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Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen
Experimental Cell Research 298 (2004) 305 – 315 www.elsevier.com/locate/yexcr
Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen Takeharu Minamitani, a Tomoki Ikuta, a Yoshinari Saito, a Gen Takebe, b Mami Sato, c Hirofumi Sawa, c Takanori Nishimura, d Fumio Nakamura, d Kazuhiko Takahashi, b Hiroyoshi Ariga, a and Ken-ichi Matsumoto a,* a
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan c Laboratory of Molecular and Cellular Pathology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan d Division of Bioresources and Bioproduction, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
b
Received 3 March 2004, revised version received 15 April 2004 Available online 18 May 2004
Abstract Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX / ) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro. D 2004 Elsevier Inc. All rights reserved. Keywords: Collagen fibrillogenesis; Knockout mouse; Tenascin-X; Type VI collagen
Introduction Extracellular matrices (ECMs) consisting of a complex network of macromolecules such as glycoproteins and proteoglycans not only provide a physical framework for cells in terms of mechanical strength, but also participate in cell proliferation, migration, differentiation, and survival. The ECM contains many adhesive proteins, including fibronectin, collagen, and laminin [1]. These proteins generally promote cell attachment or migration. In addition to these adhesive proteins, adhesion modulatory proteins are also present in the ECM and regulate the interactions between cell receptors and these adhesive proteins. Tenascin * Corresponding author. Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita, Sapporo 060-0812, Japan. Fax: +81-11-706-4988. E-mail address: [email protected] (K. Matsumoto). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.04.030
family belongs to such a family [2]. Tenascin family proteins each have a characteristic domain structure with a cysteine-rich segment at the amino terminus, followed by epidermal growth factor (EGF)-like repeats, fibronectin type III (FNIII)-like repeats, and a fibrinogen-like domain at the carboxy terminus. So far, six members of the tenascin family have been identified in various species from zebrafish to mammals: tenascin/cytotactin (tenascin-C, TNC), restictin/J1-160/180 (tenascin-R, TNR), tenascin-X (TNX), tenascin-Y (TNY), tenascin-W (TNW), and recently identified tenascin-N (TNN) [3– 7]. Tenascin family proteins modulate the adhesion of cells on the other ECM components. The cellular ligands mediating these interactions include integrins, proteoglycans and cell adhesion molecules. So far, at least six members of the integrin family that recognize TNC as a ligand have been identified. Five of these (integrins a9h1, avh3, avh6, a8h1, and a7h1) bind to the FNIII repeats; the third FNIII
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repeat of TNC was identified as a cell adhesive site interacting with integrins avh3, avh6, a8h1 through the RDG sequence, and this domain also interacts with integrin a9h1 in an RGD-independent manner [8,9]. Integrin a7h1 has been shown to interact preferentially with the alternative spliced FNIII repeat D of human TNC, leading to an increase in neurite outgrowth [10]. On the other hand, integrin avh3 also binds to the C-terminal fibrinogen-like domain of TNC [11]. Furthermore, the adhesion to FNIII repeats 9 –10 of bovine TNX was shown to be mediated by integrin avh3 in an RGD-dependent manner [12]. Recently, integrin a8h1 has been shown to recognize a FNIII repeat of TNW [7]. Many studies have indicated the important role of interaction of proteoglycan in the functions of members of the tenascin family. Several types of proteoglycans have been shown to bind to TNC. For example, neurocan, a member of the aggrecan family of chondroitin sulfate proteoglycans, has been shown to interact with the FNIII repeats of TNC [13]. Syndecan, a cell surface proteoglycan, has been shown to bind to TNC via its heparan sulfate side chains [14]. Agrin, a major brain heparan sulfate proteoglycan, has been shown to interact with TNC solely via its protein core [15]. TNX binding to glycosaminoglycans, namely heparin and dermatan sulfate chains of decorin, has also been reported [16,17]. The binding sites of them have been identified in FNIII repeats 10– 11 of bovine TNX by the use of recombinant repeats produced by bacteria. As for TNR, lecticans, a family of chondroitin sulfate proteoglycans encompassing aggrecan, versican, neurican, and brevican, have been shown to bind TNR mediated by a protein –protein interaction through the FNIII repeats 3 – 5 of TNR. Brevican had the highest affinity in the lecticans [18]. Interactions between TNR or TNC and cell surface molecules also affect cell adhesion and cell migration. Contactin/F11, an axon-associated GPI-anchored member of the immunoglobulin superfamily, can bind to both TNC [19] and TNR [20]. The interaction of TNR with F11 in in vitro cultures enhanced F11-mediated neurite outgrowth [21]. Furthermore, the h2 subunit of voltage-gated sodium channels also interacts with TNC and TNR with crucial roles in regulation of sodium channel localization and its activity [22]. On the other hand, no evidence of strong interactions between tenascin family proteins and other components of the ECM glycoproteins has been obtained. TNC did not bind to gelatin, laminin, or types I, III, IV, or V collagen [23]. The interaction of TNC with fibronectin was apparently weak. However, there have been no experiments on the interaction of other tenascins with the ECM. Currently, TNX is the largest known member of the tenascin family. Initially, the TNX gene was identified in the class III region of the major histocompatibility complex (MHC) with an overlap of the last exon of the CYP21B gene in the opposite direction [24 –26]. Thereafter, TNX protein was also purified as a new ECM glycoprotein
localized on collagen fibrils and was named flexilin [27]. TNX is expressed much more widely than other tenascins and appears specifically in skeletal muscle, heart, and skin [28 – 30]. Some findings concerning the function of TNX have been reported. TNX-deficiency has been shown to cause Ehlers – Danlos syndrome, a heritable connective tissue disorder. Symptoms of this syndrome are hyperextensible skin, hypermobile joints, hyperelastic skin, and easy bruising [31,32]. Mice with deficiency in TNX (TNX / mice) caused by homologous recombination using embryonic stem cells also showed progressive skin hyperextensibility, similar to patients with Ehlers – Danlos syndrome [33]. These results suggest that TNX plays an important structural role in the skin. Another finding is that TNX is required for impeding the invasion and metastasis of tumor cells. TNX / mice generated by us showed a high incidence of tumor invasion and metastasis following subcutaneous inoculation of B16 –BL6 melanoma cells due to the increased activities of matrix metalloproteinases (MMPs), especially those of MMP-2 and MMP-9 [34]. It has also been shown that TNX is involved in endothelial cell proliferation. TNX interacted with vascular endothelial growth factor B (VEGF-B) and enhanced cell proliferation in combination with VEGF-B [35]. Finally, we have found that TNX influences synthesis of type VI collagen at the RNA level [36]. Expression level of mRNA of type VI collagen was remarkably decreased in TNX-null fibroblasts. Transient expression of TNX in Balb3T3 cells caused an increased mRNA level of type VI collagen compared with that in empty vector and increased the promoter activity of type VI collagen a1 subunit gene. The expressions of type I collagen and other collagen fibril-associated molecules such as type XII and type XIV collagens, decorin, lumican, and fibromodulin were also affected by TNX deficiency. We therefore speculated that TNX plays a role in collagen fibrillogenesis directly and/or indirectly by mediating the levels of collagen fibril-associated proteins such as type VI collagen. To determine the roles of TNX and type VI collagen in collagen fibrillogenesis, the ability of purified TNX and type VI collagen molecules to bind directly to type I collagen was investigated in this study using a solid-phase binding assay. The ability of domain-deleted mutants of TNX to interact with type I collagen was also investigated. Finally, the effects of TNX and type VI collagen on collagen fibril formation were investigated.
