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Vol. 20, Issue 3, 846-858, February 1, 2009
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*Faculty of Medicine, Department of Anatomy and Cell Biology, and Faculty of Dentistry, McGill University, Montreal, QC, Canada H3A 2B2; and Departments of Biomolecular Chemistry and Medicine, University of Wisconsin, Madison, WI 53706
Submitted August 13, 2008;
Revised October 27, 2008;
Accepted November 17, 2008
Monitoring Editor: Jean E. Schwarzbauer
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Fibrillins are 
350 kDa in size, are disulfide-rich and glycosylated, and have a characteristic modular structure. The three human fibrillins—fibrillin-1, -2, and -3 —are encoded by different genes and are conserved at the amino acid level both in relation to one another and among species. Fibrillins are mainly composed of tandem arrays of calcium-binding epidermal growth factor-like domains interspersed with TGF-β–binding protein domains (TB/8-Cys) and hybrid domains (Kielty et al., 2005; Hubmacher et al., 2006). Mutations in fibrillins give rise to the so-called fibrillinopathies, which include Marfan syndrome and autosomal dominant Weill-Marchesani syndrome, both caused by mutations in fibrillin-1, and Beal's syndrome, caused by mutations in fibrillin-2 (Robinson et al., 2006).
High-molecular-weight, multiprotein assemblies called microfibrils are the functional units of fibrillins, which serve as a scaffold for the biogenesis of elastic fibers, confer structural integrity to individual organ systems, regulate growth factor signaling of TGF-β–bone morphogenic protein (BMP) superfamily members and provide limited elasticity to tissues (Kielty et al., 2002; Charbonneau et al., 2004; Ramirez and Dietz, 2007). Extracted microfibrils from cell culture or tissues display a typical bead-on-a-string ultrastructure, having a 50–55-nm periodicity when analyzed by electron microscopy after rotary shadowing (Keene et al., 1991; Kielty et al., 1991). Although many publications over recent years have addressed the spatial organization of fibrillins as it relates to the bead-on-a-string microfibrillar structure (Sakai et al., 1991; Reinhardt et al., 1996; Downing et al., 1996; Liu et al., 1996; Qian and Glanville, 1997; Baldock et al., 2001, 2006; Lee et al., 2004; Kuo et al., 2007), little information is available about the dynamic processes and components involved in microfibril formation. The pathogenetic relevance of this is highlighted by the fact that microfibril assembly is frequently disturbed in patients with fibrillinopathies (Tiedemann et al., 2004).
One of the early mechanisms in the multistep, cell-associated fibrillin assembly process involves N-to-C-terminal (N–C) self-interactions, whereby the interaction sites for fibrillin-1 have been mapped to the N- and C-terminal ends of the molecule (Lin et al., 2002; Marson et al., 2005; Hubmacher et al., 2008). In this process, cell-associated multimerization of the fibrillin-1 C-terminus appears to be an important determinant in generating high-affinity binding sites for the fibrillin-1 N-terminus (Hubmacher et al., 2008). In addition to linear N–C interactions, lateral homotypic interactions in different regions of the fibrillin-1 molecule may play a role in stabilizing initial multimers or the lateral associations of individual microfibrils (Trask et al., 1999; Ashworth et al., 1999; Marson et al., 2005). Although glycosaminoglycans of the heparin/heparan sulfate family have an important, but as yet undefined role in microfibril formation (Tiedemann et al., 2001; Ritty et al., 2003), it is currently not known whether other components on the cell surface or in the extracellular matrix are involved in the multifacetted fibrillin assembly process.
Fibronectin, like the fibrillins, is also a modular protein, consisting of fibronectin types I, II, and III (FNI, FNII, and FNIII) domains that confer self-assembly and ligand-binding properties (Hynes, 1985; Pankov and Yamada, 2002). Fibronectin exists in two forms: cellular fibronectin present in tissues where it is assembled into a fibrous network, and soluble plasma fibronectin, which polymerizes upon blood vessel injury. Fibronectin is secreted from cells as disulfide-bonded, soluble inactive dimers that must be activated to assemble into fibrils (Mao and Schwarzbauer, 2005). Activation occurs primarily through interaction with the cell surface 
5β1 integrin, but can be replaced by other integrins (Akiyama et al., 1989; Fogerty et al., 1990; Takahashi et al., 2007). Tension generated by the intracellular actin cytoskeleton stretches fibronectin molecules through ligated integrin receptors, leading to the exposure of cryptic self-interaction sites and subsequently to the formation of an extracellular fibronectin network (Zhong et al., 1998; Baneyx et al., 2002). There are at least four self-interaction sites present on each fibronectin subunit. The most important epitope for assembly is located in the N-terminally located five FNI domains, and this region is indispensable for fibronectin assembly (Keown-Longo and Mosher, 1985; McDonald et al., 1987; Sottile et al., 1991). This portion of the molecule is directly followed by the binding site for collagen/gelatin, which is located between FNI6 and FNI9 (Engvall et al., 1978; Balian et al., 1979; Shimizu et al., 1997). Although a plethora of reports have been published on the initial assembly mechanisms for fibronectin, virtually nothing is known about how individual fibronectin molecules are spatially oriented and organized in early and fully assembled fibronectin networks.
