Fibrillins Can Co-assemble in Fibrils, but Fibrillin Fibril Composition Displays Cell-specific Differences*

Noe L. Charbonneau, Bette J. DzambaDagger, Robert N. Ono, Douglas R. Keene, Glen M. Corson, Dieter P. Reinhardt§, and Lynn Y. Sakai

From the Department of Biochemistry and Molecular Biology, Shriners Hospital for Children, Oregon Health and Science University, Portland, Oregon 97201

Received for publication, September 9, 2002, and in revised form, November 3, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibrillins are microfibril-forming extracellular matrix macromolecules that modulate skeletal development. In humans, mutations in fibrillins result in long bone overgrowth as well as other distinct phenotypes. Whether fibrillins form independent microfibrillar networks or can co-polymerize, forming a single microfibril, is not known. However, this knowledge is required to determine whether phenotypes arise because of loss of singular or composite functions of fibrillins. Immunolocalization experiments using tissues and de novo matrices elaborated by cultured cells demonstrated that both fibrillins can be present in the same individual microfibril in certain tissues and that both fibrillins can co-polymerize in fibroblast cultures. These studies suggest that the molecular information directing fibrillin fibril formation may be similar in both fibrillins. Furthermore, these studies provide a molecular basis for compensation of one fibrillin by the other during fetal life. In postnatal tissues, fibrillin-2 antibodies demonstrated exuberant staining in only one location: peripheral nerves. This surprising finding implicates distinct functions for fibrillin-2 in peripheral nerves, because a unique feature in humans and in mice mutant for fibrillin-2 is joint contractures that resolve over time.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fibrillin-1 and fibrillin-2 are extracellular matrix proteins with highly homologous structures (1, 2). Both molecules contribute to ultrastructurally identifiable fibrils, called microfibrils, present in a variety of connective tissues (2, 3). However, because the expression of fibrillin-2 corresponds primarily to early morphogenesis, it has been proposed that fibrillin-2 regulates the early process of elastic fiber assembly, whereas fibrillin-1 provides mostly force-bearing structural support (4).

Fibrillin-2 protein synthesis has not been well documented. Fibrillin-2, first discovered by gene cloning (5), cannot be distinguished from fibrillin-1 by migration on SDS-PAGE. However, anti-peptide antibodies have been utilized for limited tissue immunolocalization studies in fetal human (2), fetal mouse (4), fetal bovine (6), and fetal rat (7). A monoclonal antibody (mAb),1 JB3, has localized fibrillin-2 in the developing heart and primary axial structures of early avian embryos (8, 9). MAb 201, specific for fibrillin-1 (3, 10, 11), demonstrated fibrillin-1 in and around Hensen's node and defined the primary axis of the early avian embryo (12). These studies have indicated that, in early embryonic and fetal tissues, the distribution of the two fibrillins is similar, with preferential accumulation of fibrillin-2 in elastic fiber-rich tissues.

Mutations in the fibrillin genes result in two related inherited diseases of connective tissue. Major phenotypic features of the Marfan syndrome (OMIM number 154700), caused by mutations in FBN1, appear in cardiovascular (aortic dilatation or aortic dissection), skeletal (long bone overgrowth), and ocular (ectopia lentis) tissues. The phenotype of congenital contractural arachnodactyly (CCA) (OMIM number 121050), caused by mutations in FBN2, overlaps with some of the skeletal features (arachnodactyly and scoliosis) of the Marfan syndrome, but cardiovascular and ocular features are usually absent. Together with expression studies, these genetic data suggest that fibrillin-2 performs a function comparable with fibrillin-1 in skeletal but not in other connective tissues. Therefore, expression of fibrillin-2 in postnatal tissues is expected to be restricted to skeletal connective tissues.

Fibrillin-2 null mice (13) are viable and fertile. However, they are born with contractures of the small and large joints, which resolve a few days after birth. This common phenotype, joint contractures, demonstrated that loss of fibrillin-2 in homozygous fbn2-null mice is equivalent to the dominant-negative effects of mutations in FBN2 in heterozygous individuals with CCA. Similarly, mice deficient in fibrillin-1 display cardiovascular and skeletal features similar to those of the Marfan syndrome (14), indicating that loss of fibrillin-1 is equivalent to the dominant-negative effects of heterozygous FBN1 mutations.

Neither fibrillin-1 deficiency nor fibrillin-2 deficiency results in early embryonic lethality, even though both fibrillins are present in the developing embryo at gastrulation. In addition to contractures, mice lacking fibrillin-2 display a limb patterning defect, bilateral syndactyly (13). However, no other obvious early fetal or embryonic phenotype has been found, despite the restriction of fibrillin-2 expression primarily to developing tissues. And, there are also no apparent abnormalities in elastic fiber assembly or morphology, even though preferential distribution to elastic fiber-rich matrices had been originally noted in expression studies.

These genetic investigations in humans and mice suggest that fibrillins perform both overlapping and unique functions. However, biochemical and morphological studies of the fibrillins, especially fibrillin-2, have not elucidated the distinctive or shared functional contributions of the fibrillins to microfibril assembly, to elastic fiber assembly and specific tissue architectures, or to cellular differentiation. The major obvious function of the fibrillins is microfibril assembly, and yet it is not known whether both fibrillins polymerize separately to form distinct independent microfibrils or co-polymerize to form microfibril heteropolymers. The unique functions of fibrillin-2, which result in joint contractures and bilateral syndactyly when lost, are not yet understood.

