs generated are shown in Figs. 1A and 3A. The primer sets used for amplification of these specific fragments are presented in supplemental Table S1. The PCR products were digested with NheI and XhoI and ligated into the pCEP-Pu expression vector (a kind gift of E. Kohfeldt, Max Planck Institut of Biochemistry, Martinsried, Germany) in-frame with the BM40 signal sequence (40Mayer U. Nischt R. Poschl E. Mann K. Fukuda K. Gerl M. Yamada Y. Timpl R. EMBO J. 1993; 12: 1879-1885Crossref PubMed Scopus (246) Google Scholar, 41Kohfeldt E. Maurer P. Vannahme C. Timpl R. FEBS Lett. 1997; 414: 557-561Crossref PubMed Scopus (201) Google Scholar). The sequences of the inserts were confirmed by automated sequencing (MWG Co., High Point, NC). The constructs were then transfected into 293-EBNA cells using the Lipofectamine 2000 reagent according to the manufacturer's instructions (Invitrogen). Transfected cells were selected in puromycin (1 μg/ml), and resistant cells were expanded into triple layer flasks. Recombinant fragments were purified from 2–3 liters of serum-free conditioned media using a nickel-nitrilotriacetic acid-agarose column according to the manufacturer's instructions (Qiagen). Bound proteins were eluted either with low pH or with 100–300 mm imidazole. If further purification was required, a mono-Q ion exchange column was used in conjunction with a Bio Cad 700E protein purification system (Applied Biosystems, Foster City, CA). Bound proteins were eluted with a linear 0–1 m NaCl gradient. Coomassie Blue staining was used to visualize the purity of the fragments, and mass spectrometry/peptide mass mapping was used to validate the recombinant LTBP1 fragments. Using this approach, we have obtained 1–2 mg of most of the recombinant LTBP1 fragments (see Figs. 1C and 3B, Coomassie Blue staining). 2T3/293-EBNA Co-culture System and Transfection of LTBP1 Constructs—A 2T3/293-EBNA co-culture system was used for transient in vitro expression of human LTBP1 constructs to determine their ability to deposit into the ECM in association with fibronectin. This co-culture system combines the advantages of the 293-EBNA cells, which express high amounts of recombinant proteins but do not produce an extensive extracellular matrix, with the 2T3 calvarial osteoblast cell line, which has a low transfection efficiency but produces an extensive ECM. 2T3 cells at 4 × 104 cells/ml and 293-EBNA cells at 4 × 103 cells/ml were plated together into eight-chamber Lab-Tek chamber slides in Dulbecco's modified Eagle's medium supplemented as described above (0.5 ml per well). 24 h later, the LTBP1 constructs were transfected into the co-culture system using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were cultured for 3 more days, and then fixed in 95% ethanol. Double immunofluorescent staining was performed as described previously (22Dallas 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 (129) Google Scholar). Recombinant LTBP1 expression was detected with anti-His monoclonal antibody. Fibronectin expression was detected using the rabbit anti-fibronectin antibody (Sigma). Appropriate combinations of Cy3-conjugated detection antibodies or biotinylated antibodies followed by streptavidin-fluorescein isothiocyanate (Jackson ImmunoResearch, West Grove, PA) were used for detection as described previously (22Dallas 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 (129) Google Scholar). The slides were viewed and photographed digitally using a Nikon E800 fluorescence microscope with cooled charge-coupled device camera driven by the AnalySIS software (Soft Imaging Systems Corp., Lakewood, CO). Incubation of 2T3 Cultures with Recombinant LTBP1 Fragments—In parallel to the co-culture transfection experiments above, 2T3 cells were also incubated with purified recombinant LTBP1 fragments (10 μg/ml) for 3 days. Cells were then fixed in 95% ethanol. Incorporation of LTBP1 fragments and co-localization of LTBP1 and fibronectin were determined by double immunofluorescence staining as described above. Heparin Sepharose Affinity Chromatography—Affinity chromatography was performed using heparin-Sepharose columns (HiTrap Heparin HP, 1 ml, Amersham Biosciences). The columns were equilibrated in 20 mm Tris-HCl, pH 7.4. Purified recombinant LTBP1 fragments (20–40 μg) were applied manually to the columns in equilibration buffer at an approximate flow rate of 0.1 ml/min, according to the manufacturer's instructions. After washing