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The Cutaneous Microfibrillar Apparatus Contains Latent Transforming Growth Factor-β Binding Protein-1 (LTBP-1) and is a Repository for Latent TGF-β1

1998; Elsevier BV; Volume: 111; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1747.1998.00339.x

1523-1747

Michael Raghunath, Christine Unsöld, Leena Bruckner‐Tuderman, Ulrich Kubitscheck, Reiner Peters, Martin Meuli,

Cell Adhesion Molecules Research

The transforming growth factors-β1 and β2 (TGF-β) stimulate synthesis of extracellular matrix proteins in vitro and appear upregulated in fibrotic conditions, in scar formation, and in wound healing. The extracellular matrix in turn might also act as a scavenger or repository for TGF-β. We therefore studied the in situ distribution of latent TGF binding protein-1 (LTBP-1) and latent TGF-β1 on extracellular matrix elements of normal human skin and skin regenerating from cultured keratinocyte autografts. We localized both LTBP-1 and latent TGF-β1 to fibrillin-containing (elastic) microfibrils. Both LTBP-1 and latent TGF-β1 were already present during the earliest stages of the de novo formation of the microfibrillar apparatus, i.e., on fusiform, randomly oriented microfibrils that later coalesced to form the typical candelabra-like structures in the papillary dermis. We show herewith that LTBP-1 exerts a dual role as a component of fibrillin-microfibrils of the skin and in targeting latent TGF-β1 to the cutaneous microfibrillar apparatus. Thus, this major connective tissue structure does not only serve as a force bearing element and scaffold for elastin deposition in the dermis, but also as an important repository for latent TGF-β in the skin. The transforming growth factors-β1 and β2 (TGF-β) stimulate synthesis of extracellular matrix proteins in vitro and appear upregulated in fibrotic conditions, in scar formation, and in wound healing. The extracellular matrix in turn might also act as a scavenger or repository for TGF-β. We therefore studied the in situ distribution of latent TGF binding protein-1 (LTBP-1) and latent TGF-β1 on extracellular matrix elements of normal human skin and skin regenerating from cultured keratinocyte autografts. We localized both LTBP-1 and latent TGF-β1 to fibrillin-containing (elastic) microfibrils. Both LTBP-1 and latent TGF-β1 were already present during the earliest stages of the de novo formation of the microfibrillar apparatus, i.e., on fusiform, randomly oriented microfibrils that later coalesced to form the typical candelabra-like structures in the papillary dermis. We show herewith that LTBP-1 exerts a dual role as a component of fibrillin-microfibrils of the skin and in targeting latent TGF-β1 to the cutaneous microfibrillar apparatus. Thus, this major connective tissue structure does not only serve as a force bearing element and scaffold for elastin deposition in the dermis, but also as an important repository for latent TGF-β in the skin. cultured epithelial autografts latent transforming growth factor β-binding protein Transforming growth factors β (TGF-β), a cytokine family of three isoforms in mammals, have profound effects on cell growth, morphology, and differentiation (Massagué, 1990Massagué J. The transforming growth factor-β family.Annu Rev Cell Biol. 1990; 6: 597-641Crossref PubMed Scopus (2948) Google Scholar;Sporn and Roberts, 1990Sporn M.B. Roberts A.B. TGF-β.Problems Prospects Cell Regul. 1990; 1: 875-882PubMed Google Scholar;Laiho and Keski-oja, 1992Laiho M. Keski-oja J. Transforming growth factors-β as regulators of cellular growth and phenotype.Crit Rev Oncog. 1992; 3: 1-12PubMed Google Scholar;Miyazono et al., 1993Miyazono K. Ichi J.H. Heldin C-H. Transforming growth factors-β: latent forms binding proteins and receptors.Growth Factors. 1993; 8: 11-22Crossref PubMed Scopus (199) Google Scholar;Rifkin et al., 1993Rifkin D.B. Kojima S. Abe M. Harpe J.G. TGF-β structure function and formation.Thromb Haemostas. 1993; 70: 177-179PubMed Google Scholar). In addition, TGF-β stimulate the synthesis of extracellular matrix (ECM) proteins. Simultaneously, they block ECM degradation by decreasing the synthesis of matrix metalloproteases and increasing the levels of protease inhibitors (Border and Ruoslahti, 1992Border W.A. Ruoslahti E. Transforming growth factor-β in diseases: the dark side of tissue repair.J Clin Invest. 