To begin to resolve these issues, detailed analyses of fibrillin-2 in developing and postnatal tissues are required. The investigations reported here demonstrate fibrillin-2 immunolocalization in fetal and postnatal human tissues, compare fibrillin-1 and fibrillin-2 protein synthesis and fibril formation in various cell types, show that both fibrillins can be found in the same microfibril, and identify cryptic epitopes in fibrillin-2, which may indicate domains important for fibrillin-2 assembly into microfibrils or binding to ligands. Of most significance, these studies suggest that the molecular information directing fibrillin fibril formation may be similar in both fibrillins and thus provide a molecular basis for compensation of one fibrillin by the other during fetal life. In addition, localization of fibrillin-2 to postnatal peripheral nerves implicates a unique function for fibrillin-2 in peripheral nerves.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production of Antibodies and Recombinant Polypeptides-- Balb/cJ mice (Jackson Laboratories, Bar Harbor, ME) and New Zealand White rabbits were immunized with purified recombinant fibrillin-2 polypeptide, rF37. MAbs were prepared by fusing splenic lymphocytes with NS-1 myeloma cells followed by cloning in selective medium, as we have previously described (3). Supernatants from wells containing hybridomas were initially screened by enzyme-linked immunosorbent assay (ELISA), using rF37 as the coated substrate. Selected wells were cloned by limiting dilution. Cloned cell lines were expanded and grown in cell culture medium containing IgG-free fetal calf serum. MAbs were purified from conditioned medium by chromatography using GammaBind Sepharose (Amersham Biosciences). Stock solutions of purified mAbs were utilized at concentrations of around 1 mg/ml in phosphate-buffered saline (PBS).

Construction of the expression vectors for recombinant fibrillin-2 polypeptides rF37 and rF33 has been described (11). Expression plasmids for the production of recombinant fibrillin-2 subdomains rF46 (coding for Ser145-Ile358), rF47 (Thr493-Leu995), rF48 (Asp359-Thr656), rF49 (Asp359-Ile808), and rF50 (Asp809-Leu995) were constructed from PCR-amplified fragments using Vent polymerase (New England Biolabs, Beverly, MA) from template pCEPSP-rF37 (11). Sense primers for constructs rF47, rF48, rF49, and rF50 were designed to introduce an NheI restriction site at the 5' end. Antisense primers for constructs rF46, rF48, and rF49 were designed to introduce the sequence for 6 C-terminal histidine residues, a stop codon, as well as an XhoI restriction site at the 3' end. For the expression constructs with the 5' end (rF46) or the 3' ends (rF47, rF50) identical to pCEPSP-rF37, sense primers and antisense primers were chosen to anneal to the noncoding regions of the expression plasmid. Each restricted PCR product was ligated directly into the episomal expression vector pCEP4/gamma 2III4, which contains the sequence for the BM40/SPARC signal peptide (15). Each insert was then verified by DNA sequencing.

PCR amplification for rF46 was done using sense primer DR69 (5'-TAAGCAGAGCTCGTTTAGTGAACCG-3') and antisense primer DR137 (5'-ACCGCTCGAGCTAGTGATGGTGATGGTGATGGATGCATCGAGAGCCATCTGTTGAGG-3'). A 670-bp NheI-XhoI restricted fragment was then subcloned into pCEP4/gamma 2III4 and designated pCEPSP-rF46. For rF47, amplification was done using sense primer DR138 (5'-CGTAGCTAGCGACAATAGATATCTGTAAGCATCATGC-3') and antisense primer DR68 (5'-TCATGTCTGGATCCGGCCTTGCC-3'). A 1537-bp NheI-XhoI fragment was then subcloned into pCEP4/gamma 2III4 and designated pCEPSP-rF47. For amplification of rF48, sense primer DR66 (5'-CGTAGCTAGCCGATCAGAGAACAGGCATGTG-3') and antisense primer DR139 (5'-ACCGCTCGAGCTAGTGATGGTGATGGTGATGAGTACAGTAACGCCCATTTGGAGC-3') were used. A 922-bp NheI-XhoI fragment was then subcloned into pCEP4/gamma 2III4 and designated pCEPSP-rF48. For rF49, primer DR66 and antisense primer DR140 (5'-ACCGCTCGAGCTAGTGATGGTGATGGTGATGAATACAGTTTCTTCCAGAGGCATCTGG-3') were used for amplification. A 1378-bp NheI-XhoI fragment was then subcloned into pCEP4/gamma 2III4 and designated pCEPSP-rF49. For rF50, sense primer DR141 (5'-CGTAGCTAGCTGACATTGATGAATGTTTAGTAAACAG-3') and primer DR68 were used for amplification. A 589-bp NheI-XhoI fragment was then subcloned into pCEP4/gamma 2III4 and designated pCEPSP-rF50.

Transfection of 293/EBNA cells was performed as described previously (10). Purification of recombinant polypeptides was achieved using chelating chromatography (10). Analyses of the recombinant polypeptides by SDS-PAGE demonstrated purities of at least 90%. Construction, transfection, purification, and characterization of recombinant fibrillin-1 polypeptides rF6, rF11, and rF18 (10), and rF23 (16) have been described.

Immunochemical Assays-- ELISA, with rF37 as the coated substrate, was used to screen hybridoma supernatants. To demonstrate antibody specificity, ELISA was performed using various recombinant polypeptides as the coated substrate. Substrates were incubated in microtiter wells at concentrations of 25 nM in 15 mM Na2CO3, 35 mM NaHCO3, pH 9.2, for 16 h at 4 °C. The wells were then washed three times with Tris-buffered saline (TBS) including 0.025% Tween 20 (Bio-Rad). 5% nonfat dry milk in TBS was incubated for 1 h at 25 °C to block nonspecific sites. Primary polyclonal antiserum was 2-fold serially diluted, beginning with an initial dilution of 1:50, in 2% nonfat dry milk in TBS, and incubated for 2 h at 25 °C. After washing the wells three times with TBS/Tween-20, horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma), diluted 1:1,000 in 2% nonfat dry milk in TBS, was incubated for 1.5 h at 25 °C. After washing again, color reaction was achieved using 1 mg/ml 5-aminosalicylic acid (Sigma) in 20 mM phosphate buffer, pH 6.8, containing 0.1% H2O2, for 4-5 min and stopped by adding 2 M NaOH. Color absorbance was determined at 492 nm using a Titertek Multiskan photometer.

For immunoblotting experiments to map epitopes recognized by mAbs, ~0.25 µg of recombinant polypeptide was loaded per lane. Western blotting was performed as described (11).

Light and Electron Microscopy-- Light and electron microscopy of tissue samples were performed as described previously (17). MAbs to peripherin and S-100 were purchased from Novocastra and used according to their instructions.