The assembly of a few extracellular matrix proteins have been demonstrated to be dependent on the presence of fibronectin, including collagen types I and III and thrombospondin-1 (McDonald et al., 1982; Sottile and Hocking, 2002; Velling et al., 2002; Li et al., 2003), fibulin-1 (Roman and McDonald, 1993; Godyna et al., 1995), fibrinogen (Pereira et al., 2002), and LTBP-1 (Dallas et al., 2005). Further studies established for collagen type I, thrombospondin-1, and LTBP-1 that a continuous assembly and supply of fibronectin is a prerequisite for matrix stability of these proteins (Sottile and Hocking, 2002; Dallas et al., 2005). These data suggest that fibronectin is a "master organizer" for the biogenesis of the extracellular matrix (Dallas et al., 2006). It is conceivable that this functional aspect of fibronectin contributes to the severe in vivo defects in mice lacking the fibronectin gene, which include mesodermal, vascular, and neural tube defects, resulting in death around embryonic day 8.5 (George et al., 1993).
Our study aimed to define the role of fibronectin in the assembly of fibrillin-1 and to characterize the molecular interactions between these two proteins. We show that the presence of a fibronectin network is essential for the assembly of fibrillin-1 in cultures of human dermal fibroblasts. Various protein-binding experiments demonstrate direct and strong interactions between fibrillins and fibronectin at the molecular level.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-rFBN1-N; synonym -rF16) or against the C-terminal half (-rFBN1-C; synonym -rF6H) of human fibrillin-1 was described in detail previously (Tiedemann et al., 2001, 2005). For some experiments, the -rFBN1-C antiserum was further purified by standard affinity chromatography using the rFBN1-C antigen. Polyclonal rabbit antibodies against the N-terminal half (-rFBN2-N) or against the C-terminal half (-rFBN2-C) of human fibrillin-2 have been characterized previously (Lin et al., 2002). A new polyclonal rabbit antiserum against the recombinant C-terminal half of human fibrillin-3 (rFBN3-C, see Proteins) has been produced and characterized by standard techniques. The mouse monoclonal anti-fibrillin-1 antibody (mAb15) was a generous gift from Dr. Lynn Sakai (Shriners Hospital for Children, Portland, OR). The mouse monoclonal anti-fibronectin antibody (FN-15) was commercially purchased (Sigma, St. Louis, MO; product F7387). FITC- and Cy3-conjugated AffiniPure goat anti-rabbit IgG (H+L), FITC-, and Cy3-conjugated AffiniPure goat anti-mouse IgG (H+L); 12-nm colloidal gold-AffiniPure donkey anti-mouse IgG (H+L); and 18-nm colloidal gold-AffiniPure donkey anti-rabbit IgG (H+L) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
To exclude cross-reactivity between the anti-fibrillin-1 antisera -rFBN1-N and -rFBN1-C with the anti-fibronectin antibody FN-15 in colocalization experiments, the antibodies were tested by standard enzyme-linked immunosorbent assays (ELISAs). The antibodies were used at appropriate concentrations, as indicated, where cross-reactivity was minimal. Additionally, absence of cross-reactivity between -rFBN1-N and -rFBN1-C with FN-15 was shown by immunofluorescence after down-regulation of the fibrillin-1 expression by small interfering RNA (siRNA) gene silencing.
Proteins
Recombinant Fibrillin Fragments.
Production of the N- and C-terminal fragments of human fibrillin-1 (rFBN1-N, rFBN1-C, respectively) and fibrillin-2 (rFBN2-N, rFBN2-C, respectively) has been described in detail previously (Jensen et al., 2001; Lin et al., 2002). To produce a new recombinant C-terminal half of human fibrillin-3 (rFBN3-C; D1321-R2684), the cDNA for human fibrillin-3 (clone KIAA1776, Nagase et al., 2001) was obtained from the Kazusa DNA Research Institute (Chiba, Japan) and cloned into the pBluescript II SK(+) plasmid (Stratagene, La Jolla, CA). The resulting plasmid pBS-FBN3 was cut with NheI x SgrAI and the 7888-base pair fragment was ligated with the complementary oligonucleotides 5'-CTAGCAGACCTGGATGAATGAATGCATCTCCCAGGAGCA-3' and 5'-CCGGTGCTTGGGAGATGCATTCATCCAGGTCTG-3' generating a NheI restriction site at the 5' end (pBS-rFBN3C-1). A PCR reaction with pBS-FBN3 as a template was performed with the sense primer 5'-AAGAACCTCATCGGTACCTTCG-3' and the antisense primer 5'-CATGAGCGGCCCGCCTATTAGTGATGGTGATGGTGATGGTGCCGAGGGGAGAGGCCATTGA-3', introducing a heptahistidine tag, two consecutive stop codons and a NotI restriction site at the 3' end. After subcloning the 1416-base pair PCR product in the pCRII-TOPO vector (Invitrogen, Carlsbad, CA), an 874-base pair SphI x NotI fragment from the resulting plasmid was ligated with the 5677-base pair SphI x NotI fragment from pBS-rFBN3C-1, resulting in pBS-rFBN3-C. This plasmid was digested with NotI x NheI, and the 4127-base pair fragment was ligated with the vector backbone of the pDNSP-rF1F plasmid (El-Hallous et al., 2007). The final expression plasmid pDNSP-rFBN3-C encodes the BM40 signal peptide for secretion, generating an artificial APLA sequence, followed by the human fibrillin-3 amino acid sequence and a heptahistidine tag (APLAD1321-R2684HHHHHHH). Transfection of human embryonic kidney cells (HEK293 cells), selection of protein-producing clones and protein purification using metal-affinity chromatography have been described in detail elsewhere (Lin et al., 2002).