1992; 90: 1-7Crossref PubMed Scopus (1015) Google Scholar). Thus, TGF-β have substantial influence on tissue composition, tissue remodelling, and wound healing. Accordingly, TGF-β appear to be prime effectors in pathologic conditions such as fibrosis and scar formation during wound healing (Border and Ruoslahti, 1992Border W.A. Ruoslahti E. Transforming growth factor-β in diseases: the dark side of tissue repair.J Clin Invest. 1992; 90: 1-7Crossref PubMed Scopus (1015) Google Scholar;Martin, 1997Martin P. Wound healing – aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3426) Google Scholar). It has long been postulated that the ECM does not only have mechanical functions but also might act as a scavenger or repository for TGF-β, thus assigning a new important role to connective tissue. It has been shown in vitro, that certain ECM proteins such as decorin, fibronectin, thrombospondin, and collagen IV, can bind active TGF-β (reviewed inTaipale and Keski-oja, 1997Taipale J. Keski-oja J. Growth factors in the extracellular matrix.Faseb J. 1997; 11: 51-59Crossref PubMed Scopus (720) Google Scholar); however, these data still await further (stoichiometric) corroboration and are currently not supported by immunohistochemical findings. TGF-β are secreted in latent, inactive complexes containing two proteins: active TGF-β and its prodomain, the TGF-β latency-associated protein (LAP or small latent TGF-β1) (Taipale and Keski-oja, 1997Taipale J. Keski-oja J. Growth factors in the extracellular matrix.Faseb J. 1997; 11: 51-59Crossref PubMed Scopus (720) Google Scholar). Most cell lines, however, secrete TGF-β as a three-protein-complex (large latent TGF-β) consisting of LAP associated with high molecular weight proteins, the best characterized of which are the latent TGF-β binding proteins (LTBP); these bind to LAP via one or more disulfide bonds. Four LTBP genes have been isolated so far (LTBP-1 through 4;Kanzaki et al., 1990Kanzaki T. Olofsson A. Morén A. TGFβ1 binding protein: a component of the large latent complex of TGFβ1 with multiple repeat sequences.Cell. 1990; 61: 1051-1061Abstract Full Text PDF PubMed Scopus (361) Google Scholar;Morén et al., 1994Morén A. Olofsson A. Stenman G. Identification and characterization of LTBP-2 a novel latent transforming growth factor β-binding protein.J Biol Chem. 1994; 269: 32469-32478Abstract Full Text PDF PubMed Google Scholar;Gibson et al., 1995Gibson M.A. Hatzinikolas G. Davis E.C. Baker E. Sutherland G.R. Mecham R.P. Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning identification of tissue isoforms and immunolocalization to elastin-associated microfibrils.Mol Cell Biol. 1995; 15: 6932-6942Crossref PubMed Google Scholar;Yin et al., 1995Yin W. Smiley E. Germiller J. Mecham R.P. Florer J.B. Wenstrup R. Bonadio J. Isolation of a novel latent transforming growth factor-β binding protein gene (LTBP-3.J Biol Chem. 1995; 270: 10147-10160Crossref PubMed Scopus (121) Google Scholar;Giltay et al., 1997Giltay R. Kostka G. Timpl R. Sequence and expression of a novel member (LTBP-4) of the family of latent transforming growth factor-beta binding proteins.FEBS Lett. 1997; 411: 164-168Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). LTBP-1 was recently shown to be incorporated into the ECM of fibroblast and osteoblast cell cultures (Taipale et al., 1992Taipale J. Koli K. Keski-oja J. Release of transforming growth factor-β1-binding protein from the pericellular matrix of cultured fibroblasts and fibrosarcoma cells by plasmin and thrombin.J Biol Chem. 1992; 267: 25378-25384Abstract Full Text PDF PubMed Google Scholar,Taipale et al., 1996Taipale J. Saharinen J. Hedman K. Keski-oja J. Latent transforming growth factor and its binding protein (LTBP-1) are components of extracellular matrix microfibrils.J Histochem Cytochem. 