Double labeling was performed using tissues incubated en bloc with pAb 9543 and mAb 48 or 143. Rabbit antibodies (to fibrillin-1) were identified using a 10-nm secondary gold conjugate. Mouse antibodies (to fibrillin-2) were identified using a 5-nm secondary gold conjugate. 18-week fetal human skin was homogenized, immunolabeled in suspension, washed extensively by centrifugation and resuspension, then absorbed onto carbon-coated grids and dried in the presence of 4% phosphotungstic acid.

Cell Cultures-- The following established human cell cultures were purchased from American Type Culture Collection: fetal lung fibroblasts, embryo fibroblasts, SW1353 (chondrosarcoma), MG63 (osteosarcoma), HT1080 (fibrosarcoma), A204 (rhabdomyosarcoma), U20S (osteosarcoma), Wish (transformed amniotic epithelial cells), and Jar (choriocarcinoma). A glioblastoma cell line, U251MG, was obtained from Dr. Eva Engvall. HaCaT human keratinocytes were obtained from Drs. Norbert Fusenig and Dirk Breitkreutz. Neonatal skin fibroblasts were derived from explant cultures of human foreskin obtained at circumcision. Adult and postnatal skin, tendon, and ligament fibroblasts were derived from explant cultures of dissected tissues obtained from surgical samples with informed consent. All of these cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (Invitrogen). Aortic smooth muscle cells were purchased from Clonetics and grown in a medium supplied by Clonetics.

Cell Culture Immunofluorescence-- Cells were plated in 4-well permanox chamber slides (Nalge Nunc International, Naperville, IL) at a concentration of 2 × 105 cells/ml. After 4 days, cells were fixed for 10 min with -20 °C acetone. After rehydration with PBS, the cells were incubated for 1 h with 10 µg/ml mAb 201 or mAb 48. The cells were washed with PBS, then labeled for 30 min with FITC-conjugated sheep anti-mouse IgG (Sigma). Labeled cells were washed with PBS, coverslipped with glycerol, 10% PBS, and viewed using a Zeiss Axiophot equipped for epifluorescence.

For double-labeling experiments, neonatal human dermal fibroblasts were examined on day 2 after passaging, using pAb 9543 together with mAbs 48 and 143 and TRITC-conjugated anti-rabbit IgG (Sigma) together with FITC-conjugated anti-mouse IgG (Sigma). In double-labeling co-culture experiments, 1 × 105 adult (age 35 years) dermal fibroblasts were plated with 1 × 105 nontransfected 293 cells, and cultures were grown and stained as described.

For testing effects of specific domains of fibrillins on fibril assembly, transfected 293 cells stably expressing recombinant fibrillin polypeptides were trypsinized, counted, and plated together with trypsinized and counted normal human dermal fibroblasts. 2 × 105 fibroblasts were mixed with 2.5 × 104, 5 × 104, 1 × 105, and 2 × 105 transfected cells in 1 ml of medium and plated into chamber slides. After allowing time for fibrillin fibrils to form (120 h), slides were fixed in methanol at -80 °C and stained with mAb 201, which recognizes fibrillin-1 at a site not contained in any of the recombinant peptides tested in these experiments.

Immunoblotting of Cell Cultures-- One million cells were plated in a 60-mm dish and grown for 72 h. The cell layers were washed with PBS and then incubated for 24 h in 3 ml of Dulbecco's modified Eagle's medium without serum. The conditioned medium was harvested and treated with 1 mM phenylmethylsulfonyl fluoride. Cell layers were washed and then scraped into 300 µl of SDS-PAGE sample buffer containing 1 mM phenylmethylsulfonyl fluoride. The medium (0.5 ml, concentrated by trichloroacetic acid precipitation, per lane) and cell layer (50 µl, per lane) were analyzed by SDS-PAGE on 3-5% gradient gels. Identical samples were analyzed by Coomassie Blue staining and by immunoblotting. For the immunoblots, samples were transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat dry milk and then incubated for 2 h with either mAb 201 or mAb 48. Blots were washed and then incubated for 2 h with a 1:1,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG. Bound antibody was visualized according to the manufacturer's instructions using either horseradish peroxidase developer 4-chloronaphthol (Bio-Rad) for mAb 201 or Super Signal substrate (Pierce, Rockford, IL) for mAb 48.

Northern Analyses-- Poly(A)+ RNA was prepared from cultured A204 and Jar cells using the FastTrack Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA (3 µg/lane) was fractionated on a 1% formaldehyde-agarose gel and blotted by capillary transfer to a nylon membrane (Hybond-N+, Amersham Biosciences). Hybridization probes of similar size were prepared from the C-terminal and 3'-untranslated regions of fibrillin-1 and fibrillin-2 to ensure specificity. For Northern analyses of A204 cells, probes were prepared by PCR using primer pairs GMC10A (5'-CGAATCACAACAGATACTTGATCG-3') and FBN1-9223AS (5'-AGCACGATTACAGTATACACACACTT-3') for fibrillin-1 and FBN2-3FS (5'-AGCCTAGAGAGTGTCGACATGG-3') and FBN2-9207AS (5'-CTAAAGAAAAACAAGTGAGTTTCC-3'). The templates were cDNA clones HFBN-10 for fibrillin-1 and 3'A (2) for fibrillin-2. The PCR products were gel purified and then radiolabeled by random primer extension in the presence of [alpha -32P]dCTP. The blots were hybridized overnight at 55 °C in aqueous hybridization buffer, then washed to a final stringency of 55 °C in 0.2× SSC, 0.1% SDS. Bound probe was detected by PhosphorImager (Amersham Biosciences). The blots were stripped and rehybridized with an actin probe for loading control. For Northern analyses of Jar and MG63 cells, probes were prepared using the PCR DIG probe synthesis kit (Roche Molecular Biochemicals, Indianapolis, IN) with primer pairs F1-9160.S (5'-GCAAGGTACAGGTGACTACC-3') and F1-9822.AS (5'-GAAGGATGCACTGGTGATCCTCTG-3') for fibrillin-1 and F2-9012.S (5'-GCATATGGCACTAAATGCAC-3') and F2-9669.AS (5'-CACAGAATGCCAGGTGTTC-3') for fibrillin-2. The blots were hybridized overnight at 50 °C in 50% (v/v) formamide in High SDS buffer according to the DIG User's Guide (Roche Molecular Biochemicals). Washing and detection with alkaline phosphatase-conjugated anti-DIG antibody and CSPD substrate were also according to the manufacturer's instructions. Blots were exposed to Kodak X-Omat film for 2 h. The blots were stripped and rehybridized with an actin probe to demonstrate equal loading. Molecular weights were estimated using an RNA ladder (Invitrogen).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of Monoclonal and Polyclonal Antibodies-- To produce monoclonal and polyclonal antibodies specific for fibrillin-2, a large recombinant fibrillin-2 polypeptide, rF37 (11) (Fig. 1), was used as immunogen. MAbs 48, 60, 72, 139, 143, 161, 171, and 205 were selected for characterization. Each of these mAbs was positive in ELISA using rF37 as coated substrate, and each immunoblotted rF37 when run on gels without disulfide-bond reducing agent, but failed to immunoblot rF37 when the sample was reduced (data not shown). No cross-reactivity was seen in ELISA or immunoblotting with recombinant polypeptide rF11, the large N-terminal half of fibrillin-1, or with rF6, the large C-terminal half of fibrillin-1 (10) (data not shown).