Fibronectin and Recombinant Fibronectin Fragments.
Purified human plasma full-length fibronectin was commercially purchased (Millipore, Bedford, MA; product FC010). Production of the recombinant fibronectin fragment FNIII1-C has been described in detail previously (Ensenberger et al., 2004). The generation of a 40-kDa, gelatin-binding fragment from human plasma fibronectin followed an established procedure (Chernousov et al., 1991). The new FNsuper70K construct spans the N-terminus through module FNIII3 plus 17 residues (GNFFKKTLPMLSYQDCS) from the following intron and is the human homolog to a splice variant found in zebrafish (Liu et al., 2003). It was produced recombinantly by modification of the pCOCO baculovirus expression vector encoding FN70K (Mosher et al., 2002; Tomasini-Johansson et al., 2006). DNA for encoding the 17-residue sequence was produced by annealing overlapping oligonucleotides, 5'-ACGCTCCGGAAACTTCTTCAAGAAGACACTTCCTATGTTATC-3' and 5'-CCATCTGCAGAACAATCCTGATAAGATAACATAGGAAGTGTCTTC-3', and extending with Taq polymerase. The resulting 67-base pair segment was digested with BspEI and PstI and used as follows. BspEI and PstI sites were added to the 3' end of DNA encoding FNIII3 by PCR amplification of a portion of the fibronectin cDNA from 5' to a natural NcoI restriction site through FNIII3 using the sense primer 5'-CACAGAACTATGATGCCGACCAG-3' and the antisense primer 5'-GAGACTGCAGTGTTCCGGAGCGTGGGGTGCCAGTGG-3'. This DNA segment was digested with NcoI and PstI and cloned into the baculovirus expression vector pCOCO encoding FN70K, extending the insert to encode the N-terminus through FNIII3. The modified pCOCO was digested with XmaI and PstI, and the insert was ligated into pGEM-4z digested with these same enzymes. The modified pGEM-4z was cleaved with BspEI and PstI and ligated to the DNA with the intron-encoding sequence, thus further extending the coding sequence to include the 17 residues. The further modified pGEM-4z was then digested with NcoI and PstI, and the insert was ligated into the baculovirus expression vector containing FN70K, resulting in the final FNsuper70K construct ending in a C-terminal affinity tag sequence (AGHHHHHH).
Cell Culture
Human skin fibroblasts (HSFs) were isolated after a standard circumcision procedure from the foreskins of healthy individuals (2–5 years of age). The procedure was approved by the local ethics committee (PED-06-054), and informed consent was obtained from the parents of the tissue donors. HSFs were used for the experiments between passage 2 and 7 and were incubated at 37°C in a 5% CO2 atmosphere with DMEM supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (Wisent, St. Bruno, QC, Canada). For some experiments, as indicated, the fibronectin present in the FCS was depleted using Gelatin Sepharose 4B as instructed by the manufacturer (GE Healthcare, Waukesha, WI), and the efficiency of the depletion was validated by immunoblotting.
