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INTRODUCTION |
Fibromodulin belongs to a family of extracellular matrix
glycoproteins/proteoglycans sharing a leucine-rich repeat
(LRR)1 structural motif (1).
The gene family includes about 10 known members and can be divided into
subfamilies based on similarities in amino acid sequences and gene
organization. One subfamily includes decorin (2) and biglycan (3),
which contains an N-terminal domain substituted with
chondroitin/dermatan-sulfate chains. These proteoglycans show 57%
protein sequence identity and are encoded by genes composed of eight
exons with exon/intron junctions in conserved positions (4, 5).
Fibromodulin (6) and lumican (7) constitute another subfamily and
exhibit 48% protein sequence identity. Their genes are composed of
three exons with conserved exon/intron junctions (8, 9). Other members
of this subfamily are keratocan (10), PRELP (11), and osteoadherin
(12). Chondroadherin represents its own family with a different
organization of the gene and a different amino acid composition
(13).
The LRR extracellular matrix glycoproteins/proteoglycans have core
proteins ranging in size between 32-42 kDa, which can be divided into
three main structural domains, the N-terminal, C-terminal, and central
domains. The N-terminal domains are least conserved, but all members of
the gene family contain four Cys residues, which form intrachain
disulfide bonds (14). The glycosaminoglycan chains in decorin and
biglycan are O-glycosidically linked to Ser residues in the
N-terminal region, providing pronounced polyanionic properties to the
proteoglycans. In analogy, the N-terminal domains in fibromodulin (15)
and most likely also in lumican carry clusters of negatively charged
Tyr sulfate residues. The C-terminal domains comprise some 50 amino
acid residues and show considerable similarities among family members.
This domain contains two Cys residues involved in an intrachain
disulfide bond leading to the formation of a 34-41-residue loop.
The common central domains constitute 60-80% of the total amino
acids. In most of the members of the family, it is composed of a
10-11-fold repeat of a 20-25-residue long LRR with preferentially Asn
and Leu residues in conserved positions. Such repeats are found in many
intracellular proteins. Studies of one of these, i.e. the
ribonuclease inhibitor, has provided important structural information.
Thus its three-dimensional structure has been determined by x-ray
crystallography (16). It was found that the LRRs form a
horseshoe-shaped coil of parallel, alternating
-helices and
-sheets stabilized by interchain hydrogen bonds. Presumably the extracellular matrix LRR glycoproteins/proteoglycans have a similar three-dimensional structure (17). There are consensus site Asn residues
in the central repeat domain for substitution with carbohydrates. In
fibromodulin four such sites appear to serve as acceptors for keratan
sulfate (15). However, in an individual molecule no more than two of
the sites carry a keratan sulfate chain (18). In lumican equivalent
positions can be substituted with carbohydrates. This substitution is
also more variable, and lumican is present as a keratan sulfate
proteoglycan primarily in the cornea, while being a classical
glycoprotein, without the repeat sulfated disaccharides, in tissues
such as skin and cartilage (9). The glycosylation of fibromodulin and
lumican is a process that appears to be tissue-specific, developmentally regulated (19), and dependent on age (20).
Decorin (21), fibromodulin (22), and lumican (23) bind to fibrillar
collagens in vitro, leading to delayed fibril formation and
the formation of thinner fibrils (24). This is most likely caused by
the binding of LRR glycoproteins/proteoglycans to the surface of the
axially growing fibril (25), which inhibits the incorporation of
additional triple helical collagen monomers. The binding of LRR
glycoproteins/proteoglycans to collagen alters the surface properties
of the fibrils and may affect the interactions between individual
collagen fibrils as well as between fibrils and matrix constituents,
other than LRR glycoproteins/proteoglycans. It has been proposed that
competitive binding and displacement of proteoglycans regulate the
growth of collagen fibrils during development (26). Interestingly, the
binding of decorin to collagen is not inhibited by fibromodulin and
vice versa, suggesting that these two members of the gene
family bind to different sites on the collagen fibril (27).
Ultrastructural analysis of collagen fibrils in tissues demonstrate
that decorin (28) and fibromodulin (29) bind to distinct and apparently
separate sites in the gap region of the D-period of the collagen fibril
in vivo. The binding site for collagen in decorin has
tentatively been localized to LRR 4-5 in the central domain (30), and
recent results suggest a critical role for a Glu residue in this region
for interaction with type I collagen (31).
The interactions between collagens and LRR glycoproteins/proteoglycans
appear to be crucial for the correct assembly of collagen fibrils and
the generation of a functional collagen matrix scaffold in
vivo. Mice lacking decorin have fragile skin with reduced tensile strength, a phenotype consistent with a malfunctioning collagen matrix
(32). Ultrastructural analysis of decorin-null mice revealed abnormal
collagen fibrils in tissues such as skin and tendon, with more coarse
and irregular collagen fiber outlines. Lumican-null mice show skin
fragility and corneal opacity (33). Electron microscopic investigations
of lumican-null mice reveal a deregulated growth of collagen fibrils
with a significant proportion of abnormally thick fibrils in skin and
cornea. The recent evaluation of biglycan-deficient mice suggests a
role for biglycan in bone homeostasis (34).