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Fig. 1.   Schematic diagram of fibrillin-1 and fibrillin-2 recombinant polypeptides used in these studies. All constructs were designed to begin and end with sequences corresponding to full domain modules.

Immunoblotting of authentic fibrillins present in cell culture media demonstrated that each mAb recognized authentic fibrillin-2 and did not cross-react with fibrillin-1. Fig. 2 shows the results obtained using fibrillin-2 mAb 48 and mAb 201, specific for fibrillin-1 (3, 10, 11). Embryo cells secrete both fibrillins into the medium and elaborate a fibrillin fibril network in the matrix (see below). Both mAb 48 and mAb 201 immunoblot authentic fibrillins secreted into the medium by embryo cells (Fig. 2B). A204 rhabdomyosarcoma cells, which secrete fibrillin-1 but not fibrillin-2 into the medium, were used to demonstrate that mAb 48 does not bind to fibrillin-1 (Fig. 2A). Identical results were obtained for the other fibrillin-2 mAbs, showing that each of the mAbs specifically recognizes authentic fibrillin-2 (data not shown).


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Fig. 2.   MAb 48 recognizes authentic fibrillin-2 but not fibrillin-1. Immunoblot analyses of conditioned media (M) and cell layers (CL) from A204 (A) and embryo cells (B) were performed with mAb 48, specific for fibrillin-2, and mAb 201, specific for fibrillin-1. MAb 48 recognized no bands in A204 cultures, even though fibrillin-1 is present in the medium as demonstrated by a positive immunoblot with mAb 201. In embryo cell-conditioned media and cell layers, mAb 48 recognized a band of the expected size for fibrillin-2, comigrating with fibrillin-1. Corresponding Coomassie Blue-stained lanes are shown in the right-hand panel. Molecular masses of marker proteins are indicated on the left in kDa.

Polyclonal antibodies, pAb 9543 (generated using rF11 as immunogen) and pAb 0868 (generated using rF37 as immunogen), demonstrated specificity for fibrillin-1 (pAb 9543) or fibrillin-2 (pAb 0868) by immunoblotting (data not shown). ELISA using rF37 and the two large N- and C-terminal halves of fibrillin-1, rF11 and rF6, as substrate showed that pAb 0868 binds only to fibrillin-2, with very little cross-reactivity to fibrillin-1 even at high concentrations (Fig. 3). Similar data were obtained demonstrating specificity of pAb 9543 for fibrillin-1 (Fig. 3).


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Fig. 3.   ELISA demonstrating specificity of polyclonal antisera. When pAb 0868 was tested against its immunogen, recombinant fibrillin-2 polypeptide rF37, a high positive titer was observed. However, even at high concentrations of antiserum, pAb 0868 showed no cross-reactivity with rF11 and rF6, recombinant fibrillin-1 polypeptides. Similarly, pAb 9543 displayed positive reactivity toward its immunogen, recombinant fibrillin-1 polypeptide rF11, and no reactivity toward rF37.

Recombinant polypeptides rF46, rF47, rF48, rF49, and rF50 (Fig. 1) were generated to map the epitopes recognized by the fibrillin-2 mAbs. Immunoblotting positioned the epitope recognized by mAb 161 to cbEGF2; by mAbs 48 and 143 to the 8Cys1/Gly-rich; mAb 171 to EGF4; and mAbs 60, 72, 139, and 205 to the four domains present in rF50 (Fig. 4A). These epitope-containing regions are indicated schematically in Fig. 4B.


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Fig. 4.   Mapping of epitopes recognized by fibrillin-2 mAbs. Immunoblot analyses (A) using recombinant subdomains of rF37 (graphed in Fig. 1) were used to map the epitopes for each of the mAbs raised to rF37. These data are shown diagrammatically in B. MAb 161 detected rF33 and rF46, but did not recognize rF47 or rF48. These data locate the epitope for mAb 161 to cbEGF2. MAb 48 and mAb 143 bound to rF32 and rF33, but not to rF46 or rF47, indicating that the epitopes for these antibodies are in 8 Cys1/glycine-rich domains. Because the epitopes for these mAbs are stabilized by disulfide bonds and because there are no cysteine residues in the glycine-rich domain, epitopes for mAb 48 and mAb 143 might reside in 8 Cys1. MAb 171 recognized rF32, rF33, and rF47, but not rF46. These data map the epitope for mAb 171 to generic EGF4. MAbs 60, 72, 139, and 205 detected rF50 and rF47, but not rF46, rF32, rF48, or rF49, mapping these epitopes to the four domains contained in rF50.