1996; 44: 875-889Crossref PubMed Scopus (185) Google Scholar;Dallas et al., 1995Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. Dual role for the latent transforming growth factor β-binding protein in storage of latent TGF-β in the extracellular matrix and as a structural matrix protein.J Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (222) Google Scholar;Oloffsson et al., 1995Oloffsson A. Ichi Jo H. Morén A. ten Dijke P. Miyazono K. Heldin C-H. Efficient association of an amino-terminally extended form of human latent transforming growth factor-β-binding protein with the extracellular matrix.J Biol Chem. 1995; 270: 31294-31297Crossref PubMed Scopus (74) Google Scholar). In vivo, LTBP-1 was localized to connective tissue surrounding gastrointestinal carcinomas (Mizoi et al., 1993Mizoi T. Ohtani H. Miyazono K. Miyazawa M. Matsuno S. Nagura H. Immunoelectron microcroscopic localisation of transforming growth factor beta 1 and latent transforming growth factor beta 1 binding protein in human gastrointestinal carcinomas: qualitative difference between cancer cells and stromal cells.Cancer Res. 1993; 53: 183-190PubMed Google Scholar), or endocardial cushion cells of the developing mouse heart (Nakajima et al., 1997Nakajima Y. Miyazono K. Katao M. Takase M. Ymagishi T. Nakamura H. Extracellular fibrillar structure of latent TGF-β-dependent endothelial-mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart.J Cell Biol. 1997; 136: 193-204Crossref PubMed Scopus (99) Google Scholar), whereas LTBP-2 was identifed in elastic microfibrils of bovine ligamentum nuchae (Gibson et al., 1995Gibson M.A. Hatzinikolas G. Davis E.C. Baker E. Sutherland G.R. Mecham R.P. Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning identification of tissue isoforms and immunolocalization to elastin-associated microfibrils.Mol Cell Biol. 1995; 15: 6932-6942Crossref PubMed Google Scholar). Here, we demonstrate that LTBP-1 is a constituent of fibrillin-containing microfibrils of normal and regenerating human skin and that the microfibrillar apparatus of the skin is an important repository for latent TGF-β. Skin biopies from normal adult volunteers and pediatric burn patients treated with cultured epithelial autografts (CEA) were used. The detailed data pertinent to these patients have been reported elsewhere (Raghunath et al., 1996Raghunath M. Bächi Th Meuli M. Altermatt S. Gobet R. Bruckner-tuderman L. Steinmann B. Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autografts: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation.J Invest Dermatol. 1996; 106: 1090-1095Crossref PubMed Scopus (61) Google Scholar). CEA sheets were prepared from scalp biopsies by a commercial laboratory (Biosurface Technology, Boston, MA). For histologic follow up after grafting, 4 mm punch biopsies were taken under general or local anesthesia after informed consent of the patients/parents. Biopsies were obtained 1 wk, 2 wk, 3–4 wk, 1 mo, 2 mo, 4–5 mo, 17 mo, and 24 mo after transplantation. The mouse monoclonal antibody MoAb69 (kindly provided by Dr. Robert Glanville, Shriners Hospital for Crippled Children, Portland, OR) is specific for the peptic fragment PF3 of fibrillin representing its unique C-terminus (Maddox et al., 1989Maddox B.K. Sakai L.Y. Keene D.R. Glanville R.W. Connective tissue microfibrils Isolation and characterization of three large pepsin-resistant domains of fibrillin.J Biol Chem. 1989; 264: 21381-21385Abstract Full Text PDF PubMed Google Scholar); it has no resemblance to regions found in LTBP. The rabbit anti-serum Ab39 (provided by Dr. Kohei Miyazono, The Cancer Institute, Tokyo, Japan) was raised against native LTBP-1 purified from human platelets (Kanzaki et al., 1990Kanzaki T. Olofsson A. Morén A. TGFβ1 binding protein: a component of the large latent complex of TGFβ1 with multiple repeat sequences.Cell. 1990; 61: 1051-1061Abstract Full Text PDF PubMed Scopus (361) Google Scholar;Miyazono et al., 1991Miyazono K. Olofsson A. Colosetti P. Heldin C-H. A role of the latent TGF-β1-binding protein in the assembly and secretion of TGF-β1.Embo J. 1991; 10: 1091-1101Crossref PubMed Scopus (399) Google Scholar); it does not cross-react with LTBP-2 (Morén et al., 1994Morén A. Olofsson A. Stenman G. Identification and characterization of LTBP-2 a novel latent transforming growth factor β-binding protein.J Biol Chem. 1994; 269: 32469-32478Abstract Full Text PDF PubMed Google Scholar). Because LTBP contain protein modules that are structurally very similar to fibrillins-1 and -2 (reviewed inSaharinen et al., 1996Saharinen J. Taipale J. Keski-oja J. Association of the small latent transforming growth factor-β with an eight cysteine repeat of its binding protein LTBP-1.Embo J. 1996; 15: 245-253Crossref PubMed Scopus (174) Google Scholar), we performed experiments to exclude cross-reactivity of Ab39 with fibrillin. Therefore, 