Gene Silencing Experiments
Duplexed siRNAs were purchased and included Hs_FN1_7 (target: human FN1; 5'-CCCGGTTGTTATGACAATGGA-3') and Hs_FBN1_1 (target: human FBN1; 5'-ACCGGTTTACCCGTTGATATT-3'; Qiagen, Chatsworth, CA). The "AllStars Negative Control" siRNA was used as control in all siRNA gene-silencing experiments (Qiagen). This siRNA has no homology to any known mammalian gene and has been validated using the Affymetrix GeneChip (Qiagen). The siRNAs (20 nM) were transfected into HSFs in suspension (1 x 105 cells/ml) using Lipofectamine 2000 (Invitrogen) in DMEM (with fibronectin-depleted FCS and without antibiotics) according to the supplier's instructions. Transfected and nontransfected control HSFs were seeded either at 3.75 x 104 cells/well in an eight-well chamber slide (0.7-cm2 growth area; BD Biosciences, San Jose, CA) for immunofluorescence experiments or at 5.0 x 105 cells/well in six-well plates (9.6-cm2 growth area; Corning Costar, Acton, MA) for production of 1) serum-free conditioned medium used for immunoblotting experiments, 2) fibronectin-depleted conditioned medium to test assembly of exogenous fibrillin-1, and 3) cells for total RNA extraction. The medium containing the transfection solution was replaced by DMEM (with fibronectin-depleted FCS) 24 h after cell seeding (300 µl DMEM/well for eight-well chamber slides; 2 ml DMEM/well for six-well plates). For immunofluorescence assays, cells were immunostained 2–4 d after seeding. For immunoblotting, the DMEM culture medium in six-well plates was replaced by serum-free DMEM (2 ml/well) 48 h after cell seeding and was harvested after an additional 48-h incubation period, and 0.33-ml aliquots were analyzed after precipitation with 10% trichloroacetic acid by standard Western blotting under nonreduced conditions. Cell layers were then washed with 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.47 mM KH2PO4, pH 7.4 (phosphate-buffered saline [PBS]), immediately after harvesting the serum-free medium and used for RNA extraction (see below). To test the efficiency of all siRNA gene-silencing experiments, the silencing effect was monitored up to 12 d after siRNA transfection by immunofluorescence and immunoblotting (not shown), establishing the full extent of gene silencing up to days 4–5 after transfection and an 50% remaining efficiency at about day 7 after the siRNA transfection.
RNA Extraction, Reverse Transcription, and Real-Time PCR
Total RNA was extracted from HSFs in six-well plates 96 h after siRNA transfection (see Gene Silencing Experiments) using RNeasy Plus Mini Kit as instructed by the supplier (Qiagen). After spectrophotometric quantification, 200 ng total RNA from each sample was reverse-transcribed in a 20-µl volume with random hexamers and Superscript III using the manufacturer's protocol (Invitrogen). Subsequently, cDNA samples corresponding to 50 ng total RNA (5 µl of the RT-PCR reaction) were used in real-time PCR analysis with Taqman Gene Expression Assays according to the manufacturer's protocol (Applied Biosystems, Foster City, CA; 7500 Fast Real-Time PCR System). The Taqman probes used include Hs01549940_m1 FN1 and Hs00171191_m1 FBN1 corresponding to the human gene for human fibronectin (FN1) and human fibrillin-1 (FBN1), respectively. Taqman probe Hs99999905_m1 GAPDH corresponding to the human gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control. Relative quantification was determined by setting the signals from the AllStars siRNA negative control divided by the signal for GAPDH to 100% (Applied Biosystems; 7500 System software v1.3.0). For each total RNA sample, reverse transcription and real-time PCR reactions were repeated four times.
Immunofluorescence Microscopy
For single or double immunofluorescence experiments in eight-well chamber slides, cells were either prepared as described under Gene Silencing Experiments using single or double siRNA transfected cells as indicated, or nontransfected cells were seeded at 7.5 x 104 cells/well for various time periods (24–48 h). After two washes with PBS (general washing buffer), the cells were fixed in ice-cold 70% methanol/30% acetone. Cells were washed again, blocked for 30 min with PBS including 10% normal goat serum (PBS-G; Jackson ImmunoResearch Laboratories,) and incubated with primary antibodies for 90 min diluted in PBS-G. The primary anti-fibrillin-1 antibodies were -rFBN1-C (1:1000) or mAb15 (1:500) and anti-fibronectin antibody FN-15 (1:1000; see Antibodies). After washing, the cells were incubated for 60 min with either FITC- and/or Cy3-labeled secondary antibodies (1:200 diluted in PBS-G) and washed again. The cells were then incubated with 4'-6-diamidino-2-phenylindole (DAPI; 1 µg/ml in PBS; Invitrogen) for a nuclear counterstain, washed again, cover-slipped, and examined using an Axioskop 2 microscope (Zeiss, Thornwood, NY). For visualization of the fluorescent signals, an Axiocam camera was used with AxioVision software version 3.1.2.1 (Zeiss).
Peptide Inhibition and Protein Binding Monitored by Immunofluorescence
The established, functional upstream domain (FUD) of Streptococcus pyogenes, which efficiently inhibits fibronectin assembly (Tomasini-Johansson et al., 2001; Ensenberger et al., 2004), and its inactive mutant Del29 lacking amino acid residue I29 (Zhou et al., 2008), were added at 500 nM to HSFs at the time of seeding (7.5 x 104 cells/well) into eight-well chamber slides. After incubation for 48 h, the cells were processed and stained with anti-fibrillin-1 and anti-fibronectin antibodies as described in Immunofluorescence Microscopy.
For binding assays of recombinant fibrillin fragments to protein networks in cell culture, rFBN1-N and rFBN1-C were covalently labeled with Cy3 Mono NHS-ester (GE Healthcare) according to the manufacturer's instructions. The proteins were then dialyzed extensively against TBS. HSFs were seeded at 7.5 x 104 cells/well in eight-well chamber slides and grown for 24 h. The Cy3-labeled proteins were added to the culture medium for 24 h at 10 µg/ml, and after washing with PBS, the cells were stained for fibronectin and processed as described in Immunofluorescence Microscopy. Labeling with the Cy3-conjugated proteins was detected using the Cy3 filter set of the fluorescence microscope.