Materials and methods Animals The generation of TNX / mice by homologous recombination using embryonic stem (ES) cells has been described previously [34]. The mice were housed in laminar flow cabinets under specific pathogen-free conditions. All
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animal experiments were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Hokkaido University. Cell culture Human embryo kidney 293T cells were grown at 37jC in a 5% CO2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui) supplemented with 10% fetal bovine serum (FBS), penicillin (10 units/ml) and streptomycin (10 Ag/ml). Preparation of a TNX antibody affinity column To generate a column prepared with TNX antibody, rabbit M11 polyclonal antibodies, which reacted with the 11th FNIII repeat of mouse TNX [34], were purified. Ten milliliters of polyclonal antiserum from rabbit was added to an equal volume of 0.9% NaCl and was then precipitated with 40% ammonium sulfate by stirring for 1 h at 4jC. After centrifugation for 20 min at 13,000 g, the pellet was dissolved in 2 ml of 0.9% NaCl and dialyzed overnight against 20 mM sodium phosphate buffer (pH 7.4). The concentrated sample was centrifuged and passed over a DEAE-cellulose (DE-52) column (Whatman, Springfield Mill, UK). The flow-through was subsequently collected as IgG fractions. The IgG fraction was dialyzed against 0.1 M sodium carbonate-bicarbonate buffer (pH 9.0) and 0.5 M NaCl. The purified M11 antibodies were coupled to CNBractivated Sepharose 4B (Amersham Biosciences, Piscataway, NJ, USA). Cloning, expression, and purification of mouse TNX and deletion mutants We previously constructed pSecFTNX-2, which encodes a short alternatively spliced form lacking M3 and M15 – M22 FNIII repeats [37]. Domain-deleted mutants of TNX, pDeltaEGF, which lacks the 18.5 EGF-like repeats, and pDeltaFB, which lacks a fibrinogen-like domain, were generated using the polymerase chain reaction (PCR) method and cloned in the pHMCMV6 vector. Briefly, pDeltaEGF and pDeltaFB were constructed as follows. For the pDeltaEGF construction, the region encoding the fibrinogen-like domain was inserted into the pHMCMV6 vector, yielding pHMCMV6 N B3 N . On the other hand, the pSecFDEFB plasmid, which lacks the 18.5 EGF-like repeats and a fibrinogen-like domain, was constructed by a gene engineering technique. Subsequently, a 6.8-kb fragment from pSecFDEFB obtained by digestion with restriction enzyme NheI were inserted into the NheI site of pHMCMV6NB3N, yielding pDeltaEGF. As for the pDeltaFB construction, a 0.6-kb fragment from pSecFDEFB obtained by double digestion with restriction enzymes NheI and NotI, which encodes the most 3V portion of the FNIII-
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like repeats, was subcloned into the pHMCMV6 vector, yielding pHMCMV6NEFBN. Subsequently, a 8.6-kb fragment from pSecFTNX-2 obtained by digestion with restriction enzyme NheI were inserted into the NheI site of pHMCMV6NEBFN, yielding pDeltaFB. Subconfluent 293T cells were transfected with pSecFTNX-2, pDeltaEGF, or pDeltaFG expression vectors by the calcium phosphate coprecipitation method [38], and cells were incubated for 24 h. Five 109 cells were washed with phosphate-buffered saline (PBS) and collected by centrifugation for 3 min at 700 g at 4jC. The resulting pellet was then suspended in 6 ml of lysis buffer (20 mM Tris – HCl, pH 7.5, 0.5 M NaCl and 1% NP-40) and homogenized in an ultrasonic homogenizer. The homogenate was rotated for 20 min at 4jC and then centrifuged at 13,000 g for 5 min at 4jC. The supernatants were collected. The sample was precipitated with 50% ammonium sulfate by stirring for 1 h at 4jC. After centrifugation for 20 min at 13,000 g, the pellet was dissolved in 20 mM Tris – HCl (pH 7.5) and 0.5 M NaCl and then dialyzed overnight against the same buffer. The preparation was subjected to gel filtration on Sephacryl S-300. Subsequently, the fractions containing TNX, DEGF, or DFB protein were adsorbed to a TNX antibody affinity column for 12 h at 4jC, since TNX, DEGF and DFB each contain the 11th FNIII repeat of mouse TNX. The TNX antibody affinity column was washed extensively with 20 mM Tris – HCl (pH 7.5) and 0.5 M NaCl before eluting TNX, DEGF or DFB with 3 M NaSCN and 0.5 M NaCl. Then, the fractions containing TNX, DEGF, or DFB were dialyzed overnight against PBS and were run by SDS-polyacrylamide gel (PAGE) under reducing conditions. They were detected by Western blot analysis with an anti-FLAG M2 monoclonal antibody (Sigma-Aldrich, Tokyo, Japan) as described previously [35], since these proteins each have a FLAG-tag at the N-terminus, and visualized by Coomassie brilliant blue staining. Isolation of type VI collagen Type VI collagen was isolated from bovine placenta by established procedures [39]. Bovine placenta (25 g, wet weight) was homogenized and extracted with 500 ml of PBS for 24 h at 4jC in the presence of protease inhibitors EDTA (5 mM), phenylmethylsulphonyl fluoride (PMSF) (1 mM), and N-ethylmaleimide (5 mM). After centrifugation at 3160 g for 20 min at 4jC, the pellet was extracted twice with 200 ml of 1 M NaCl and 0.1 M Tris –HCl (pH 7.5) with the above protease inhibitors for 12 h at 4jC. Residual materials were further extracted with 150 ml of 6 M guanidine HCl and 0.1 M Tris –HCl (pH 7.5) with the above protease inhibitors for 8 h at 4jC. After centrifugation at 32,300 g for 20 min at 4jC, the supernatant was dialyzed extensively against 0.05 M acetic acid and lyophilized. Subsequently, the sample was dissolved in 5 ml of 100 mM NaCl, 1 mM EDTA, 50 mM Tris –HCl (pH 7.2) and 0.2% SDS and then passed
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over a Superose 6 gel filtration column [40]. Then, the fractions containing type VI collagen were dialyzed overnight against PBS and analyzed by 7.5% SDS-PAGE containing 3.6 M urea under reducing conditions and detected by Western blot analysis with anti-type VI collagen polyclonal antibodies (LSL, Tokyo, Japan).
At least two mice of each genotype were used for measurement of collagen fibril diameter and density. Micrographs (4/group) from nonoverlapping regions of the dorsal skin were taken from cross-sections. The distribution of collagen fibril diameters was calculated using NIH image. Eight areas in a square (1 Am2) were chosen from dorsal skin of each genotype for determination of density.
Solid-phase assays Ninety-six-well microtiter plates (Maxisorp, Nunc, Tokyo, Japan) were coated overnight at 4jC with 0.5 Ag of TNX, DEGF or DFB protein. After washing the wells with PBS three times, they were saturated with Block Ace (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) at room temperature for 2 h and then incubated with various amounts of type I collagen (BD Biosciences, Bedford, MA, USA) or purified type VI collagen, fibronectin (Chemicon International Inc., Temecula, CA, USA) or laminin (Harbor Bio-products, Norwood, MA, USA) for a further 2 h. Wells were washed with PBS eight times and then incubated for 1 h with polyclonal antibodies specific for type I collagen (LSL), type VI collagen (LSL), fibronectin (Chemicon International Inc.) or laminin (LSL) and then for 1 h with anti-rabbit IgG-conjugated peroxidase (Amersham Biosciences). After washing with PBS eight times, bound peroxidase was detected with H2O2 and 2,2-azino-bis (3ethylbenthiazoline-6-sulfonic acid). The absorbance was measured at 405 nm. Collagen fibril formation assay Type I collagen (BD Biosciences) was dissolved in 0.5 M acetic acid at the concentration of 2 mg/ml. Three hundred Ag of type I collagen was added to 0.6 ml of fibrillogenesis buffer (0.12 M NaCl and 30 mM sodium phosphate, pH 7.3). TNX, type VI collagen, DEGF, or DFB protein dissolved in PBS was added to the sample, and the final volume was brought to 1.0 ml. This mixture was transferred to a cuvette equilibrated at 30jC. The process of fibrillogenesis was measured as turbidity change by monitoring the change in absorbance at 400 nm at 30-min or 1-h intervals. Electron microscopy Immediately after cervical dislocation, caudal dorsal skin from age-matched wild-type and TNX / mice was dissected out. The dorsal skin was immersed in 2% glutaraldehyde (TAAB, UK) and processed for transmission electron microscopy as previously described [41]. Briefly, samples were rinsed in 7% sucrose and post-fixed in 1% osmium tetroxide (Merck, NJ) in 0.1 M phosphate buffer, and thereafter dehydrated in graded acetones and embedded in Epon (TAAB, UK). Ultrathin sections of 0.1 Am in thickness were stained with 3% uranyl acetate and 0.2% lead citrate, and they were examined under a Hitachi H7100 electron microscope (Hitachinaka, Ibaraki, Japan).