2 liters of serum-free Waymouth medium conditioned by embryonic human dermal fibroblasts were precipitated with 40% saturated ammonium sulfate. The precipitate was dissolved in 40 ml of 50 mM Tris/HCl pH 7.4 and extensively dialyzed against several changes of the same buffer at 4°C. Aliquots were loaded on a 4% sodium dodecyl sulfate-polyacrylamide gel and proteins were separated under nonreducing conditions and then transferred for 4–6 h at 495 mV (watts unlimited) onto nitrocellulose membranes using 25 mM Tris, 192 mM glycine, 0.1% (wt/vol) sodium dodecyl sulfate, and 20% (vol/vol) methanol. The membranes were blocked with 2% non-fat dry milk and incubated with MoAb69 1:200 and Ab39 1:400, respectively, over night at room temperature, washed thoroughly in phosphate-buffered saline, and incubated with a peroxidase-coupled goat anti-rabbit IgG (Kirkegaard & Perry, Gaithersburg, MD) 1:2000 or peroxidase-coupled swine anti-mouse IgG (Dako, Glostrup, Denmark) 1:500 for 2 h at room temperature. Enzyme reactions were performed with chloronaphthol. In parallel experiments, fibroblasts were metabolically labeled with 35S-Met/Cys and culture medium used for radioimmunoprecipitation with Ab39 (Taipale et al., 1994Taipale J. Miyazono K. Heldin C-H. Keski-oja J. Latent transforming growth facor β1-associates to fibroblast extracellular matrix via latent TGF-β binding protein.J Cell Biol. 1994; 124: 171-181Crossref PubMed Scopus (360) Google Scholar) and MoAb69. We further used rabbit antibody Ab96 (kindly provided by K. Miyazono) and goat antibodies against β-LAP (R&D Systems; Minneapolis, MN). Five micrometer cryostat sections were fixed in acetone at –20°C for 10 min and air dried. Non-specific binding sites were blocked for 30 min with phosphate-buffered saline/1% bovine serum albumin/5% normal swine serum/5% normal goat serum (Dako). This solution was replaced by polyclonal rabbit anti-sera Ab39 against LTBP-1 and Ab96 against TGF-β1 precursor in dilutions of 1:200. Either antibody was used in combination with MoAb69 against fibrillin diluted 1:20. These primary antibodies were detected with biotinylated swine anti-rabbit IgG 1:50 (Dako), streptavidin-DTAF 1:100 (Jackson Immunoresearch, West Grove, PA), and a Texas Red coupled goat anti-mouse antibody 1:50 (Jackson Immunoresearch), respectively. For triple immunofluorescence analyses we used MoAb69 1:20/donkey anti-mouse antibody-Cy3 1:100 (Jackson Immunoresearch), goat anti-LAP (1:100)/donkey anti-rabbit-Texas Red 1:100, and Ab39 1:400/swine anti-rabbit-fluoroscein isothiocyanate 1:30 (Dako). Preparations were mounted in Mowiol (Hoechst) in Tris/HCl pH 8.6 and examined using a LSM 410 invert device (Carl Zeiss, Oberkochen, Germany) combined with two HeNe lasers (543/633 nm) and an argon laser (488 nm) for multicolor fluorescence. In some instances cryosections of normal human skin were incubated before immunostaining with 0.1 U human plasmin (Sigma, St. Louis, MO) per ml in serum-free MEM/HEPES for 1 h at 37°C according toDallas et al., 1995Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. Dual role for the latent transforming growth factor β-binding protein in storage of latent TGF-β in the extracellular matrix and as a structural matrix protein.J Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (222) Google Scholar. Photographs were taken on Tmax 3200 ASA (Kodak), using a Axiophot (Zeiss, Jena, Germany) epifluorescence equipment. The monoclonal antibody MoAb69 recognizes the carboxyterminus of fibrillin-1 (Maddox et al., 1989Maddox B.K. Sakai L.Y. Keene D.R. Glanville R.W. Connective tissue microfibrils Isolation and characterization of three large pepsin-resistant domains of fibrillin.J Biol Chem. 1989; 264: 21381-21385Abstract Full Text PDF PubMed Google Scholar), which is unique to this protein (Ramirez, 1996Ramirez F. Fibrillin mutations in Marfan syndrome and related phenotypes.Curr Opin Genet Dev. 1996; 6: 309-315Crossref PubMed Scopus (100) Google Scholar). Accordingly, a single band representing fibrillin at the expected molecular weight of ≈320 kDa was obtained under nonreducing conditions Figure 1a. No other protein was detected with this antibody. Ab39, in contrast, did not cross-react with fibrillin, but specifically recognized a broad band of ≈200 kDa Figure 1. This is in accordance with published work (Kanzaki et al., 