Immunogold Staining and Electron Microscopy
HSFs were prepared, grown, fixed, and stained with antibodies as described in Immunofluorescence Microscopy with the following alterations. PBS including 10% normal donkey serum (PBS-D; Jackson ImmunoResearch Laboratories) was used as blocking and antibody dilution buffer. The primary anti-fibrillin-1 antibodies were -rFBN1-C (1:100) and anti-fibronectin antibody FN-15 (1:100) diluted in PBS-D (see Antibodies). Twelve- and 18-nm gold-conjugated secondary antibodies were used diluted at 1:20 in PBS-D. After antibody incubation, the cells were fixed again with 2% glutaraldehyde and 1% OsO4 and embedded in EPON epoxy resin. Ultrathin sections were stained with 4% aqueous uranyl acetate and Reynold's lead citrate following an established procedure (Sasaki et al., 1996). Sections were then examined with a FEI Tecnai 12, 120 kV electron microscope (Eindhoven, Hillsboro, OR) equipped with a Gatan 792 Bioscan 1k x 1k wideangle multiscan CCD camera (Pleasanton, CA).
Assembly of Exogenous Fibrillin-1
To produce conditioned cell culture media containing fibrillin-1 and fibronectin, HSFs were seeded in six-well plates (Corning Costar, Acton, MA) at a density of 5.0 x 105 cells/well in DMEM. After 48 h, the medium was replaced by serum-free DMEM, and after an additional 48-h incubation period, the conditioned medium was harvested. The production of fibronectin-depleted (but fibrillin-1 containing) conditioned medium was achieved in a similar manner except that 1) the HSFs were transfected with siRNA Hs_FN1_7 as described in Gene Silencing Experiments and 2) the transfection medium was replaced by DMEM after 24 h. Serum-free medium production as described above was started after an additional 24 h. Both, the normal and the fibronectin-depleted conditioned media were concentrated up to 30-fold by ultrafiltration (Centriplus YM-30; Millipore). Aliquots of the concentrated media (12.5 µl) were analyzed by standard Western blotting under nonreducing conditions using 1:500 diluted mAb15 to detect fibrillin-1 and 1:1000 diluted FN-15 to detect fibronectin.
To analyze the assembly properties of exogenously added fibrillin-1 to HSFs, the background level of endogenously produced fibrillin-1 or fibronectin was reduced by gene silencing with siRNA Hs_FBN1_1 or Hs_FN1_7 in eight-well chamber slides as described in Gene Silencing Experiments. The transfection medium was replaced 24 h after transfection with DMEM containing 10% fibronectin-depleted FCS (300 µl/well), which was again replaced after additional 24 h with 300 µl/well of unconcentrated, 15- or 30-fold concentrated conditioned fibronectin-containing or fibronectin-depleted media. After 48 h of incubation with the conditioned media, the networks were visualized by immunofluorescence (see Immunofluorescence Microscopy) after exposure to mAb15 (diluted 1:500) to detect fibrillin-1, and FN-15 (diluted 1:1000) to detect fibronectin. The analysis of colocalization of exogenously added fibrillin-1 and fibronectin (in the form of conditioned medium) was identical to this procedure except that both fibrillin-1 and fibronectin expression was reduced by siRNA gene silencing simultaneously with siRNA Hs_FBN1_1 and Hs_FN1_7 and the -rFBN1-C antiserum (diluted 1:1000) was used for detection of fibrillin-1. For these experiments, the conditioned medium applied was 15-fold concentrated and contained both fibrillin-1 and fibronectin. In addition, a nonconditioned DMEM was used as a control.
Protein Interaction and Inhibition Assays
Solid-phase binding assays were performed as described previously in detail with minor modifications (Lin et al., 2002). In brief, 10 µg/ml (100 µl) of either human plasma full-length fibronectin, proteolytic fragment FN40K, recombinant fibronectin fragments FNsuper70K, or FNIII1-C were immobilized in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl (TBS) for 16 h at 4°C in 96-well plates (Maxisorp; Nalge Nunc International, Naperville, IL) and blocked with 5% (wt/vol) nonfat milk in TBS including 2 mM CaCl2. Washing after this and all subsequent steps was performed with TBS including 2 mM CaCl2 and 0.05% Tween 20. Serial dilutions (1:2) of fibrillin fragments in TBS including 2 mM CaCl2 and 2% (wt/vol) nonfat milk (binding buffer) were incubated for 2 h at 22°C with the immobilized proteins. To detect bound ligands, the wells were incubated for 90 min with polyclonal antisera (diluted 1:1000) for rFBN1-N, rFBN1-C, rFBN2-N, rFBN2-C, and rFBN3-C (see Antibodies), followed by a 90-min incubation with horseradish peroxidase–conjugated goat anti-rabbit antibody (diluted 1:800) followed by the color reaction. In experiments to determine calcium dependency, the blocking, binding, and washing buffers outlined above were modified to include either 5 mM CaCl2 or 10 mM EDTA. For inhibition experiments, the above described interaction assay was modified as follows. After blocking nonspecific binding sites, serial dilutions (1:2; starting at 10 µg/ml) of gelatin from porcine skin (Sigma; product G1890) in binding buffer were incubated for 2 h with the immobilized proteins, followed by an incubation of recombinant fibrillin fragments at constant concentrations (75 µg/ml the fibrillin C-termini and 100 µg/ml the fibrillin N-termini).