Immunolocalization of Fibrillin-2 in Developing Tissues-- Although all of the mAbs recognized authentic monomeric fibrillin-2, only mAbs 48 and 143 consistently bound to the polymeric microfibrillar form of fibrillin-2 in tissues. MAb 60 yielded partial tissue distribution patterns. Electron microscopic immunolocalization of either mAb 48 or 143 demonstrated specific labeling of microfibrils in both bovine (Fig. 5, A-C) and human (Fig. 5D) tissues. In the youngest fetal calves obtained (5-7 inches crown to rump, equivalent to second trimester gestation), periodic labeling of fibrillin-2 along an individual microfibril could be discerned (Fig. 5C). PAb 0868, like pAb 9543, can be used to stain tissues from a variety of different species, including human, bovine, mouse, and frog. In tadpoles, around stage 40, pAb 0868 labeled single fibrils in the skin (Fig. 5E) and around muscles in the trunk; pAb 9543 did not label these fibrils.


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Fig. 5.   Fibrillin-2 immunolocalizes to microfibrils in fetal skin and perichondrium. MAb 143 directed gold labeling to microfibrils in fetal calf (13 inches, crown to rump) skin (A) and to early elastin-associated microfibrils in fetal calf (5 inches, crown to rump) perichondrium (B). Occasionally, labeling of microfibrils demonstrated a regular periodicity: in C, mAb 143-labeled microfibrils in fetal calf (5 inches, crown to rump) perichondrium. In D, mAb 48 was used to localize fibrillin-2 to microfibrils in fetal human (18 weeks) skin. PAb 0868 identified individual microfibrils composed of fibrillin-2, without fibrillin-1, in Xenopus tadpole (around stage 40) skin (E). All bars = 100 nm.

Immunofluorescence of all fetal tissues examined displayed comparable distribution patterns and fluorescence signals for fibrillin-1 and fibrillin-2, with the exception of lung (data not shown). Blood vessels in the fetal lung demonstrated similar distributions early in gestation, but fibrillin-2 staining in the lung vasculature was lost midway through gestation. Fibrillin-2 immunofluorescence was abundant in skin (Fig. 6A) and in all connective tissue sheaths of the peripheral nerve (Fig. 6B) of 20-week fetal human tissues.


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Fig. 6.   Fibrillin-2 is abundant in peripheral nerves in postnatal tissues. In fetal tissues, fibrillin-2 is ubiquitously distributed. Revealing patterns identical to fibrillin-1, human fetal (20 weeks) skin (A), and peripheral nerve (B) were stained with mAb 48 or mAb 143. After birth, scant but detectable amounts of fibrillin-2 can be found in tissues. However, compared with the faint staining of human skin (the arrow points at staining of the dermal-epidermal junction in C), fibrillin-2 immunofluorescence of peripheral nerve was robust (C, E, and G). The same structures stained by mAbs for fibrillin-2 were identified by monoclonal antibodies to peripherin (D, F, and H) and S-100 (data not shown). Sections in C and D were from an extra human toe, age 5. Sections in E-H were from an 8-year-old peroneal nerve. Bar = 50 µm.

Immunolocalization of Fibrillin-2 in Postnatal Tissues-- In postnatal tissues, there was very little fibrillin-2 immunofluorescence. In neonatal foreskin, scant fibrillin-2 immunofluorescence was observed, primarily close to the dermal-epidermal junction (data not shown). In surgically discarded tissues obtained from children (ages 3-10 years), fibrillin-2 was just detectable in skin (Fig. 6C, arrow), perichondrium and tendon, but not in skeletal muscle or blood vessels (data not shown). However, exuberant fibrillin-2 immunofluorescence was found in one tissue, peripheral nerve (Fig. 6, C, E, and G), which costained with antibodies to peripherin (Fig. 6, D, F, and H) and S-100 (data not shown). In these samples, fibrillin-1 antibodies stained all areas of the peripheral nerve connective tissue, in a manner similar to Fig. 6B (data not shown). In contrast, incomplete staining of peripheral nerves by fibrillin-2 antibodies was observed (compare Fig. 6, E and G, with Fig. 6B), suggesting that expression of fibrillin-2 may be declining with age in this tissue.

Secretion of Fibrillins by Cells in Culture-- Fibroblasts and other mesenchymal cells in culture secrete fibrillin-1 into the medium and deposit fibrillin-1 into the matrix as fibrils (3, 18). To compare secretion of fibrillins into the medium and incorporation of fibrillins into the matrix, a variety of cultured cells were examined by immunoblotting of media and immunofluorescence of matrices. We used standardized cell culture conditions that were originally developed to examine fibrillin-1 fibril formation by fibroblasts from individuals with Marfan syndrome, compared with fibroblasts from control individuals (19). The results of these experiments are summarized in Table I.

                              
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Table I
Expression of fibrillins in cultured cells

Immunoblotting of media samples revealed that all cell cultures secreted detectable amounts of both fibrillins, with the exception of A204 cells, a rhabdomyosarcoma cell line that appears to secrete only fibrillin-1, and Jar cells, a choriocarcinoma cell line that appears to secrete only fibrillin-2. Northern blots of these cell lines were consistent with the immunoblotting data (Fig. 7, A and B). HaCaT cells preferentially assemble fibrillin-1 into the matrix and secrete fibrillin-2 into the medium (18).


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Fig. 7.   Northern analyses of FBN1 and FBN2 expression in Jar and A204 cells. Preparations of Jar cell RNA contained relatively more FBN2 than FBN1 mRNA (A), whereas FBN1 mRNA was more abundant than FBN2 mRNA in A204 RNA (B). Actin probes on the same blots demonstrate equal loading of samples.

When the cell culture matrices were examined by immunofluorescence, variability was found in the extent to which the cultured cells incorporated fibrillin-1, fibrillin-2, or both, into fibrils. Representative examples of the results are shown in Fig. 7. Embryo cells (Fig. 8, A and B) assembled both fibrillins into extensive fibrillar networks. These cells also secreted large and apparently equivalent amounts of fibrillins into the media (Table I and Fig. 2B). MG63 osteosarcoma cells, secreting smaller but also apparently equivalent amounts of fibrillins, demonstrated incorporation of fibrillin-1 into an extensive network of fibrils (Fig. 8C) with only occasional scattered fibrillin-2 containing fibrils in the matrix (Fig. 8D). Secreting even smaller amounts of fibrillins, U2OS osteosarcoma cells, however, assembled some fibrillin-2 fibrils (Fig. 8F), but did not appear to assemble fibrillin-1 fibrils, although fibrillin-1 was observed in the culture in a punctate pattern (Fig. 8E). SW1353 chondrosarcoma cells, which secreted large amounts of fibrillin-1 into the medium, also assembled abundant fibrillin-1 fibrils (Fig. 8G); however, although fibrillin-2 was observed in the medium, no fibrillin-2 fibrils were found in the matrix (Fig. 8H).