1990Kanzaki T. Olofsson A. Morén A. TGFβ1 binding protein: a component of the large latent complex of TGFβ1 with multiple repeat sequences.Cell. 1990; 61: 1051-1061Abstract Full Text PDF PubMed Scopus (361) Google Scholar;Taipale et al., 1994Taipale J. Miyazono K. Heldin C-H. Keski-oja J. Latent transforming growth facor β1-associates to fibroblast extracellular matrix via latent TGF-β binding protein.J Cell Biol. 1994; 124: 171-181Crossref PubMed Scopus (360) Google Scholar). In radioimmunoprecipitation, Ab39 precipitated an identical band Figure 1b without any evidence for coprecipitation of fibrillin. MoAb69 did not work well for immunoprecipitation in our hands (not shown). In normal skin, LTBP-1 was identified on both the papillary and the reticular part of the cutaneous microfibrillar apparatus. This was confirmed by the almost complete colocalization of LTBP-1 and fibrillin, the major component of the microfibrillar apparatus Figure 2a, b. In addition, LTBP-1 was also present in the walls of some blood vessels and smooth muscle cells of m. arrectores pilorum where fibrillin was not identified (data not shown). In regenerating skin, fibrillin and LTBP-1 colocalized extensively to the same ECM structures from which the microfibrillar apparatus was gradually reconstituted (Raghunath et al., 1996Raghunath M. Bächi Th Meuli M. Altermatt S. Gobet R. Bruckner-tuderman L. Steinmann B. Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autografts: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation.J Invest Dermatol. 1996; 106: 1090-1095Crossref PubMed Scopus (61) Google Scholar) Figure 2c, d, e and f, starting from the first fusiform fibrils 7 d after grafting Figure 2c to a near normal appearance 17–24 mo after grafting Figure 2f. The microfibrillar apparatus of normal skin extensively stained with MoAb69 against fibrillin and Ab96 against β-LAP. Superimposed confocal images revealed extensive colocalization on individual microfibrillar bundles Figure 3a, b). As an example of another force-bearing ECM structure that is solely composed of microfibrils, we used the suspensory ligament of the primate (cynomolgus monkey) and the human eye lens. We found brilliant immunostaining for both fibrillin and TGF-β1 precursor Figure 3c. Triple immunofluorescence analyses with a goat antibody against the TGF-β1 propeptide (LAP) allowed us to localize both LTBP-1 and latent TGF-β1 to the same individual fibrillin-containing microfibrils Figure 3d. In contrast, we could not detect LTBP-1 on ciliary zonules of the monkey eye lens Figure 4, which suggests a different composition of this microfibrillar structure but also that the antibody Ab39 does not cross-react with fibrillin.Figure 4Fibrillin but not LBTP-1 is present on zonule fibrils of the cynomolgus monkey eye. Conventional immunofluorescence detection of fibrillin (a) and LTBP-1 (b) in equatorial cryosection sections of cynomolgus monkey eye. L, lens; C, lens capsule; ZF, zonule fibril. Fibrillin is present on zonule fibrils, whereas LTBP-1 cannot be detected. Scale bars: 25 μm.View Large Image Figure ViewerDownload (PPT) In regenerating skin, TGF-β1 precursor colocalized extensively with the microfibrillar elements throughout the entire process of the de novo formation of the microfibrillar apparatus Figure 5. There were also structures exclusively positive for TGF-β1 precursor and not for fibrillin, such as some blood vessels Figure 5e and m. arrectores pilorum Figure 5f. In the epidermis TGF-β1 precursor could be detected to a varying degree in the cytoplasm of keratinocyte layers with a predominance in the stratum granulosum Figure 5c and occasionally in basal and suprabasal layers (compare with Figure 3. Because the activation of latent TGF-β can be achieved by proteolytic treatment, we treated normal human skin cryosections with plasmin. This led to a substantial reduction of the fibrillar staining for latent TGF-β1 precursor of the microfibrillar apparatus and smooth muscle Figure 6b, c. In contrast, fibrillin immunostaining appeared unchanged and partially slightly enhanced Figure 6e, f. Skin regeneration following transplantation of CEA offers a unique opportunity to observe the complex processes transforming the initial arrangement of confluent keratinocyte cultures placed onto a highly variable, surgically created wound bed into an