Results Production and purification of recombinant TNX, domain-deleted proteins, and type VI collagen After two successive purifications by gel filtration and TNX antibody affinity columns, purified TNX, DEGF (EGF-like domain-deleted TNX), and DFB (fibrinogen-like domain-deleted TNX) proteins (Fig. 1A) were analyzed by 7.5% SDS-PAGE, confirming the excellent level of purity for each protein (Fig. 1Ba). To further characterize the molecular mass of each protein, it was resolved by 3.5% SDS-PAGE. Consequently, each protein was electrophoresed according to its estimated molecular mass, namely, at molecular weights of 340, 275, and 312 kDa for TNX, DEGF and DFB, respectively (Fig. 1Bb). On the other hand, type VI collagen was isolated by guanidinium extraction from bovine placenta followed by gel chromatography on Superose 6. The isolated proteins were resolved on 7.5% SDS-PAGE containing 3.6 M urea and stained with Coomassie brilliant blue (Fig. 1Ca). The immunoblot of this gel was stained with type VI collagen-specific antibodies (Fig. 1Cb). It has been reported that type VI collagen consists of a 200-kDa subunit as the a3(VI) chain and two 140-kDa subunits as a1(VI) and a2(VI) chains [39,40]. A crossreacted band at more than 200 kDa in Fig. 1Cb would correspond to an alternative splicing isoform of the a3(VI) chain as described previously [42]. Purified TNX, domaindeleted TNX proteins, DEGF and DFB, and type VI collagen were used for the following assay. TNX binds to type I collagen but not to type VI collagen Interaction between TNX and type I or type VI collagen was investigated by solid-phase binding assays. First, to confirm that our solid-phase binding assays are well accomplished, the interaction between type VI and type I collagens was performed by our solid-phase binding assay (Fig. 2A). The ability of type VI collagen to bind to type I collagen has been reported [43]. As expected, interaction between type VI and type I collagens was ascertained, indicating that our solid-phase binding system is well accomplished. Next, the interaction between TNX and type I or type VI collagen was examined using TNX as the immobilized substrate with type I or type VI collagen in the soluble phase. As shown in Fig. 2B, the interaction between TNX and type I collagen was dose-dependent and saturable. On the other hand, TNX did
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Fig. 1. SDS-polyacrylamide electrophoresis analysis of purified TNX recombinant proteins and type VI collagen extracted from the placenta. (A) Models of recombinant TNX and domain-deleted mutants, DEGF and DFB. Each recombinant protein is depicted as a linear array from the N-terminus to C-terminus showing the central domain (segment of circle), heptad repeats (wavy line), EGF-like repeats (diamonds), FNIII-like repeats (rectangles) and the fibrinogen-like domain (circle), and the designation of each protein is indicated. (B) Corresponding purified recombinant TNX proteins. (a) The purified recombinant proteins were run on a 7.5% SDS-PAGE after reduction and stained with Coomassie brilliant blue. To the left of the gel, positions of the molecular mass markers are indicated in kDa. (b) The samples were also loaded on 3.5% SDS-PAGE under reducing conditions and then transferred to a nitrocellulose membrane and developed with anti-FLAG monoclonal antibodies. (C) SDS-PAGE containing 3.6 M urea of type VI collagen from the placenta. (a) The purified samples were run under reducing conditions on a 7.5% SDS-PAGE containing 3.6-M urea and stained with Coomassie brilliant blue. (b) Immunoblot with antibodies against type VI collagen. The samples were resolved on a 7.5% SDS-PAGE containing 3.6 M urea under reducing conditions and then transferred to a nitrocellulose membrane and stained with antibodies against type VI collagen. As indicated by arrowheads, the 200-kDa subunit corresponds to the a3(VI) chain and the two 140-kDa subunits correspond to a1(VI) and a2(VI) chains.
not bind to type VI collagen (Fig. 2C). Solid-phase binding assays were also performed to examine TNX binding to other ECM components such as fibronectin and laminin. As shown in Fig. 2D, no TNX binding to fibronectin could be demonstrated. As for TNX binding to laminin, it would seem that very weak binding or almost no binding was observed (Fig. 2E). Next, to inspect which domain of TNX is involved in the binding to type I collagen, purified two domain-deleted proteins, DEGF and DFB, were used as the immobilized substrate for the solid-phase binding assay (Fig. 2F). As shown in Fig. 2F, both DEGF and DFB bound to type I collagen efficiently, indicating that the EGF-like domain and fibrinogen-like domain of TNX are not necessary for the interaction. Probably, the FNIII-like domain would be necessary for the interaction of TNX with type I collagen. Effects of TNX and type VI collagen on in vitro type I collagen fibril formation Since TNX and type VI collagen bind to type I collagen, we considered the possibility that they could affect the rate and/or quantity of collagen fibril formation. We investigated the effects of increasing amounts of TNX (Fig. 3A) and type
VI collagen (Fig. 3B) on type I collagen fibril formation. As shown in Fig. 3A, TNX increased the rate and the final quantity of fibril formation based on the absorbance at 400 nm in a concentration-dependent manner. On the other hand, type VI collagen increased the rate of fibril formation in a concentration-dependent manner but did not alter the final quantity of fibril formation (Fig. 3B). Next, we examined the role of the domains of TNX in type I collagen fibril formation by using the domain-deleted mutants DEGF and DFB. Interestingly, DEGF and DFB increased the final quantity of fibril formation as TNX did, but the initial rate of collagen fibril formation was decreased by the addition of DEGF or DFB compared with that in the case of TNX. This result indicates that EGF-like and fibrinogen-like domains of TNX are involved in the initial rate of fibril formation (Fig. 3C). Furthermore, the effect of combining TNX with type VI collagen on fibril formation was investigated. The combination of TNX and type VI collagen resulted in an additive effect, especially in the rate of fibril formation. In the case of the combination of type VI collagen and domain-deleted mutants, DEGF or DFB, the initial rate of fibril formation suppressed by the domain-deleted mutant was restored by the effect of type VI collagen.
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Fig. 2. Solid-phase assays of interactions between TNX and ECM components. Wells were each coated with 0.5 Ag of type VI collagen (A), TNX (B, C, D, and E), DEGF (F) or DFB (F). After saturation with Block Ace, various amounts of type I collagen (A, B, and F), type VI collagen (C), fibronectin (D), or laminin (E) were added. Bound type I collagen (A, B, and F), type VI collagen (C), fibronectin (D), or laminin (E) was detected by each specific antibody. Results are expressed as means F SE of duplicated experiments.
These results indicate that TNX is involved in the rate and quantity of fibril formation, whereas type VI collagen is involved only in the rate of fibril formation. Collagen fibril diameter distributions during development of TNX / skin The formation of collagen fibrils during dorsal skin development in TNX / mice was analyzed by electron microscopy (Fig. 4A). The fibrils from TNX / mice showed abnormality during development compared to those from wild-type controls. Although no difference was found between the shapes of fibrils from wild-type mice and those from TNX / mice, transmission electron micrographs of
transverse sections showed a higher abundance of fibrils with large diameters in the later stages of development, best seen at 3 months postnatal in the TNX / mice (Fig. 4A). We analyzed the fibril diameters quantitatively during development in the mutant mice (Fig. 4B). To characterize the diameter distributions, all of the fibril diameters (n = 785– 1765) were plotted in a histogram for each group at each developmental point. At 1 month postnatal, the TNX / mice had fibril diameter distributions with a mean diameter of 56.7 F 14.7 nm that were similar to that observed from wild-type skin with a diameter of 61.8 F 14.5 nm. In contrast, at 3 months postnatal, TNX / skin showed an increase in the number of fibrils with large diameters, with a mean diameter of 61.4 F 14.2 nm, compared with those of
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Fig. 3. Collagen fibril formation assay. Effect of TNX (A) or type VI collagen (B) on type I collagen fibril formation. A 300-Ag portion of type I collagen and various amount of TNX (A) or type VI collagen (B) were combined in 1 ml of saline/phosphate buffer, equilibrated at 30jC. In the case of TNX, PBS (control), 0.2 or 0.5 Ag of TNX was added. In the case of type VI collagen, PBS (control), 0.2 or 2 Ag of type VI collagen was added. After mixing, absorbance at 400 nm was monitored at 30-min or 1-h intervals. (C) Effects of TNX mutants, DEGF or DFB, on fibril formation. A 300-Ag portion of type I collagen and 0.5 Ag each of TNX, DEGF, DFB or PBS (control) were combined in 1 ml of saline/phosphate buffer. After mixing, collagen fibril formation was monitored at 30jC. (D) Effect of combining TNX with type VI collagen on fibril formation. About 0.5 Ag each of TNX, DEGF, or DFB, 2 Ag of type VI collagen, or 0.5 Ag each of TNX, DEGF, or DFB in combination with 2 Ag of type VI collagen, or PBS (control) was added in 1 ml of saline/phosphate buffer. After mixing, collagen fibril formation was monitored at 30jC.
normal skin, with a mean diameter of 47.3 F 13.4 nm. At 6 months postnatal, TNX / skin fibrils had diameter distributions distinctly different from those of the normal skin. At 6 months postnatal, the normal skin demonstrated a substantial increase in the number of fibrils with small diameters. In contrast, there was no increase in the number of fibrils with small diameters in TNX / skin. The increase in TNX / fibril diameter appears to be the result of a relative decrease in the smaller fibril population [56.9 F 25.3 nm (wild-type skin) versus 66.4 F 20.1 nm (TNX / skin)]. On the other hand, no significant difference between the density of collagen fibrils of TNX / skin and that of normal skin was found during skin development [number of collagen fibrils in an area of 1 Am2 in wild-type skin versus that in
TNX / skin: 160.9 F 30.3 versus 155.2 F 35.7 at 1 month postnatal (n = 12), 162.5 F 47.9 versus 139.0 F 28.3 at 3 months postnatal (n = 27) and 114.2 F 21.9 versus 119.3 F 19.3 at 6 months postnatal (n = 42)]. Taken together, these results indicate that the skin of TNX / mice exhibits an increased number of large collagen fibrils, but no difference in the density of collagen fibrils compared with that of wildtype mice.