Size-Exclusion Chromatography and Analysis of the Fibrillin C-terminal Multimers
The multimerization of the fibrillin-2 (rFBN2-C) and fibrillin-3 (rFBN3-C) recombinant C-terminal halves were analyzed by Superose 6 gel filtration chromatography (100-ml column on a high-precision ÄktaPurifier 10; GE Healthcare) as described in detail for the fibrillin-1 C-terminal construct rFBN1-C (Hubmacher et al., 2008). Recombinant protein, 2.7 mg, previously purified by metal-chelating chromatography were loaded onto the column equilibrated with TBS, including 2 mM CaCl2 at a flow rate of 0.5 ml/min. Starting at the void volume (37 ml), the eluted proteins were collected in 1-ml fractions. Aliquots of the fractions were separated by SDS gel electrophoresis, in the presence or absence of 10 mM dithiothreitol (DTT), on 4–20% acrylamide gradient gels that were then analyzed by silver staining. The molar concentrations of rFBN1-C, rFBN2-C, and rFBN3-C were calculated as described in detail previously with an estimated average of 10 molecules per multimer (Hubmacher et al., 2008 in Supplemental Information).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The fibroblasts were treated with specific siRNA oligonucleotides for human fibrillin-1 or fibronectin and the extracellular assembly and expression of these matrix proteins were analyzed by indirect immunofluorescence, real-time PCR, and Western blotting (Figure 1). The extracellular networks produced by the respective siRNA-treated fibroblasts were significantly reduced as demonstrated by indirect immunofluorescence (Figure 1A). Fibronectin was normally assembled by fibroblasts treated with siRNA specific for fibrillin-1, demonstrating that fibronectin assembly is not dependent on the expression and assembly of fibrillin-1. However, when fibronectin expression, and consequently extracellular fibronectin assembly, was abolished by siRNA treatment, then the assembly of fibrillin-1 was significantly suppressed. As controls, the expression levels of mRNA (Figure 1B) and the protein content of the conditioned medium (Figure 1C) were significantly reduced by each siRNA as demonstrated by real-time PCR and Western blotting, respectively. In summary, these data show that endogenous fibronectin must be expressed by the fibroblasts as a prerequisite for the organization of fibrillin-1 into a fibrillar matrix.
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). Blocking formation of the fibronectin network by the peptide FUD resulted in complete inhibition of fibrillin-1 network formation, whereas the control peptide Del29 had no effect on either fibronectin or fibrillin-1 network formation (Figure 2A). Control experiments demonstrated that the expression and secretion of fibronectin and fibrillin-1 were unaffected by the peptides used (Figure 2B). In summary, these data demonstrate that the presence of assembled fibronectin is a critical prerequisite for the assembly of fibrillin-1.
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). Because the C-terminal half of fibrillins contain interaction epitopes for heparin/heparan sulfate (Tiedemann et al., 2001), the potential to inhibit the fibrillin–fibronectin interaction by heparin was tested. Heparin did not inhibit this interaction in concentrations up to 200 µg/ml (13 µM), indicating different binding epitopes (data not shown). Recently, we demonstrated that C-terminal fibrillin-1 fragments have the propensity to multimerize into structures with similarity to the beads found in the bead-on-a-string–structured, extracted microfibrils (Hubmacher et al., 2008). It was further shown that this multimerization significantly increases the avidity for the self-interaction of the fibrillin-1 C-terminus with the N-terminus. Gel filtration chromatography of C-terminal fibrillin-2 and -3 fragments demonstrated that they have similar propensities to multimerize into reducible high-molecular-weight assemblies similar to the fibrillin-1 C-terminus (Figure 7A). The multimers and monomers of each fibrillin C-terminal half were tested as soluble ligands in solid-phase binding assays with immobilized full-length plasma fibronectin (Figure 7B). The results demonstrate that only the C-terminal multimers interact strongly with fibronectin, whereas the monomers do not interact.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Downing et al., 1996; Liu et al., 1996; Reinhardt et al., 1996; Qian and Glanville, 1997; Baldock et al., 2001; Lee et al., 2004; Baldock et al., 2006; Kuo et al., 2007), relatively little information is available about mechanisms and components involved in microfibril formation. Revealing these mechanisms is ultimately critical for the understanding of the pathobiology of microfibrillopathies associated with mutations in fibrillins. In this study, we have 1) found that fibronectin is an essential component in the assembly of fibrillin-1 into microfibrils and 2) analyzed in detail the molecular interactions of fibrillin-1, -2, and -3 with fibronectin.