almost normally structured skin. This regeneration process includes formation of a neo-epidermis from CEA, formation of a neodermis from wound bed connective tissue, and, very importantly, the de novo formation of the dermo–epidermal junction (Raghunath and Meuli, 1997Raghunath M. Meuli M. Cultured epithelial autografts: diving from surgery into matrix biology.Ped Surg Int. 1997; 12: 478-483Crossref PubMed Scopus (13) Google Scholar). We have recently characterized the morphogenetic stages of the regeneration of the microfibrillar apparatus in skin regenerating from CEA (Raghunath et al., 1996Raghunath M. Bächi Th Meuli M. Altermatt S. Gobet R. Bruckner-tuderman L. Steinmann B. Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autografts: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation.J Invest Dermatol. 1996; 106: 1090-1095Crossref PubMed Scopus (61) Google Scholar). This ECM structure consists of a perpendicular fiber system traversing the papillary dermis and a horizontal system in the reticular dermis. Thus, it connects the dermo–epidermal junction with the deeper dermis. It is a scaffold for elastin deposition and presumably plays a role in the organization of the papilla/rete ridge pattern (Raghunath et al., 1996Raghunath M. Bächi Th Meuli M. Altermatt S. Gobet R. Bruckner-tuderman L. Steinmann B. Fibrillin and elastin expression in skin regenerating from cultured keratinocyte autografts: morphogenesis of microfibrils begins at the dermo-epidermal junction and precedes elastic fiber formation.J Invest Dermatol. 1996; 106: 1090-1095Crossref PubMed Scopus (61) Google Scholar). The main components of 10–12 nm microfibrils have been identified as fibrillins 1 and 2 (reviewed inRamirez, 1996Ramirez F. Fibrillin mutations in Marfan syndrome and related phenotypes.Curr Opin Genet Dev. 1996; 6: 309-315Crossref PubMed Scopus (100) Google Scholar). These proteins share a number of features with LTPB, including multiple EGF-like repeats, an RGD sequence, and a novel eight-cyteine repeat motif that is unique to this family of proteins (Saharinen et al., 1996Saharinen J. Taipale J. Keski-oja J. Association of the small latent transforming growth factor-β with an eight cysteine repeat of its binding protein LTBP-1.Embo J. 1996; 15: 245-253Crossref PubMed Scopus (174) Google Scholar). Accordingly, it has been suggested that there may be a gene family of fibrillin-like proteins that are involved in the formation of microfibrils and that have important structural roles in maintaining tissue integrity and organization (Dallas et al., 1995Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. Dual role for the latent transforming growth factor β-binding protein in storage of latent TGF-β in the extracellular matrix and as a structural matrix protein.J Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (222) Google Scholar). In accordance with this hypothesis we found that LTBP-1 is a constitutive element of the cutaneous microfibrillar apparatus during its entire regeneration process. This extends earlier reports on the colocalization of LTBP-1 with ECM fibrils deposited in culture, such as bone cells (Dallas et al., 1995Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. Dual role for the latent transforming growth factor β-binding protein in storage of latent TGF-β in the extracellular matrix and as a structural matrix protein.J Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (222) Google Scholar), human fibroblasts (Taipale et al., 1996Taipale J. Saharinen J. Hedman K. Keski-oja J. Latent transforming growth factor and its binding protein (LTBP-1) are components of extracellular matrix microfibrils.J Histochem Cytochem. 1996; 44: 875-889Crossref PubMed Scopus (185) Google Scholar), or endocardial cushion (Nakajima et al., 1997Nakajima Y. Miyazono K. Katao M. Takase M. Ymagishi T. Nakamura H. Extracellular fibrillar structure of latent TGF-β-dependent endothelial-mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart.J Cell Biol. 1997; 136: 193-204Crossref PubMed Scopus (99) Google Scholar). The LTBP-1 carrying ECM fibrils in cell culture were partially characterized by immunoelectron microscopy as fibronectin fibrils (Taipale et al., 1996Taipale J. Saharinen J. Hedman K. Keski-oja J. Latent transforming growth factor and its binding protein (LTBP-1) are components of extracellular matrix microfibrils.J Histochem Cytochem. 