Discussion In this work, we showed that TNX and type VI collagen, the expression of which is regulated by TNX, bind to type I
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Fig. 4. Transmission electron microscopic analyses of dorsal skin from wild-type and TNX / mice during development. (A) Transmission electron micrographs. Shown are transverse (a, c, e, g, i, and k) and longitudinal (b, d, f, h, j, and l) sections of dorsal skin from wild-type (+/+) and TNX / ( / ) mice at different developmental stages: 1 month (a, b, c, and d), 3 months (e, f, g, and h), and 6 months (i, j, k, and l) postnatal. Scale bar, 100 nm. (B) Collagen fibril diameter distributions during development of wild-type and TNX / mice. Collagen fibril diameter distributions are presented as histograms for the dorsal skin of wild-type (+/+) and TNX / ( / ) mice at 1 month (a), 3 months (b) and 6 months (c) postnatal. Four areas from each genotype were randomly selected, and diameters of collagen fibrils present in those areas were measured. (C) Comparison of fibril densities of dorsal skin from wild-type (+/+) and TNX / ( / ) mice at 1 month (a), 3 months (b) and 6 months (c) postnatal. Eight square areas (1 Am2) were randomly selected, and numbers of fibrils present in those areas were counted.
collagen and affect type I collagen fibril formation, TNX being involved in both the rate and quantity of collagen fibril formation and type VI collagen playing a role in the rate of collagen fibril formation. Our earlier studies revealed that the expression of type VI collagen in TNX-null fibroblasts is significantly downregulated at the mRNA and protein levels compared with that in wild-type fibroblasts [36]. It is known that type VI collagen is often associated with collagen fibers forming an interconnecting meshwork [44]. In addition, the mRNA expression levels of other collagen fibril-associated molecules such as decorin were also affected by TNX deficiency. These results suggest that TNX mediates collagen fibrillogenesis via regulation of synthesis of collagen fibril-associated molecules. Taking this indirect effect of TNX on collagen fibrillogenesis into consideration together with the findings in the
present study that TNX directly interacts with type I collagen and regulates type I collagen fibril formation, we speculate that TNX is involved in collagen fibrillogenesis directly and indirectly. Actually, the TNX / mice generated by us showed the skin hyperextensibility and reduced tensile strength (our unpublished observations). The function of each domain of TNX has not yet been investigated fully. As an exception, it has been reported that the FNIII repeats 9 and 10 of bovine TNX interact with integrin avh3 [12] and that heparin and dermatan sulfate chains of decorin bind to the FNIII repeats 10 and 11 of bovine TNX [16,17], indicating that the FNIII domain of TNX is involved in the interactions of cells and extracellular matrices. In the present study, analysis of the involvement of domain-deleted mutants of TNX in collagen fibril formation revealed that EGF-like and fibrinogen-like domains of TNX
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participate in the rate of collagen fibril formation but not in the quantity of fibril formation, demonstrating that the rate and quantity of fibril formation are regulated by distinct domains of TNX. Furthermore, results of solid-phase binding assays indicated that these domains do not take part in the interaction with type I collagen but, rather, the remaining part of TNX, for example, FNIII-like domain, is involved in the binding to type I collagen. Further study is needed to determine the region of TNX involved in collagen fibril formation. Based on observation of the skin of TNX / mice by electron microscopy, collagen fibrillogenesis of the skin of TNX / mice was found to be impaired; in the later stage of development, at 3 months postnatal, the number of fibrils with large diameters was increased in the skin of TNX / mice compared with that in wild-type skin. However, there was no significant difference between the density of collagen fibrils of TNX / skin and that of normal skin. These observations concerning collagen fibrils in the skin of TNX / mice are not consistent with the results of Mao et al. [33]. They reported that in independently derived TNX / mice collagen fibrils were normal size and shape but that the density of fibrils in the skin was reduced. This discrepancy may be due to the differences in genetic background, age, or location from which skin specimens were obtained. In addition to the observation on the density of collagen fibrils, they also showed reduced collagen content, suggesting that the defect in TNX is not in collagen fibrillogenesis but resides in collagen deposition. In contrast, our biochemical evaluation and electron microscopic observation in this paper indicates that TNX is involved in collagen fibrillogenesis directly and indirectly. An in vitro collagen I fibril formation assay is often used to examine the ability of a certain molecule to effect collagen fibrillogenesis [45]. Based on the turbidity curve, in vitro type I collagen fibril formation was divided into three different phases: a lag phase in which there is no detectable change in turbidity, a growth phase in which turbidity is increased drastically, and a plateau phase in which there is a constant level of turbidity. These three phases in in vitro collagen fibril formation coincide with the three phases in in vivo collagen fibrillogenesis. The lag period in in vitro fibril formation would correspond to an early step, the period of molecular assembly of collagen monomers into fibril intermediates, and a stabilization step, the period of stabilization of fibril intermediates with fibrilassociated macromolecules in in vivo collagen fibrillogenesis. The growth phase in in vitro fibril formation would correspond to a fusion step in which fusion of the fibril intermediates occurs to generate mature collagen fibrils [46]. Thus, if only one molecule were involved in collagen fibrillogenesis in vivo, the results from in vitro collagen fibril formation would be consistent with those from in vivo collagen fibrillogenesis. However, many collagen fibril-associated molecules are actually involved in collagen fibrillogenesis in vivo. As shown in Fig. 3,
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TNX acts on collagen fibril formation positively, whereas TNX-deficient mice have larger fibrils on average than do wild-type animals as shown in Fig. 4. The in vitro results regarding collagen fibril formation apparently contradict the in vivo results. As analogous example that the results from in vitro and in vivo fibrillogenesis do not consist, fibromodulin has been reported [45,47]. This might be due to the fact that the phenomenon observed in in vivo collagen fibrillogenesis is the comprehensive outcome of the effects of many collagen fibril-associated molecules such as type VI collagen and decorin, whose expressions are influenced by TNX deficiency [36]. The interaction between TNX and type I collagen and the effect of TNX on collagen fibrillogenesis demonstrated in this study might have important biological implications in terms of understanding Ehlers – Danlos syndrome in pathological situations. The Ehlers –Danlos syndrome is a genetically heterogeneous connective tissue disorder. It has been known that Ehlers – Danlos syndrome is caused by mutations of fibrillar collagen metabolism in which the genes encoding type I, III, and V collagens, as well as many collagen processing enzymes are involved. Recently, it has been reported that some patients with Ehlers –Danlos syndrome have mutations in the TNX gene and they show the recessive pattern of inheritance [32]. However, the mechanism by which TNX deficiency causes the disease remained to be resolved. The facts that TNX is localized on collagen fibrils [27] and binds to type I collagen with affecting the collagen fibril formation as shown in this paper, and influences synthesis of other extracellular matrices such as type VI collagen [36], suggest important roles of TNX in collagen fibrillogenesis. Loss of TNX would cause alteration of macromolecular organization of collagen matrix elements, leading to the Ehlers –Danlos syndrome. Elucidation of the molecular basis of TNX in collagen fibrillogenesis is indispensable for new therapeutic strategies for Ehlers –Danlos syndrome. Acknowledgments We thank Kiyomi Takaya for her technical assistance. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] J.C. Adams, F.M. Watt, Regulation of development and differentiation by the extracellular matrix, Development 117 (1993) 1183 – 1198. [2] H.P. Erickson, M.A. Bourdon, Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors, Annu. Rev. Cell Biol. 5 (1989) 71 – 92. [3] R. Chiquet-Ehrismann, C. Hagios, K. Matsumoto, The tenascin gene family, Perspect. Dev. Neurobiol. 2 (1994) 3 – 7. [4] C. Hagios, M. Koch, J. Spring, M. Chiquet, R. Chiquet-Ehrismann, Tenascin-Y: a protein of novel domain structure is secreted by differ-
