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
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ABSTRACT |
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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.
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.
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/
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/
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
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 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 [ 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).
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).
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).
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.
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.
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.
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.
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).
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).
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).
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).
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.
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 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2III4, which contains the sequence for the
BM40/SPARC signal peptide (15). Each insert was then verified by DNA sequencing.
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/
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/
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/
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/
2III4 and designated pCEPSP-rF50.
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.
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.
-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
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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.
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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.
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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.
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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.
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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.
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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.
Expression of fibrillins in cultured cells
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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.
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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.
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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.
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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.
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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
v
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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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.
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REFERENCES |
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