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Fig. 8.   Immunofluorescence localization of fibrillin-1 and fibrillin-2 in cell cultures. Standardized cell cultures of embryo (A and B), MG63 osteosarcoma (C and D), U2OS osteosarcoma (E and F), and SW1353 chondrosarcoma (G and H) were labeled with either mAb 201 recognizing fibrillin-1 (A, C, E, and G) or mAb 48 specific for fibrillin-2 (B, D, F, and H). Whereas some cells assembled extensive networks of both fibrillins (A and B), other cultures assembled more of one fibrillin than the other (C-F) or formed exclusively fibrillin-1 fibrils (G and H). Bar = 50 µm.

Formation of Fibrillin-1/Fibrillin-2 Heteropolymeric Fibrils-- Electron microscopic immunolocalization of fetal human skin tissue and fetal human skin homogenates revealed the presence of both fibrillins within the same microfibril. Double labeling with secondary antibody gold conjugates specific for rabbit (large gold particles) or for mouse (small gold particles) IgG and a combination of pAb 9543 and mAb 48 (Fig. 9, A and C-E) or mAb 143 (Fig. 9B) resulted in the presence of both large and small gold conjugates on individual microfibrils. Similar fibrillin-1 and fibrillin-2 double labeling of microfibrils was also identified in fetal bovine skin, fetal bovine ligament, and fetal bovine perichondrium (data not shown).


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Fig. 9.   Co-immunolocalization of fibrillin-1 and fibrillin-2 to microfibrils in tissue samples. Elastin-associated microfibrils in 18 week human fetal skin contained both fibrillin-1 (pAb 9543, large gold particles) and also fibrillin-2 (mAb 48, small gold particles) (A). Occasionally, single microfibrils labeled with antibodies to both fibrillins were seen (B). In tissue homogenates, extracted microfibrils were labeled with antibodies to fibrillin-1 and fibrillin-2: both gold conjugates decorated single microfibrils (C and D) as well as groups of elastin-associated microfibrils (E). Bars = 100 nm.

To examine the initial formation of fibrillin fibrils, cultures of fetal human fibroblasts were selected for double labeling experiments. Fibroblasts were passaged into chamber slides, and immunofluorescence was performed. When fibrillin fibrils were first observed (on day 2 after passaging), both fibrillin-1 and fibrillin-2 antibodies labeled the same fibrils (Fig. 10, A and B). In control experiments, single labeling with FITC conjugates displayed no fluorescence when viewed using the rhodamine filters, and single labeling with rhodamine conjugates displayed no fluorescence when viewed using the FITC filters (data not shown).


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Fig. 10.   Co-assembly of fibrillin-1 and fibrillin-2 by fibroblast cell cultures and co-cultures. In A and B, neonatal human dermal fibroblasts were double-labeled with fibrillin-1-specific pAb 9543 (detected by TRITC-anti-rabbit IgG) (A) and with fibrillin-2-specific mAbs 48/143 (detected by FITC-anti-mouse IgG) (B). De novo assembled fibrillin fibrils stained equally with both fibrillin probes. In C-F, co-cultures of human embryonic kidney 293 cells were plated with adult human dermal fibroblasts, and immunofluorescence was performed using fibrillin-1-specific pAb 9543 (detected by TRITC-anti-rabbit IgG) (C and E) and fibrillin-2-specific mAbs 48/143 (detected by FITC-anti-mouse IgG) (D and F). Fibrillin-2 produced by the 293 cells was incorporated into fibrillin-1 containing fibrils assembled by the fibroblasts. Bar = 50 µm.

Co-cultures between human epithelial cells and mouse dermal fibroblasts previously showed that secreted human fibrillin-1 and human fibrillin-2 can be incorporated into fibrils by mouse fibroblasts (18). However, in those studies, the composition of the mouse fibrillin fibrils was unknown. Here, adult dermal fibroblasts that neither assembled nor secreted fibrillin-2 were used (data not shown), along with human embryonic kidney 293 cells. 293 cells secreted relatively small but equivalent amounts of fibrillin-1 and fibrillin-2 but failed to assemble fibrillin fibrils (data not shown). Double labeling of de novo fibrillin fibril formation in co-cultures of these cells demonstrated fibrils containing both fibrillin-1 and fibrillin-2 (Fig. 10, C and D and E and F).

Using a different approach to determine whether fibrillin-1 and fibrillin-2 can co-polymerize, dermal fibroblasts were co-cultured with 293 cells transfected with fibrillin-1 expression constructs rF23, rF18, or rF36 (represented schematically in Fig. 1) or with fibrillin-2 expression construct rF37. N-terminal fibrillin constructs rF37 (Fig. 11, A-C) and rF23 (Fig. 11, D and E) inhibited fibrillin-1 fibril formation in a dose-dependent manner. Other expression constructs like rF18 (Fig. 10F) or rF36 (data not shown) did not inhibit fibrillin-1 fibril formation.


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Fig. 11.   N-terminal regions of fibrillins inhibit fibrillin-1 fibril formation in co-cultures. In co-cultures of fibroblasts and 293 cells transfected with comparable N-terminal regions of fibrillin-1 (rF23) and fibrillin-2 (rF37), a dose-dependent inhibition of fibrillin-1 fibril formation was observed. 2 × 105 fibroblasts were incubated with 5 × 104 (A), 1 × 105 (B), and 2 × 105 (C) rF37-transfected cells. When fibroblasts were incubated with the least numbers of rF23-transfected cells (2.5 × 104, D, and 5 × 104, E), fibrillin fibril formation was potently inhibited. Fibrillin-1 fibrils were visualized using mAb 201, which binds to fibrillin-1 in a region between cbEGF7 and hybrid 2 (30). In control co-cultures using other transfected cell lines, fibrillin-1 fibrils were easily visualized. rF18, even at the highest concentration of transfected cells to fibroblasts (1:1, 2 × 105 fibroblasts to 2 × 105 transfected 293 cells), failed to inhibit fibrillin fibril formation (F). Bar = 50 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Microfibrils are polymers of multiple individual molecules of fibrillin. The fibrillin composition of these polymers has been unspecified so far. In theory, microfibrils could be 1) heteropolymers of both fibrillins; 2) distinct fibrillin homopolymers with similar tissue distribution and bundled together into heteropolymeric microfibrillar fibers; or 3) distinct fibrillin homopolymers with distinct tissue distributions. Each of these architectural possibilities carries important functional implications for understanding fibrillin pathophysiology.