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Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen Takeharu Minamitani, a Tomoki Ikuta, a Yoshinari Saito, a Gen Takebe, b Mami Sato, c Hirofumi Sawa, c Takanori Nishimura, d Fumio Nakamura, d Kazuhiko Takahashi, b Hiroyoshi Ariga, a and Ken-ichi Matsumoto a,* a
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Department of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan c Laboratory of Molecular and Cellular Pathology, Graduate School of Medicine, Hokkaido University, Sapporo 060-8638, Japan d Division of Bioresources and Bioproduction, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
b
Received 3 March 2004, revised version received 15 April 2004 Available online 18 May 2004
Abstract Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX / ) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro. D 2004 Elsevier Inc. All rights reserved. Keywords: Collagen fibrillogenesis; Knockout mouse; Tenascin-X; Type VI collagen
Introduction Extracellular matrices (ECMs) consisting of a complex network of macromolecules such as glycoproteins and proteoglycans not only provide a physical framework for cells in terms of mechanical strength, but also participate in cell proliferation, migration, differentiation, and survival. The ECM contains many adhesive proteins, including fibronectin, collagen, and laminin [1]. These proteins generally promote cell attachment or migration. In addition to these adhesive proteins, adhesion modulatory proteins are also present in the ECM and regulate the interactions between cell receptors and these adhesive proteins. Tenascin * Corresponding author. Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita, Sapporo 060-0812, Japan. Fax: +81-11-706-4988. E-mail address: [email protected] (K. Matsumoto). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.04.030
family belongs to such a family [2]. Tenascin family proteins each have a characteristic domain structure with a cysteine-rich segment at the amino terminus, followed by epidermal growth factor (EGF)-like repeats, fibronectin type III (FNIII)-like repeats, and a fibrinogen-like domain at the carboxy terminus. So far, six members of the tenascin family have been identified in various species from zebrafish to mammals: tenascin/cytotactin (tenascin-C, TNC), restictin/J1-160/180 (tenascin-R, TNR), tenascin-X (TNX), tenascin-Y (TNY), tenascin-W (TNW), and recently identified tenascin-N (TNN) [3– 7]. Tenascin family proteins modulate the adhesion of cells on the other ECM components. The cellular ligands mediating these interactions include integrins, proteoglycans and cell adhesion molecules. So far, at least six members of the integrin family that recognize TNC as a ligand have been identified. Five of these (integrins a9h1, avh3, avh6, a8h1, and a7h1) bind to the FNIII repeats; the third FNIII
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repeat of TNC was identified as a cell adhesive site interacting with integrins avh3, avh6, a8h1 through the RDG sequence, and this domain also interacts with integrin a9h1 in an RGD-independent manner [8,9]. Integrin a7h1 has been shown to interact preferentially with the alternative spliced FNIII repeat D of human TNC, leading to an increase in neurite outgrowth [10]. On the other hand, integrin avh3 also binds to the C-terminal fibrinogen-like domain of TNC [11]. Furthermore, the adhesion to FNIII repeats 9 –10 of bovine TNX was shown to be mediated by integrin avh3 in an RGD-dependent manner [12]. Recently, integrin a8h1 has been shown to recognize a FNIII repeat of TNW [7]. Many studies have indicated the important role of interaction of proteoglycan in the functions of members of the tenascin family. Several types of proteoglycans have been shown to bind to TNC. For example, neurocan, a member of the aggrecan family of chondroitin sulfate proteoglycans, has been shown to interact with the FNIII repeats of TNC [13]. Syndecan, a cell surface proteoglycan, has been shown to bind to TNC via its heparan sulfate side chains [14]. Agrin, a major brain heparan sulfate proteoglycan, has been shown to interact with TNC solely via its protein core [15]. TNX binding to glycosaminoglycans, namely heparin and dermatan sulfate chains of decorin, has also been reported [16,17]. The binding sites of them have been identified in FNIII repeats 10– 11 of bovine TNX by the use of recombinant repeats produced by bacteria. As for TNR, lecticans, a family of chondroitin sulfate proteoglycans encompassing aggrecan, versican, neurican, and brevican, have been shown to bind TNR mediated by a protein –protein interaction through the FNIII repeats 3 – 5 of TNR. Brevican had the highest affinity in the lecticans [18]. Interactions between TNR or TNC and cell surface molecules also affect cell adhesion and cell migration. Contactin/F11, an axon-associated GPI-anchored member of the immunoglobulin superfamily, can bind to both TNC [19] and TNR [20]. The interaction of TNR with F11 in in vitro cultures enhanced F11-mediated neurite outgrowth [21]. Furthermore, the h2 subunit of voltage-gated sodium channels also interacts with TNC and TNR with crucial roles in regulation of sodium channel localization and its activity [22]. On the other hand, no evidence of strong interactions between tenascin family proteins and other components of the ECM glycoproteins has been obtained. TNC did not bind to gelatin, laminin, or types I, III, IV, or V collagen [23]. The interaction of TNC with fibronectin was apparently weak. However, there have been no experiments on the interaction of other tenascins with the ECM. Currently, TNX is the largest known member of the tenascin family. Initially, the TNX gene was identified in the class III region of the major histocompatibility complex (MHC) with an overlap of the last exon of the CYP21B gene in the opposite direction [24 –26]. Thereafter, TNX protein was also purified as a new ECM glycoprotein
localized on collagen fibrils and was named flexilin [27]. TNX is expressed much more widely than other tenascins and appears specifically in skeletal muscle, heart, and skin [28 – 30]. Some findings concerning the function of TNX have been reported. TNX-deficiency has been shown to cause Ehlers – Danlos syndrome, a heritable connective tissue disorder. Symptoms of this syndrome are hyperextensible skin, hypermobile joints, hyperelastic skin, and easy bruising [31,32]. Mice with deficiency in TNX (TNX / mice) caused by homologous recombination using embryonic stem cells also showed progressive skin hyperextensibility, similar to patients with Ehlers – Danlos syndrome [33]. These results suggest that TNX plays an important structural role in the skin. Another finding is that TNX is required for impeding the invasion and metastasis of tumor cells. TNX / mice generated by us showed a high incidence of tumor invasion and metastasis following subcutaneous inoculation of B16 –BL6 melanoma cells due to the increased activities of matrix metalloproteinases (MMPs), especially those of MMP-2 and MMP-9 [34]. It has also been shown that TNX is involved in endothelial cell proliferation. TNX interacted with vascular endothelial growth factor B (VEGF-B) and enhanced cell proliferation in combination with VEGF-B [35]. Finally, we have found that TNX influences synthesis of type VI collagen at the RNA level [36]. Expression level of mRNA of type VI collagen was remarkably decreased in TNX-null fibroblasts. Transient expression of TNX in Balb3T3 cells caused an increased mRNA level of type VI collagen compared with that in empty vector and increased the promoter activity of type VI collagen a1 subunit gene. The expressions of type I collagen and other collagen fibril-associated molecules such as type XII and type XIV collagens, decorin, lumican, and fibromodulin were also affected by TNX deficiency. We therefore speculated that TNX plays a role in collagen fibrillogenesis directly and/or indirectly by mediating the levels of collagen fibril-associated proteins such as type VI collagen. To determine the roles of TNX and type VI collagen in collagen fibrillogenesis, the ability of purified TNX and type VI collagen molecules to bind directly to type I collagen was investigated in this study using a solid-phase binding assay. The ability of domain-deleted mutants of TNX to interact with type I collagen was also investigated. Finally, the effects of TNX and type VI collagen on collagen fibril formation were investigated.