The assembly of fibrillins likely requires many steps leading from individual fibrillin molecules to the final macromolecular assemblies in tissues—a process that includes self-assembly (Trask et al., 1999; Lin et al., 2002; Marson et al., 2005; Hubmacher et al., 2008), disulfide and transglutaminase cross-link formation (Qian and Glanville, 1997; Reinhardt et al., 2000), fibril elongation and others mechanisms (for review see Tiedemann et al., 2004). Fibrillins require the presence of cells to multimerize, and mesenchymal cells such as fibroblasts or smooth muscle cells, which can be readily manipulated, are an ideal system to analyze fibrillin assembly on the molecular level.
In a candidate approach using primary human dermal fibroblasts from penile foreskin, we reduced the expression levels of extracellular proteins and membrane receptors by siRNA gene silencing approaches and screened for effects on fibrillin assembly. Among the targets tested, only fibronectin down-regulation had a negative impact on fibrillin assembly. Down-regulation of the fibronectin mRNA expression and peptide inhibition of extracellular fibronectin assembly similarly resulted in a significant disruption of fibrillin-1 assembly, demonstrating that the extracellular assembly of fibronectin is an absolute requirement for fibrillin-1 assembly. Fibronectin has previously been demonstrated to be essential for the organization of some matrix components, including collagen types I and III and thrombospondin-1 (McDonald et al., 1982; Sottile and Hocking, 2002; Velling et al., 2002; Li et al., 2003), fibulin-1 (Roman and McDonald, 1993; Godyna et al., 1995), fibrinogen (Pereira et al., 2002), and LTBP-1 (Dallas et al., 2005). With fibronectin having a key role in the assembly of another important extracellular fibril system, the fibrillin-containing microfibrils, it becomes increasingly evident that fibronectin is a master orchestrator for the organization of various matrix components. In the case of collagen type I, thrombospondin-1, and LTBP-1, it has been shown that fibronectin is required for both, as an initiator for assembly and as a key element in subsequent matrix stability (Sottile and Hocking, 2002; Dallas et al., 2005). For fibrillin assembly, it is clear from our study that fibronectin is required to initiate fibrillin assembly. Whether or not fibronectin regulates downstream stability of assembled premature or mature microfibrils remains to be established.
We have now demonstrated that soluble fibrillin-1 from fibroblasts in conditioned medium assembles into typical networks when reapplied to fibroblast cultures. These results correlate with data from others showing that fibrillin-1 produced from epithelial cells can be assembled when added to fibroblast cultures (Dzamba et al., 2001). Here, we show that assembly of exogenously added fibrillin-1 only occurs when either endogenously produced or exogenously added fibronectin was assembled into a network. We conclude that the crucial interactions for assembly between fibrillin-1 and fibronectin do not occur in the late secretory pathway or in cell invaginations that are not accessible to exogenously added fibrillin. We suggest that the critical interactions occur either on, or close to, the cell surface.
N–C self-interaction of fibrillins is considered one of the first events in fibrillin assembly (Lin et al., 2002; Marson et al., 2005). Recently, we have shown that multimerization of the fibrillin-1 C-terminus is a prerequisite for high-affinity binding to the fibrillin-1 N-terminus, whereby the C-terminal self-interaction epitope is located within the last three calcium-binding epidermal growth factor-like domains (Hubmacher et al., 2008). Here, we demonstrate that recombinant C-terminal expression constructs of fibrillin-2 and -3 also have the intrinsic property of forming high-molecular-weight, disulfide-bonded multimers similar to those found previously for fibrillin-1. Interestingly, only the C-terminal multimers of all three fibrillins, but not the monomers, interacted strongly with fibronectin. These findings, together with our previous report on the fibrillin-1 N–C self-interaction properties, suggest that low-affinity binding sites to either itself or to fibronectin, which are situated in the C-terminal region of all fibrillins, become high-affinity binding sites upon multimerization. This multimerization occurs early in the fibrillin assembly process and requires cells, although it is not clear whether it occurs during late secretory stages or directly on the cell surface (Hubmacher et al., 2008 in Supplemental Material). Typically, the secretion and assembly of fibronectin by fibroblasts occurs much faster compared with the assembly process of fibrillins (Wartiovaara et al., 1974; Hollister et al., 1990), suggesting that a fully or partially assembled fibronectin network is already present outside the cells once fibrillin multimers appear, or are generated, on the cell surface. These fibrillin multimers might then be directly deposited onto the existing fibronectin fibrils through direct molecular interactions. This notion is supported by our colocalization studies performed at the light microscopic and the ultrastructural level, suggesting colocalization of fibrillin-1 with fibronectin in relatively early stages of fibrillin assembly. Speculatively, this interaction may align the premature fibrillin assemblies, which could then permit interaction with other fibrillin assemblies deposited onto the fibronectin scaffold, thus leading to the cognate directionality of fibrillins in microfibrils. Binding of fibrillin to fibronectin may also induce conformational changes within individual fibrillin molecules in order to proceed with the subsequent assembly process. In this regard, the intrinsic protein disulfide isomerase activity present in the C-terminus of fibronectin may help to form proper intermolecular disulfide bonds in early fibrillin assemblies (Langenbach and Sottile, 1999; Reinhardt et al., 2000; Hubmacher et al., 2008). Recently, it has been reported that LTBP-1 and -4 are initially deposited, independent of the presence of fibrillin-1, onto fibronectin fibrils and later switch to different fibril systems including fibrillin-containing microfibrils (Dallas et al., 2005; Kantola et al., 2008). In this light, binding of fibrillin to fibronectin may not only promote fibrillin assembly but may be a mechanism to load microfibrils with LTBP-1 and -4, which both bind to fibrillins (Isogai et al., 2003).