1996; 44: 875-889Crossref PubMed Scopus (185) Google Scholar). Using monoclonal antibodies to fibronectin and fibrillin we could confirm in hyperconfluent fibroblast cultures colocalization of LTBP-1 with the pericellular fibronectin matrix, but also with fibrillin-containing microfibrils that are devoid of fibronectin (unpublished data). The localization of LTBP-1 to the cutaneous microfibrillar apparatus clearly points to its role in storing latent TGF-β in this ECM compartment Figure 7. We have demonstrated this role by showing that, like LTBP-1, latent TGF-β1 was consistently associated with the cutaneous microfibrils of the normal and the regenerating microfibrillar apparatus. Remarkably, like LTBP-1, latent TGF-β1 was associated with fibrillin-microfibrils as soon as they were discernible during the regeneration of the microfibrillar apparatus. This graphically demonstrates that even very small aggregates of fibrillin-microfibrils can store latent TGF-β1 via LTBP-1 without being a part of a large supramolecular structure yet. This would allow for storage of latent TGF-β also during embryogenesis. We could detect latent TGF-β1, but not LTBP-1, in ciliary zonules of the eye lens, structures that are solely composed of fibrillin-containing microfibrils. This does not only highlight compositional differences between the microfibrillar assemblies in different tissues, but also suggests the presence of another LTBP targeting latent TGF-β to these ECM structures. At this point we cannot rule out direct interactions between latent TGF-β and fibrillin itself. Probably, LTBP-1 plays a skin-specific role targeting latent TGF-β. We also found colocalization of LTPB-1 and latent TGF-β on tissue structures besides the microfibrillar apparatus such as smooth muscle cells and keratinocytes (which were negative for fibrillin). We could not observe LTBP-1 in normal epidermis and only occasionally in the neoepidermis of regenerating skin. The possible role of keratinocytes as a source for LTBP-1 (and thus large latent TGF-β1 complex) synthesis, however, warrants further investigation. Latent TGF-β is activated by heat, acid, or alkaline treatment, binding to thrombospondin, deglycosylation, proteolysis (Taipale and Keski-oja, 1997Taipale J. Keski-oja J. Growth factors in the extracellular matrix.Faseb J. 1997; 11: 51-59Crossref PubMed Scopus (720) Google Scholar), or irradiation (Barcellos-hoff et al., 1994Barcellos-hoff M.H. Derynck R. Tsang ML-S. Weatherbee J.A. Transforming growth factor-β activation in irradiated murine mammary gland.J Clin Invest. 1994; 93: 892-899Crossref PubMed Scopus (351) Google Scholar). The most likely physiologic route of activation involves the proteolytic cleavage of LAP by plasmin or other proteases (Taipale and Keski-oja, 1997Taipale J. Keski-oja J. Growth factors in the extracellular matrix.Faseb J. 1997; 11: 51-59Crossref PubMed Scopus (720) Google Scholar). It has been recently shown that LTBP complexed to TGF-β can be released from the ECM of cultured fibroblasts or bone cells, respectively, by plasmin treatment (Taipale et al., 1994Taipale J. Miyazono K. Heldin C-H. Keski-oja J. Latent transforming growth facor β1-associates to fibroblast extracellular matrix via latent TGF-β binding protein.J Cell Biol. 1994; 124: 171-181Crossref PubMed Scopus (360) Google Scholar;Dallas et al., 1995Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. Dual role for the latent transforming growth factor β-binding protein in storage of latent TGF-β in the extracellular matrix and as a structural matrix protein.J Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (222) Google Scholar). This is in keeping with our findings that latent TGF-β1 is removed from the microfibrillar apparatus after plasmin treatment. In contrast, the fibrillin immunostaining pattern was unchanged, if not slightly enhanced. We also noted no significant reduction of the LTBP-1 signal after plasmin digest (unpublished) and therefore speculate that Ab39 might preferentially recognize the aminoterminal portion of LTBP-1 that is retained in association with microfibrils following plasmin treatment. It has been recently proposed that the sequestration of latent TGF-β to the ECM in large pools – as we have shown here – is a prerequiste for the activation by cell surface-associated plasmin (Nunes et al., 1997Nunes I. Gelizes P-E. Metz C.N. Rifkin D.B. Latent transforming growth factor -β binding domains involved in activation and transglutaminase-dependendent cross-linking of latent transforming growth factor-β.J Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (329) Google Scholar). 