Materials and methods Animals The generation of TNX / mice by homologous recombination using embryonic stem (ES) cells has been described previously [34]. The mice were housed in laminar flow cabinets under specific pathogen-free conditions. All
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animal experiments were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Hokkaido University. Cell culture Human embryo kidney 293T cells were grown at 37jC in a 5% CO2 humidified atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui) supplemented with 10% fetal bovine serum (FBS), penicillin (10 units/ml) and streptomycin (10 Ag/ml). Preparation of a TNX antibody affinity column To generate a column prepared with TNX antibody, rabbit M11 polyclonal antibodies, which reacted with the 11th FNIII repeat of mouse TNX [34], were purified. Ten milliliters of polyclonal antiserum from rabbit was added to an equal volume of 0.9% NaCl and was then precipitated with 40% ammonium sulfate by stirring for 1 h at 4jC. After centrifugation for 20 min at 13,000 g, the pellet was dissolved in 2 ml of 0.9% NaCl and dialyzed overnight against 20 mM sodium phosphate buffer (pH 7.4). The concentrated sample was centrifuged and passed over a DEAE-cellulose (DE-52) column (Whatman, Springfield Mill, UK). The flow-through was subsequently collected as IgG fractions. The IgG fraction was dialyzed against 0.1 M sodium carbonate-bicarbonate buffer (pH 9.0) and 0.5 M NaCl. The purified M11 antibodies were coupled to CNBractivated Sepharose 4B (Amersham Biosciences, Piscataway, NJ, USA). Cloning, expression, and purification of mouse TNX and deletion mutants We previously constructed pSecFTNX-2, which encodes a short alternatively spliced form lacking M3 and M15 – M22 FNIII repeats [37]. Domain-deleted mutants of TNX, pDeltaEGF, which lacks the 18.5 EGF-like repeats, and pDeltaFB, which lacks a fibrinogen-like domain, were generated using the polymerase chain reaction (PCR) method and cloned in the pHMCMV6 vector. Briefly, pDeltaEGF and pDeltaFB were constructed as follows. For the pDeltaEGF construction, the region encoding the fibrinogen-like domain was inserted into the pHMCMV6 vector, yielding pHMCMV6 N B3 N . On the other hand, the pSecFDEFB plasmid, which lacks the 18.5 EGF-like repeats and a fibrinogen-like domain, was constructed by a gene engineering technique. Subsequently, a 6.8-kb fragment from pSecFDEFB obtained by digestion with restriction enzyme NheI were inserted into the NheI site of pHMCMV6NB3N, yielding pDeltaEGF. As for the pDeltaFB construction, a 0.6-kb fragment from pSecFDEFB obtained by double digestion with restriction enzymes NheI and NotI, which encodes the most 3V portion of the FNIII-
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like repeats, was subcloned into the pHMCMV6 vector, yielding pHMCMV6NEFBN. Subsequently, a 8.6-kb fragment from pSecFTNX-2 obtained by digestion with restriction enzyme NheI were inserted into the NheI site of pHMCMV6NEBFN, yielding pDeltaFB. Subconfluent 293T cells were transfected with pSecFTNX-2, pDeltaEGF, or pDeltaFG expression vectors by the calcium phosphate coprecipitation method [38], and cells were incubated for 24 h. Five 109 cells were washed with phosphate-buffered saline (PBS) and collected by centrifugation for 3 min at 700 g at 4jC. The resulting pellet was then suspended in 6 ml of lysis buffer (20 mM Tris – HCl, pH 7.5, 0.5 M NaCl and 1% NP-40) and homogenized in an ultrasonic homogenizer. The homogenate was rotated for 20 min at 4jC and then centrifuged at 13,000 g for 5 min at 4jC. The supernatants were collected. The sample was precipitated with 50% ammonium sulfate by stirring for 1 h at 4jC. After centrifugation for 20 min at 13,000 g, the pellet was dissolved in 20 mM Tris – HCl (pH 7.5) and 0.5 M NaCl and then dialyzed overnight against the same buffer. The preparation was subjected to gel filtration on Sephacryl S-300. Subsequently, the fractions containing TNX, DEGF, or DFB protein were adsorbed to a TNX antibody affinity column for 12 h at 4jC, since TNX, DEGF and DFB each contain the 11th FNIII repeat of mouse TNX. The TNX antibody affinity column was washed extensively with 20 mM Tris – HCl (pH 7.5) and 0.5 M NaCl before eluting TNX, DEGF or DFB with 3 M NaSCN and 0.5 M NaCl. Then, the fractions containing TNX, DEGF, or DFB were dialyzed overnight against PBS and were run by SDS-polyacrylamide gel (PAGE) under reducing conditions. They were detected by Western blot analysis with an anti-FLAG M2 monoclonal antibody (Sigma-Aldrich, Tokyo, Japan) as described previously [35], since these proteins each have a FLAG-tag at the N-terminus, and visualized by Coomassie brilliant blue staining. Isolation of type VI collagen Type VI collagen was isolated from bovine placenta by established procedures [39]. Bovine placenta (25 g, wet weight) was homogenized and extracted with 500 ml of PBS for 24 h at 4jC in the presence of protease inhibitors EDTA (5 mM), phenylmethylsulphonyl fluoride (PMSF) (1 mM), and N-ethylmaleimide (5 mM). After centrifugation at 3160 g for 20 min at 4jC, the pellet was extracted twice with 200 ml of 1 M NaCl and 0.1 M Tris –HCl (pH 7.5) with the above protease inhibitors for 12 h at 4jC. Residual materials were further extracted with 150 ml of 6 M guanidine HCl and 0.1 M Tris –HCl (pH 7.5) with the above protease inhibitors for 8 h at 4jC. After centrifugation at 32,300 g for 20 min at 4jC, the supernatant was dialyzed extensively against 0.05 M acetic acid and lyophilized. Subsequently, the sample was dissolved in 5 ml of 100 mM NaCl, 1 mM EDTA, 50 mM Tris –HCl (pH 7.2) and 0.2% SDS and then passed
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over a Superose 6 gel filtration column [40]. Then, the fractions containing type VI collagen were dialyzed overnight against PBS and analyzed by 7.5% SDS-PAGE containing 3.6 M urea under reducing conditions and detected by Western blot analysis with anti-type VI collagen polyclonal antibodies (LSL, Tokyo, Japan).
At least two mice of each genotype were used for measurement of collagen fibril diameter and density. Micrographs (4/group) from nonoverlapping regions of the dorsal skin were taken from cross-sections. The distribution of collagen fibril diameters was calculated using NIH image. Eight areas in a square (1 Am2) were chosen from dorsal skin of each genotype for determination of density.
Solid-phase assays Ninety-six-well microtiter plates (Maxisorp, Nunc, Tokyo, Japan) were coated overnight at 4jC with 0.5 Ag of TNX, DEGF or DFB protein. After washing the wells with PBS three times, they were saturated with Block Ace (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) at room temperature for 2 h and then incubated with various amounts of type I collagen (BD Biosciences, Bedford, MA, USA) or purified type VI collagen, fibronectin (Chemicon International Inc., Temecula, CA, USA) or laminin (Harbor Bio-products, Norwood, MA, USA) for a further 2 h. Wells were washed with PBS eight times and then incubated for 1 h with polyclonal antibodies specific for type I collagen (LSL), type VI collagen (LSL), fibronectin (Chemicon International Inc.) or laminin (LSL) and then for 1 h with anti-rabbit IgG-conjugated peroxidase (Amersham Biosciences). After washing with PBS eight times, bound peroxidase was detected with H2O2 and 2,2-azino-bis (3ethylbenthiazoline-6-sulfonic acid). The absorbance was measured at 405 nm. Collagen fibril formation assay Type I collagen (BD Biosciences) was dissolved in 0.5 M acetic acid at the concentration of 2 mg/ml. Three hundred Ag of type I collagen was added to 0.6 ml of fibrillogenesis buffer (0.12 M NaCl and 30 mM sodium phosphate, pH 7.3). TNX, type VI collagen, DEGF, or DFB protein dissolved in PBS was added to the sample, and the final volume was brought to 1.0 ml. This mixture was transferred to a cuvette equilibrated at 30jC. The process of fibrillogenesis was measured as turbidity change by monitoring the change in absorbance at 400 nm at 30-min or 1-h intervals. Electron microscopy Immediately after cervical dislocation, caudal dorsal skin from age-matched wild-type and TNX / mice was dissected out. The dorsal skin was immersed in 2% glutaraldehyde (TAAB, UK) and processed for transmission electron microscopy as previously described [41]. Briefly, samples were rinsed in 7% sucrose and post-fixed in 1% osmium tetroxide (Merck, NJ) in 0.1 M phosphate buffer, and thereafter dehydrated in graded acetones and embedded in Epon (TAAB, UK). Ultrathin sections of 0.1 Am in thickness were stained with 3% uranyl acetate and 0.2% lead citrate, and they were examined under a Hitachi H7100 electron microscope (Hitachinaka, Ibaraki, Japan).