In addition, to the strong interactions of the fibrillin C-terminal multimers with fibronectin, we observed additional interactions mediated through the fibrillin-1 N-terminus, which were not observed for the fibrillin-2 N-terminus; the fibrillin-3 N-terminal half was not available for our studies. This interaction was relatively weak with full-length fibronectin as a binding ligand, but was much more pronounced using recombinant or proteolytic fibronectin fragments. Nevertheless, similar labeling intensities of the fibronectin network were observed when fluorescently labeled recombinant N- and C-terminal fibrillin-1 fragments were added to fibroblast culture medium. These results indicate that a weak fibronectin binding site for the fibrillin-1 N-terminus becomes more readily available once fibronectin is assembled into a network. Potentially, the binding site becomes fully available only after mechanical stretching of the fibronectin dimer on the cell surface via its well-established interaction with integrin 5β1 (Zhong et al., 1998; Baneyx et al., 2002). It is also possible that the site is more exposed in recombinant or proteolytic fragments as compared with the full-length (nonpolymerized) fibronectin, potentially explaining the observed differences in the in vitro binding of the fibrillin-1 N-terminal fragment to full-length versus recombinant or proteolytic fibronectin fragments. The fact that the corresponding fibrillin-2 N-terminal fragment did not bind, or bound only very little, to fibronectin might reflect differences in how fibrillin-1 and -2 require fibronectin for assembly. It has been shown that fibrillin-1 and -2 frequently coassemble into the same microfibrils (Charbonneau et al., 2003). Given that the fibrillin-2 N-terminus also does not efficiently mediate self-interaction with its C-terminus (Lin et al., 2002), the current results can be speculatively interpreted as indicating that fibrillin-1 may be a carrier for fibrillin-2 coassembly, when they are expressed in the same context.
With recombinant and proteolytic fibronectin fragments, we have localized the major binding site for the C-terminal binding epitope of all three fibrillins and an N-terminal binding epitope for fibrillin-1 to the region in fibronectin spanning domains FNI6–9. One or more additional minor binding sites for the fibrillin-1 N-terminus may be localized further C-terminal. This major binding region also mediates collagen/gelatin binding (Engvall et al., 1978; Balian et al., 1979; Shimizu et al., 1997). It has been shown that all six modules in the gelatin-binding region contribute either directly or indirectly, through domain stabilization effects, to gelatin binding, whereby FNI6 is likely involved in indirect contributions rather than directly (Ingham et al., 1989; Pickford et al., 2001; Katagiri et al., 2003; Millard et al., 2005; Pagett et al., 2005). In our study, we observed that the interactions of both the N- and the C-terminal fibrillin fragments with the gelatin-binding region of fibronectin or fibronectin fragments could be efficiently inhibited by gelatin. This positions the major fibrillin binding site between domains FNII1 and FNI9. Further studies are required to determine whether the fibrillin-binding region spans this entire region similar to gelatin binding or whether more discrete epitopes mediate this interaction. Along these lines it will be also important to characterize the relationship of the fibrillin binding site(s) in fibronectin to that of other matrix molecules including collagens that have also been shown to be organized by fibronectin (Sottile and Hocking, 2002).
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work. 
Address correspondence to: Dieter Reinhardt (dieter.reinhardt{at}mcgill.ca)
Abbreviations used: DTT, dithiothreitol; HSF, human skin fibroblasts; LTBP, latent transforming growth factor—binding protein; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; TGF-β, transforming growth factor-β.
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 V. Hucthagowder, E. Morava, U. Kornak, D. J. Lefeber, B. Fischer, A. Dimopoulou, A. Aldinger, J. Choi, E. C. Davis, D. N. Abuelo, et al. Loss-of-function mutations in ATP6V0A2 impair vesicular trafficking, tropoelastin secretion and cell survival Hum. Mol. Genet., June 15, 2009; 18(12): 2149 - 2165. [Abstract] [Full Text] [PDF]
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) and multimeric (
) states with immobilized full-length plasma fibronectin. Data represent means of duplicates; error bars, SDs. Note that only the fibrillin multimers bind to fibronectin but not the monomers.
). Efficient immobilization of fibronectin fragments was verified by ELISA assays (see Supplemental Figure S3). Data points represent means of duplicates; error bars, SDs.