1The role of plasmin has been highlighted by transgenic mice expressing human apolipoprotein(a). These animals show an inhibited plasminogen activation and, in turn, a lack of TGF-β activation in the aortic wall. This is currently believed to underly the proliferation of vascular smooth muscle cells in the aortic wall heralding arteriosclerosis (Grainger et al., 1994Grainger D.J. Kemp P.R. Liu A.C. Lawn R.M. Metcalfe J.C. Activation of transforming growth factor β is inhibited in transgenic apolipoprotein (a) mice.Nature. 1994; 370: 460-462Crossref PubMed Scopus (334) Google Scholar). Following current concepts (Nunes et al., 1997Nunes I. Gelizes P-E. Metz C.N. Rifkin D.B. Latent transforming growth factor -β binding domains involved in activation and transglutaminase-dependendent cross-linking of latent transforming growth factor-β.J Cell Biol. 1997; 136: 1151-1163Crossref PubMed Scopus (329) Google Scholar;Taipale and Keski-oja, 1997Taipale J. Keski-oja J. Growth factors in the extracellular matrix.Faseb J. 1997; 11: 51-59Crossref PubMed Scopus (720) Google Scholar) it is conceivable that a migrating or sedentary cell receives local and repetitive doses of active TGF-β derived from microfibrils upon focal cell surface proteolysis in conjunction with focal contacts to the ECM via integrin receptors. It is worth noting that fibrillin-1 facilitates cell contacts by an RGD sequence that is recognized by αvβ3 integrin of dermal fibroblasts (Sakamoto et al., 1996Sakamoto H. Broekelmann T. Cheresh D.A. Ramirez F. Rosenbloom J. Mecham R.P. Cell-type specific recognition of RGD- and non-RGD containing cell binding domains in fibrillin-1.J Biol Chem. 1996; 271: 4916-4922Crossref PubMed Scopus (146) Google Scholar). This study demonstrates that LTBP-1 is a structural ECM protein involved in the formation of the cutaneous microfibrillar apparatus. Based on distribution and reasonable sequence homologies, LTBP-1 qualifies as a new member of the fibrillin family of ECM proteins. Our data also underline the recently proposed dual role of LTBP-1 in targeting latent TGF-β to the ECM (Dallas et al., 1995Dallas S.L. Miyazono K. Skerry T.M. Mundy G.R. Bonewald L.F. Dual role for the latent transforming growth factor β-binding protein in storage of latent TGF-β in the extracellular matrix and as a structural matrix protein.J Cell Biol. 1995; 131: 539-549Crossref PubMed Scopus (222) Google Scholar). This will be important with regard to therapeutic effects of irradiation or phototherapy, and also microfibrillopathies based on fibrillin mutations such as the Marfan syndrome or the tight skin mouse phenotype (Ramirez, 1996Ramirez F. Fibrillin mutations in Marfan syndrome and related phenotypes.Curr Opin Genet Dev. 1996; 6: 309-315Crossref PubMed Scopus (100) Google Scholar;Kielty et al., 1998Kielty C.M. Raghunath M. Siracusa L. Sheratt M.J. Shuttleworth C.A. Peters R. Jimenez S. The tight skin mouse: demonstration of mutant fibrillin-1 production and microfibrillar abnormalities.J Cell Biol. 1998; 140: 1-8Crossref PubMed Scopus (98) Google Scholar). Finally, these findings will be important for therapeutic approaches to modulate the activation of latent TGF-β in the quest for anti-scarring agents. We thank Drs. Rita Gobet and Stefan Altermatt, Pediatric Burn Center University Children's Hospital Zürich, for providing skin biopsies. MoAb69 was generously given by Dr. Robert Glanville, Shriners Hospital for Crippled Children, Portland, Oregon. Ab39 and Ab96 were donated by Dr. Kohei Miyazono, The Cancer Institute, Department of Biochemistry, Tokyo. We thank Sylvia Teich, H. Esche, Andrea Wissel, and Dr. Jan-Dirk Fautek for excellent technical assistance. Mathias Tschödrich-Rotter was of invaluable help with the confocal laser scanning microscope. M.R. was funded by the Deutsche Forschungsgemeinschaft (DFG grant 447/3–1 and 3–2) and is indebted to Drs Hans Kresse and Peter Bruckner, Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, for kind support. This work was supported by the IKF Münster, Project D5.