Results Production and purification of recombinant TNX, domain-deleted proteins, and type VI collagen After two successive purifications by gel filtration and TNX antibody affinity columns, purified TNX, DEGF (EGF-like domain-deleted TNX), and DFB (fibrinogen-like domain-deleted TNX) proteins (Fig. 1A) were analyzed by 7.5% SDS-PAGE, confirming the excellent level of purity for each protein (Fig. 1Ba). To further characterize the molecular mass of each protein, it was resolved by 3.5% SDS-PAGE. Consequently, each protein was electrophoresed according to its estimated molecular mass, namely, at molecular weights of 340, 275, and 312 kDa for TNX, DEGF and DFB, respectively (Fig. 1Bb). On the other hand, type VI collagen was isolated by guanidinium extraction from bovine placenta followed by gel chromatography on Superose 6. The isolated proteins were resolved on 7.5% SDS-PAGE containing 3.6 M urea and stained with Coomassie brilliant blue (Fig. 1Ca). The immunoblot of this gel was stained with type VI collagen-specific antibodies (Fig. 1Cb). It has been reported that type VI collagen consists of a 200-kDa subunit as the a3(VI) chain and two 140-kDa subunits as a1(VI) and a2(VI) chains [39,40]. A crossreacted band at more than 200 kDa in Fig. 1Cb would correspond to an alternative splicing isoform of the a3(VI) chain as described previously [42]. Purified TNX, domaindeleted TNX proteins, DEGF and DFB, and type VI collagen were used for the following assay. TNX binds to type I collagen but not to type VI collagen Interaction between TNX and type I or type VI collagen was investigated by solid-phase binding assays. First, to confirm that our solid-phase binding assays are well accomplished, the interaction between type VI and type I collagens was performed by our solid-phase binding assay (Fig. 2A). The ability of type VI collagen to bind to type I collagen has been reported [43]. As expected, interaction between type VI and type I collagens was ascertained, indicating that our solid-phase binding system is well accomplished. Next, the interaction between TNX and type I or type VI collagen was examined using TNX as the immobilized substrate with type I or type VI collagen in the soluble phase. As shown in Fig. 2B, the interaction between TNX and type I collagen was dose-dependent and saturable. On the other hand, TNX did
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Fig. 1. SDS-polyacrylamide electrophoresis analysis of purified TNX recombinant proteins and type VI collagen extracted from the placenta. (A) Models of recombinant TNX and domain-deleted mutants, DEGF and DFB. Each recombinant protein is depicted as a linear array from the N-terminus to C-terminus showing the central domain (segment of circle), heptad repeats (wavy line), EGF-like repeats (diamonds), FNIII-like repeats (rectangles) and the fibrinogen-like domain (circle), and the designation of each protein is indicated. (B) Corresponding purified recombinant TNX proteins. (a) The purified recombinant proteins were run on a 7.5% SDS-PAGE after reduction and stained with Coomassie brilliant blue. To the left of the gel, positions of the molecular mass markers are indicated in kDa. (b) The samples were also loaded on 3.5% SDS-PAGE under reducing conditions and then transferred to a nitrocellulose membrane and developed with anti-FLAG monoclonal antibodies. (C) SDS-PAGE containing 3.6 M urea of type VI collagen from the placenta. (a) The purified samples were run under reducing conditions on a 7.5% SDS-PAGE containing 3.6-M urea and stained with Coomassie brilliant blue. (b) Immunoblot with antibodies against type VI collagen. The samples were resolved on a 7.5% SDS-PAGE containing 3.6 M urea under reducing conditions and then transferred to a nitrocellulose membrane and stained with antibodies against type VI collagen. As indicated by arrowheads, the 200-kDa subunit corresponds to the a3(VI) chain and the two 140-kDa subunits correspond to a1(VI) and a2(VI) chains.
not bind to type VI collagen (Fig. 2C). Solid-phase binding assays were also performed to examine TNX binding to other ECM components such as fibronectin and laminin. As shown in Fig. 2D, no TNX binding to fibronectin could be demonstrated. As for TNX binding to laminin, it would seem that very weak binding or almost no binding was observed (Fig. 2E). Next, to inspect which domain of TNX is involved in the binding to type I collagen, purified two domain-deleted proteins, DEGF and DFB, were used as the immobilized substrate for the solid-phase binding assay (Fig. 2F). As shown in Fig. 2F, both DEGF and DFB bound to type I collagen efficiently, indicating that the EGF-like domain and fibrinogen-like domain of TNX are not necessary for the interaction. Probably, the FNIII-like domain would be necessary for the interaction of TNX with type I collagen. Effects of TNX and type VI collagen on in vitro type I collagen fibril formation Since TNX and type VI collagen bind to type I collagen, we considered the possibility that they could affect the rate and/or quantity of collagen fibril formation. We investigated the effects of increasing amounts of TNX (Fig. 3A) and type
VI collagen (Fig. 3B) on type I collagen fibril formation. As shown in Fig. 3A, TNX increased the rate and the final quantity of fibril formation based on the absorbance at 400 nm in a concentration-dependent manner. On the other hand, type VI collagen increased the rate of fibril formation in a concentration-dependent manner but did not alter the final quantity of fibril formation (Fig. 3B). Next, we examined the role of the domains of TNX in type I collagen fibril formation by using the domain-deleted mutants DEGF and DFB. Interestingly, DEGF and DFB increased the final quantity of fibril formation as TNX did, but the initial rate of collagen fibril formation was decreased by the addition of DEGF or DFB compared with that in the case of TNX. This result indicates that EGF-like and fibrinogen-like domains of TNX are involved in the initial rate of fibril formation (Fig. 3C). Furthermore, the effect of combining TNX with type VI collagen on fibril formation was investigated. The combination of TNX and type VI collagen resulted in an additive effect, especially in the rate of fibril formation. In the case of the combination of type VI collagen and domain-deleted mutants, DEGF or DFB, the initial rate of fibril formation suppressed by the domain-deleted mutant was restored by the effect of type VI collagen.
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Fig. 2. Solid-phase assays of interactions between TNX and ECM components. Wells were each coated with 0.5 Ag of type VI collagen (A), TNX (B, C, D, and E), DEGF (F) or DFB (F). After saturation with Block Ace, various amounts of type I collagen (A, B, and F), type VI collagen (C), fibronectin (D), or laminin (E) were added. Bound type I collagen (A, B, and F), type VI collagen (C), fibronectin (D), or laminin (E) was detected by each specific antibody. Results are expressed as means F SE of duplicated experiments.
These results indicate that TNX is involved in the rate and quantity of fibril formation, whereas type VI collagen is involved only in the rate of fibril formation. Collagen fibril diameter distributions during development of TNX / skin The formation of collagen fibrils during dorsal skin development in TNX / mice was analyzed by electron microscopy (Fig. 4A). The fibrils from TNX / mice showed abnormality during development compared to those from wild-type controls. Although no difference was found between the shapes of fibrils from wild-type mice and those from TNX / mice, transmission electron micrographs of
transverse sections showed a higher abundance of fibrils with large diameters in the later stages of development, best seen at 3 months postnatal in the TNX / mice (Fig. 4A). We analyzed the fibril diameters quantitatively during development in the mutant mice (Fig. 4B). To characterize the diameter distributions, all of the fibril diameters (n = 785– 1765) were plotted in a histogram for each group at each developmental point. At 1 month postnatal, the TNX / mice had fibril diameter distributions with a mean diameter of 56.7 F 14.7 nm that were similar to that observed from wild-type skin with a diameter of 61.8 F 14.5 nm. In contrast, at 3 months postnatal, TNX / skin showed an increase in the number of fibrils with large diameters, with a mean diameter of 61.4 F 14.2 nm, compared with those of
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Fig. 3. Collagen fibril formation assay. Effect of TNX (A) or type VI collagen (B) on type I collagen fibril formation. A 300-Ag portion of type I collagen and various amount of TNX (A) or type VI collagen (B) were combined in 1 ml of saline/phosphate buffer, equilibrated at 30jC. In the case of TNX, PBS (control), 0.2 or 0.5 Ag of TNX was added. In the case of type VI collagen, PBS (control), 0.2 or 2 Ag of type VI collagen was added. After mixing, absorbance at 400 nm was monitored at 30-min or 1-h intervals. (C) Effects of TNX mutants, DEGF or DFB, on fibril formation. A 300-Ag portion of type I collagen and 0.5 Ag each of TNX, DEGF, DFB or PBS (control) were combined in 1 ml of saline/phosphate buffer. After mixing, collagen fibril formation was monitored at 30jC. (D) Effect of combining TNX with type VI collagen on fibril formation. About 0.5 Ag each of TNX, DEGF, or DFB, 2 Ag of type VI collagen, or 0.5 Ag each of TNX, DEGF, or DFB in combination with 2 Ag of type VI collagen, or PBS (control) was added in 1 ml of saline/phosphate buffer. After mixing, collagen fibril formation was monitored at 30jC.
normal skin, with a mean diameter of 47.3 F 13.4 nm. At 6 months postnatal, TNX / skin fibrils had diameter distributions distinctly different from those of the normal skin. At 6 months postnatal, the normal skin demonstrated a substantial increase in the number of fibrils with small diameters. In contrast, there was no increase in the number of fibrils with small diameters in TNX / skin. The increase in TNX / fibril diameter appears to be the result of a relative decrease in the smaller fibril population [56.9 F 25.3 nm (wild-type skin) versus 66.4 F 20.1 nm (TNX / skin)]. On the other hand, no significant difference between the density of collagen fibrils of TNX / skin and that of normal skin was found during skin development [number of collagen fibrils in an area of 1 Am2 in wild-type skin versus that in
TNX / skin: 160.9 F 30.3 versus 155.2 F 35.7 at 1 month postnatal (n = 12), 162.5 F 47.9 versus 139.0 F 28.3 at 3 months postnatal (n = 27) and 114.2 F 21.9 versus 119.3 F 19.3 at 6 months postnatal (n = 42)]. Taken together, these results indicate that the skin of TNX / mice exhibits an increased number of large collagen fibrils, but no difference in the density of collagen fibrils compared with that of wildtype mice.