the columns with equilibration buffer, bound material was eluted with 1 m NaCl. The eluted volumes were fractionated into 0.5-ml aliquots. The amounts of recombinant LTBP1 fragments in each fraction were determined by ELISA, and the protein was verified by SDS-PAGE and/or Coomassie Blue staining. Solid-phase Binding Assays—Solid-phase binding assays to measure binding of LTBP1 fragments to fibronectin or fibronectin fragments were performed on 96-well ELISA high binding plates (Corning Costar, Acton, MA). Plasma fibronectin, superfibronectin, or fibronectin fragments (100 nm) (see Fig. 4C), diluted in phosphate-buffered saline (PBS) containing 2 mm calcium, were immobilized on high binding ELISA plates overnight at 4 °C. All subsequent steps were performed at room temperature. The wells were washed three times with PBS-T washing buffer (PBS containing 0.05% Tween 20). Blocking of nonspecific binding sites was performed by incubating for 1–2 h with 200 μl of binding buffer (PBS, pH 7.4, plus 5% nonfat dry milk and either 2 mm CaCl2 or 10 mm EDTA). The wells were incubated for 2 h with recombinant LTBP1 fragments at a starting concentration of 100 or 150 nm, then diluted serially 1:2 in binding buffer (100 μl per well). The plates were then washed three times with washing buffer, and the wells were incubated for 1 h with 100 μl of mouse anti-His monoclonal antibody (1:1000, diluted in binding buffer). After washing three times, the wells were incubated with 100 μl of donkey anti-mouse horseradish peroxidase conjugate (1:1000 diluted in binding buffer, Jackson ImmunoResearch). After washing, the plates were reacted with O-phenylenediamine using 4-mg substrate tablets, according to the manufacturer's instructions (Sigma). The plates were read on an ELX800 plate reader (Biotek Instruments Inc., Winooski, VT) at 490 nm after treatment with 25 μl of 3 m HCl. Background subtraction was performed using a blank control that was coated with 100 nm bovine serum albumin (BSA) in place of the fibronectin or fibronectin fragments. For solid-phase binding assays to examine binding of LTBP1 fragments to heparin, heparin coupled to BSA was used (Sigma). 100 nm heparin-BSA in PBS, pH 7.4, was coated onto plates overnight at 4 °C. All subsequent steps were performed at room temperature as described above. Effects of Heparin and β-d-Xylosides on LTBP1 Deposition in the ECM—FRC cells were seeded at 2 × 104 cells per cm2 of growth area in 8-chamber Lab-Tek slides or 6-well plates (Nalge Nunc International, Rochester, NY) in the presence of heparin (0.1–0.5 mg/ml), chondroitin 6-sulfate (CS, 0.1–0.5 mg/ml), heparin-BSA (0.01–0.1 mg/ml), 4-methylumbelliferyl-β-d-xylopyranoside (0.1 mm), or p-nitrophenyl-β-d-xylopyranoside (0.2 mm). After overnight adherence, the cells were washed twice with PBS and then incubated with culture medium containing treatments for 3–6 days. Media was changed every 3 days. Cells in Lab-Tek slides were immunostained for LTBP1 and fibronectin as described above. To determine the relative amounts of secreted LTBP1 in the conditioned media, the cultures were changed to serum-free culture media (plus treatments) on day 5. After a further 24 h, the conditioned medium was harvested and concentrated 10- to 20-fold using a Centricon membrane (30-kDa cut-off). To test the LTBP1 levels in the ECM, plasmin digestions of detergent-insoluble matrix pellets were performed as described previously (11Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (233) Google Scholar, 22Dallas 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 (129) Google Scholar). Both the conditioned media and plasmin-digested matrix samples were normalized to cell number, counted from triplicate wells, prior to loading on 4–20% gradient SDS-PAGE gels (Bio-Rad). Gel electrophoresis and Western blotting were performed as described previously (11Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. J. Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (233) Google Scholar, 22Dallas 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 (129) Google Scholar) using polyclonal antibody Ab-K to detect LTBP1. TGF-β Assays—A commercial ELISA (TGF-β1 Emax, Promega, Madison, WI) was used for measurement of TGF-β1 in serum-free conditioned media samples collected over 48 h or in guanidine-HCl extracts of the ECM, prepared as described elsewhere (42Pfeilschifter J. Laukhuf F. Mu

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