In postnatal tissues, where fibrillin-2 is largely absent, microfibrils are likely fibrillin-1 homopolymers. In the cell culture studies reported here, certain cell lines assembled fibrillin-1 fibrils that did not contain fibrillin-2 (Table I; Fig. 9, G and H). In other cell lines, fibrillin-2 fibrils were assembled that did not appear to contain fibrillin-1 (Fig. 9, E and F). In Xenopus tadpoles (around stage 40), microfibrils in the skin and around trunk skeletal muscle contained fibrillin-2, but not fibrillin-1 (Fig. 5E). These data indicate that both fibrillin-1 and fibrillin-2 can form independent fibril polymers. As independent fibrils, fibrillins can be expected to perform distinct functions.

Examination of fetal bovine and human tissues (digits, limbs, and axial skeletal tissues; ocular tissues; vascular and lung tissues) collected midway through gestation revealed no differences between fibrillin-1 and fibrillin-2 in tissue distribution or in the morphological patterns of distribution. However, temporal differences in the distribution of fibrillin-1 and fibrillin-2 were detected in lung, a finding consistent with previously published data (4). A study of early fetal human tissues (5-12 weeks gestation) also found equivalent distributions of fibrillin-1 and fibrillin-2 in skin, central nervous system and meninges, heart and aorta, lung, and peripheral nervous system, but identified distinct differences in developing liver, kidney, axial skeleton, and ribs (20). These tissue distribution studies indicate that fibrillins can be co-distributed as well as uniquely distributed, consistent with the notion that fibrillins can form distinct fibril polymers. However, these studies cannot rule in or rule out the formation of heteropolymeric fibrillin fibrils.

To address whether individual microfibrils can be assembled from molecules of fibrillin-1 and fibrillin-2, double labeling experiments were performed using fetal tissues as well as fibroblast cell cultures. In tissues where fibrillins appeared to be co-distributed (fetal skin, ligament, and perichondrium), extensive immunoelectron microscopic analyses revealed microfibrils that were composed of both fibrillins. Results from fibroblast cell cultures, which allow visualization of the de novo formation of fibrillin fibrils, indicated co-polymerization of fibrillin-1 and fibrillin-2. In these latter studies, complete co-localization was demonstrated. These data together with the immunoelectron microscopic studies of microfibrils in tissues support the hypothesis that fibrillins can form heteropolymeric microfibrils.

Co-cultures of adult dermal fibroblasts and 293 cells also indicated that fibrillin-1 and fibrillin-2 can co-assemble fibrils. In addition, these experiments showed that exogenous fibrillin-2 was co-assembled by the fibroblast as easily as the fibroblast-secreted fibrillin-1. Co-cultures utilizing transfected 293 cells identified regions in fibrillins that can inhibit fibril formation. N-terminal domains of both fibrillins were able to inhibit the formation of fibrillin-1 fibrils, whereas other domains did not affect fibrillin-1 fibril formation. These data confirm that fibrillin-1 and fibrillin-2 can co-polymerize. In addition, these data implicate N-terminal domains as crucial interactive sites in fibrillin fibril assembly and suggest the hypothesis that fibrillin-1 and fibrillin-2 may utilize homologous domains to mediate fibril assembly. We are currently using this approach to exactly specify these interactive sites and test this hypothesis.

Until now, demonstration of fibrillin fibrils produced by cells in culture has been limited primarily to dermal fibroblasts. However, some cells, like epithelial cells, deposit fibrillin into a nonfibrillar matrix (described as "pericellular" or "punctate" in Table I) and may do so because they lack cellular factors or receptors required for fibrillin fibril formation (18). These cellular factors or receptors may be specific for fibrillin-1 or fibrillin-2. For example, SW1353 chondrosarcoma cells, which can assemble fibrillin-1 into fibrils, may lack cellular factors required for assembly of fibrillin-2 into fibrils. Thus, in contrast to fibroblasts, other cell types may display variable incorporation of fibrillins into fibrils or nonfibrillar matrices, consistent with an emerging hypothesis that fibrillin fibril formation is a cell-regulated process that can distinguish between the fibrillins.

Fibrillin-2 contains two RGD sites, whereas fibrillin-1 contains one RGD sequence. Integrin receptors for fibrillin-1 include alpha vbeta 3 (21, 22). It is unknown whether integrins or other cell surface molecules are involved in the fibrillin fibril assembly process. However, recently the importance of heparan-sulfate proteoglycans to fibrillin assembly has been demonstrated (23). Our data support the hypothesis that cellular factors are required for fibrillin fibril assembly and furthermore, suggest that these cellular factors may interact differentially with fibrillin-1 and fibrillin-2. Thus, whereas fibrillins contain homologous functional domains for fibril formation, cells can be expected to perform important roles in the determination of fibril architecture in tissues.

All mAbs generated to the recombinant fibrillin-2 polypeptide, rF37, also recognized fibrillin-2 secreted into medium by cells. This, together with the fact that mAb epitopes are dependent on intact disulfide bonds, demonstrates that the mAbs recognize naturally occurring epitopes in fibrillin-2. Although all of the mAbs immunoblotted authentic fibrillin-2 secreted into fibroblast medium, only mAbs 48 and 143 recognized fibrillin-2 in the fibroblast matrix. In fibroblast cultures, no immunofluorescence (neither fibrillar, punctate, pericellular, nor diffuse) was detected with the other antibodies. These results indicate that certain regions of fibrillin-2, which are available in the monomeric molecule, become cryptic when fibrillin-2 is deposited into the extracellular matrix. In fibroblast cultures, the process whereby these regions are made cryptic occurs immediately upon deposition into the matrix. Because assembly of fibrillin occurs quickly in the extracellular space (24), these cryptic epitopes may implicate certain domains in the assembly of fibrillin-2.