Discussion In this work, we showed that TNX and type VI collagen, the expression of which is regulated by TNX, bind to type I
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Fig. 4. Transmission electron microscopic analyses of dorsal skin from wild-type and TNX / mice during development. (A) Transmission electron micrographs. Shown are transverse (a, c, e, g, i, and k) and longitudinal (b, d, f, h, j, and l) sections of dorsal skin from wild-type (+/+) and TNX / ( / ) mice at different developmental stages: 1 month (a, b, c, and d), 3 months (e, f, g, and h), and 6 months (i, j, k, and l) postnatal. Scale bar, 100 nm. (B) Collagen fibril diameter distributions during development of wild-type and TNX / mice. Collagen fibril diameter distributions are presented as histograms for the dorsal skin of wild-type (+/+) and TNX / ( / ) mice at 1 month (a), 3 months (b) and 6 months (c) postnatal. Four areas from each genotype were randomly selected, and diameters of collagen fibrils present in those areas were measured. (C) Comparison of fibril densities of dorsal skin from wild-type (+/+) and TNX / ( / ) mice at 1 month (a), 3 months (b) and 6 months (c) postnatal. Eight square areas (1 Am2) were randomly selected, and numbers of fibrils present in those areas were counted.
collagen and affect type I collagen fibril formation, TNX being involved in both the rate and quantity of collagen fibril formation and type VI collagen playing a role in the rate of collagen fibril formation. Our earlier studies revealed that the expression of type VI collagen in TNX-null fibroblasts is significantly downregulated at the mRNA and protein levels compared with that in wild-type fibroblasts [36]. It is known that type VI collagen is often associated with collagen fibers forming an interconnecting meshwork [44]. In addition, the mRNA expression levels of other collagen fibril-associated molecules such as decorin were also affected by TNX deficiency. These results suggest that TNX mediates collagen fibrillogenesis via regulation of synthesis of collagen fibril-associated molecules. Taking this indirect effect of TNX on collagen fibrillogenesis into consideration together with the findings in the
present study that TNX directly interacts with type I collagen and regulates type I collagen fibril formation, we speculate that TNX is involved in collagen fibrillogenesis directly and indirectly. Actually, the TNX / mice generated by us showed the skin hyperextensibility and reduced tensile strength (our unpublished observations). The function of each domain of TNX has not yet been investigated fully. As an exception, it has been reported that the FNIII repeats 9 and 10 of bovine TNX interact with integrin avh3 [12] and that heparin and dermatan sulfate chains of decorin bind to the FNIII repeats 10 and 11 of bovine TNX [16,17], indicating that the FNIII domain of TNX is involved in the interactions of cells and extracellular matrices. In the present study, analysis of the involvement of domain-deleted mutants of TNX in collagen fibril formation revealed that EGF-like and fibrinogen-like domains of TNX
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participate in the rate of collagen fibril formation but not in the quantity of fibril formation, demonstrating that the rate and quantity of fibril formation are regulated by distinct domains of TNX. Furthermore, results of solid-phase binding assays indicated that these domains do not take part in the interaction with type I collagen but, rather, the remaining part of TNX, for example, FNIII-like domain, is involved in the binding to type I collagen. Further study is needed to determine the region of TNX involved in collagen fibril formation. Based on observation of the skin of TNX / mice by electron microscopy, collagen fibrillogenesis of the skin of TNX / mice was found to be impaired; in the later stage of development, at 3 months postnatal, the number of fibrils with large diameters was increased in the skin of TNX / mice compared with that in wild-type skin. However, there was no significant difference between the density of collagen fibrils of TNX / skin and that of normal skin. These observations concerning collagen fibrils in the skin of TNX / mice are not consistent with the results of Mao et al. [33]. They reported that in independently derived TNX / mice collagen fibrils were normal size and shape but that the density of fibrils in the skin was reduced. This discrepancy may be due to the differences in genetic background, age, or location from which skin specimens were obtained. In addition to the observation on the density of collagen fibrils, they also showed reduced collagen content, suggesting that the defect in TNX is not in collagen fibrillogenesis but resides in collagen deposition. In contrast, our biochemical evaluation and electron microscopic observation in this paper indicates that TNX is involved in collagen fibrillogenesis directly and indirectly. An in vitro collagen I fibril formation assay is often used to examine the ability of a certain molecule to effect collagen fibrillogenesis [45]. Based on the turbidity curve, in vitro type I collagen fibril formation was divided into three different phases: a lag phase in which there is no detectable change in turbidity, a growth phase in which turbidity is increased drastically, and a plateau phase in which there is a constant level of turbidity. These three phases in in vitro collagen fibril formation coincide with the three phases in in vivo collagen fibrillogenesis. The lag period in in vitro fibril formation would correspond to an early step, the period of molecular assembly of collagen monomers into fibril intermediates, and a stabilization step, the period of stabilization of fibril intermediates with fibrilassociated macromolecules in in vivo collagen fibrillogenesis. The growth phase in in vitro fibril formation would correspond to a fusion step in which fusion of the fibril intermediates occurs to generate mature collagen fibrils [46]. Thus, if only one molecule were involved in collagen fibrillogenesis in vivo, the results from in vitro collagen fibril formation would be consistent with those from in vivo collagen fibrillogenesis. However, many collagen fibril-associated molecules are actually involved in collagen fibrillogenesis in vivo. As shown in Fig. 3,
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TNX acts on collagen fibril formation positively, whereas TNX-deficient mice have larger fibrils on average than do wild-type animals as shown in Fig. 4. The in vitro results regarding collagen fibril formation apparently contradict the in vivo results. As analogous example that the results from in vitro and in vivo fibrillogenesis do not consist, fibromodulin has been reported [45,47]. This might be due to the fact that the phenomenon observed in in vivo collagen fibrillogenesis is the comprehensive outcome of the effects of many collagen fibril-associated molecules such as type VI collagen and decorin, whose expressions are influenced by TNX deficiency [36]. The interaction between TNX and type I collagen and the effect of TNX on collagen fibrillogenesis demonstrated in this study might have important biological implications in terms of understanding Ehlers – Danlos syndrome in pathological situations. The Ehlers –Danlos syndrome is a genetically heterogeneous connective tissue disorder. It has been known that Ehlers – Danlos syndrome is caused by mutations of fibrillar collagen metabolism in which the genes encoding type I, III, and V collagens, as well as many collagen processing enzymes are involved. Recently, it has been reported that some patients with Ehlers –Danlos syndrome have mutations in the TNX gene and they show the recessive pattern of inheritance [32]. However, the mechanism by which TNX deficiency causes the disease remained to be resolved. The facts that TNX is localized on collagen fibrils [27] and binds to type I collagen with affecting the collagen fibril formation as shown in this paper, and influences synthesis of other extracellular matrices such as type VI collagen [36], suggest important roles of TNX in collagen fibrillogenesis. Loss of TNX would cause alteration of macromolecular organization of collagen matrix elements, leading to the Ehlers –Danlos syndrome. Elucidation of the molecular basis of TNX in collagen fibrillogenesis is indispensable for new therapeutic strategies for Ehlers –Danlos syndrome. Acknowledgments We thank Kiyomi Takaya for her technical assistance. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. References [1] J.C. Adams, F.M. Watt, Regulation of development and differentiation by the extracellular matrix, Development 117 (1993) 1183 – 1198. [2] H.P. Erickson, M.A. Bourdon, Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors, Annu. Rev. Cell Biol. 5 (1989) 71 – 92. [3] R. Chiquet-Ehrismann, C. Hagios, K. Matsumoto, The tenascin gene family, Perspect. Dev. Neurobiol. 2 (1994) 3 – 7. [4] C. Hagios, M. Koch, J. Spring, M. Chiquet, R. Chiquet-Ehrismann, Tenascin-Y: a protein of novel domain structure is secreted by differ-
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