Mapping the epitopes of monoclonal antibodies revealed that the 8Cys1/Gly-rich domains in fibrillin-2 are fully accessible in microfibrils, whereas other flanking domains (cbEGF 2 and EGF 4) are not available for binding. Similar studies using mAbs specific for fibrillin-1 have demonstrated that 8Cys1 in fibrillin-1 (recognized by mAb 26) is also fully accessible in tissue microfibrils. In contrast to cbEGF2 in fibrillin-2, cbEGF2 in fibrillin-1 (recognized by mAb 78) is available in tissue microfibrils (data not shown). The region (represented by rF50) around the second hybrid domain, recognized by mAbs 60, 72, 139, and 205, is also (predominantly) cryptic in tissues and fibroblast matrices. Comparable fine mapping by antibodies has not been performed in fibrillin-1.

Our identification of domains containing cryptic epitopes is consistent with other studies of microfibril assembly. Domains around the proline-rich region of fibrillin-1 and the glycine-rich region of fibrillin-2 may direct fibrillin dimer formation (25, 26). Cryptic epitopes may also result from interactions with proteins other than fibrillin. A binding site for decorin has been located near the proline-rich region of fibrillin-1, but comparable interactions near the glycine-rich region of fibrillin-2 are absent (27). The same segments between the proline-rich/glycine-rich region and the second 8 Cys domain have been reported to also mediate binding between elastin and fibrillins (28). A binding site for fibulin-2 has been identified in the N-terminal region of fibrillin-1 (16). No interactions have yet been reported to occur around the region of the second hybrid domain in either fibrillin. Identification of these cryptic epitopes may therefore implicate domains contained in rF50 in as yet unknown interactions.

The postnatal expression and tissue distribution of fibrillin-2 has not been described previously. Because mutations in FBN2 result in CCA, it has been assumed that fibrillin-2 is present in affected skeletal tissues. Major features of the Marfan skeletal phenotype (long bone overgrowth accompanied by scoliosis and chest deformities) are also common in CCA, although these features are usually less severe in CCA. These genetic similarities suggest that fibrillin-2 may perform a role comparable with that of fibrillin-1 in the perichondrium and growth plate cartilage (11). Fibrillin-1 and fibrillin-2 may impact growth of the skeleton during fetal stages when both molecules are abundant in skeletal tissues, as well as during postnatal growth when fibrillin-1 appears to be more plentiful than fibrillin-2. In this investigation, we found that, in contrast to weak expression in tissues like skin, tendon, and perichondrium, fibrillin-2 is abundant around peripheral nerves. This localization of fibrillin-2 to peripheral nerves may explain why individuals with mutations in FBN2 are affected by joint contractures that resolve over time. Congenital contractures can be caused by neuropathic abnormalities affecting peripheral nerves (29). If the fate of fibrillin-2 is to be lost with age in nerves, then one might expect the effects of mutant fibrillin-2 to be lost with time, if these effects do not result in persistent structural changes in the joint.

Based upon studies in mice (14), it has been proposed that aortic aneurysm and scoliosis develop when normal levels of fibrillin dip below a critical threshold. If microfibrils in certain tissues (like perichondrium) are heteropolymers of fibrillin-1 and fibrillin-2, then dominant-negative effects of a mutation in either fibrillin could result in reducing the level of fibrillin microfibrils and in similar phenotypes. This could be the case in skeletal tissues, because overlapping skeletal phenotypes occur in Marfan syndrome and CCA. In contrast, if microfibrils are distinct homopolymers, then it might be expected that loss of one fibrillin would lead to the same phenotypic features resulting from a dominant-negative allele. However, loss of fibrillin-2 in mice does not seem to result in long bone overgrowth and scoliosis (13), suggesting that these features in CCA are rather because of dominant-negative effects of fibrillin-2 on heteropolymeric fibrils containing fibrillin-1 and fibrillin-2. Hence, it can now be hypothesized that functions which, when lost, result in long bone overgrowth may be ascribed primarily to fibrillin-1 or to composite functions performed by both fibrillins within a heteropolymeric context.

In peripheral nerves, both fibrillins are present during fetal development and in early postnatal life. And yet, mutations in human fibrillin-2 and loss of fibrillin-2 in mice result in joint contractures, whereas only certain severe cases of neonatal Marfan syndrome display joint contractures. This might suggest that in peripheral nerves, fibrillin-2 performs a distinct function, implying that microfibrils are composed primarily of fibrillin-2 polymers. In the future, tissue-specific variations in the fibrillin composition of microfibrils (homopolymers, heteropolymers, or combinations of both) should be elucidated and related to distinct or composite fibrillin functions.

    ACKNOWLEDGEMENTS

We thank Dr. Susanne Reber-Mueller for help with the Northern analyses, Dr. Francesco Ramirez for the 3'A FBN2 plasmid, Dr. Jan Christian at Oregon Health and Science University for providing Xenopus tadpoles, and Sara Tufa for outstanding technical assistance.

    FOOTNOTES

* This work was supported by grants from the Shriners Hospitals for Crippled Children (to L. Y. S. and D. R. K.), National Institutes of Health Grant AR46811 (to L. Y. S.), and Deutsche Forschungsgemeinschaft Grants Re1021/3-1 and 4-1 (to D. P. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: Dept. of Cell Biology, University of Virginia, Box 800732 Health Sciences Center, Charlottesville, VA 22908.

§ Current address: Dept. of Medical Molecular Biology, University of Lübeck, D-23538 Lübeck, Germany.

To whom correspondence should be addressed: Shriners Hospital for Children, 3101 S.W. Sam Jackson Park Road, Portland, OR 97201. Tel.: 503-221-3436; Fax: 503-221-3451; E-mail: LYS@SHCC.org.

Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M209201200

    ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; CCA, congenital contractural arachnodactyly; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; pAb, polyclonal antibody; FITC, fluorescein isothiocyanate; EGF, epidermal growth factor; TRITC, tetramethylrhodamine isothiocyanate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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