We have generated fibromodulin-deficient mice to study a possible role
for fibromodulin in the assembly of collagen matrices. Here we show
that a null mutation in the fibromodulin gene leads to abnormal
collagen fibrils in tendons. In addition, the absence of fibromodulin
leads to an increase in lumican, which is associated with decreased
lumican mRNA levels.
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MATERIALS AND METHODS |
Construction of the Targeting Vector and Generation of Chimeric
Mice--
A bovine fibromodulin cDNA (6) was used to isolate
several overlapping genomic clones from a cosmid mouse 129/sv library (kindly provided by J. S. Mudgett, Merck, Rahway, NJ). A
8-kilobase pair EcoRI/SalI fragment that includes
exon 2 was subcloned in pBluescript KS II (Stratagene, La Jolla, CA). A
BamHI site located 5 kilobase pairs from the 5' end of the
genomic fragment was used for the insertion of a phosphoglycerate
kinase-neomycin resistance cassette (pGKNeo) (35), which interrupts the
coding sequence in exon 2 at a Trp residue located in position 8 of the
mature mouse fibromodulin (GenBankTM accession number
X94998). The resistance cassette pGKNeo was blunt end ligated into the
BamHI site in the opposite transcriptional orientation of
the fibromodulin gene. The targeting vector also contained a herpes
simplex virus thymidine kinase cassette, pIC19R/MC1-Tk (36), which was
blunt end ligated in the NaeI site of pBluescript KSII prior
to insertion of the genomic fragment.
R1 embryonic stem cells (37) were grown and electroporated as described
previously (38). Briefly, the targeting vector (45 µg) was linearized
with NotI and used in the electroporation of 3 × 107 embryonic stem cells. The cells were selected with 0.4 mg/ml G418 (Life Technologies, Inc.) and 0.2 µM FIAU
(Bristol-Myers Squibb, Wallingford, CT). Individual clones were picked
and isolated DNA was screened by Southern blotting after digestion with
EcoRI. Filters were hybridized with a 300-bp
EcoRI/SalI external probe to identify targeted
clones (see Fig. 1).
Two individually targeted ES cell clones were used to generate chimeric
mice as described (38). Chimeric males were mated with C57/B6 mice, and
males with germ line transmission were further bred with 129/sv females
to establish an inbred strain of fibromodulin-null mice.
RNA and Protein Transfer Blot Analysis--
Total RNA was
isolated from mouse tails or tendons using guanidine isothiocyanate.
After CsCl gradient centrifugation, RNA was subjected to agarose gel
electrophoresis in formaldehyde, and fibromodulin mRNA was detected
on the filters as described previously (6).
Proteins were extracted from tails using 4 M guanidine HCl,
0.05 M sodium acetate, pH 5.8, containing the protease
inhibitor Complete (Boehringer Mannheim) as described (39). After
48 h at 4 °C, the extracts were cleared by centrifugation, and
the supernatant was subjected to precipitation with 9 volumes of 95% ethanol. The protein precipitates were reprecipitated twice with 9 volumes of ethanol after resuspension in 0.05 M sodium
acetate, pH 5.8. Finally the precipitates were dissolved in
SDS-polyacrylamide gel electrophoresis sample buffer and
electrophoresed on a 10% SDS-polyacrylamide gel. Proteins were
transferred to nitrocellulose filters by diffusion and detected using a
rabbit antiserum against bovine fibromodulin as described (6). Bound
antibodies were visualized with an ECL kit (Amersham Pharmacia Biotech)
or 125I-labeled goat anti-rabbit IgG, a generous gift from
Dr. B. Åkerström (Department of Cell and Molecular Biology, Lund
University, Lund, Sweden) (40).
Immunological Methods--
Antisera against intact rat decorin
(41) and bovine fibromodulin (6) were used in protein transfer blots,
ELISA, and immunostainings of mouse tissues. An antiserum against mouse
lumican was prepared in rabbits using a bacterially produced
lumican/glutatione-thio-transferase fusion protein as described (41). A
MscI/StuI fragment from exon 2 of the mouse
lumican gene was ligated in pGEX-3X (Amersham Pharmacia Biotech). This
DNA encodes a 66-residue-long peptide, corresponding to residues 19-84
in the mouse sequence (GenBankTM accession number
AF013262). Fusion protein was produced and purified as recommended by
the manufacturer.
ELISA was performed as described previously (39). Microtiter plates
were coated with recombinant mouse decorin, mouse fibromodulin, and
mouse lumican fusion proteins, which were produced in bacteria and
purified as recommended by the manufacturers.
A mouse lumican/maltose-receptor fusion protein was prepared by
ligating a MscI/StuI genomic fragment (see above
under "Immunological Methods") in pMALp (New England Biolabs).
Recombinant mouse lumican fusion protein was isolated on
amylose-Sepharose.
A full-length mouse decorin cDNA was prepared using mRNA from
mouse tail. The mRNA was converted to cDNA with oligo(dT)
primers and reverse transcriptase (Boehringer Mannheim). The
single-stranded cDNA was amplified using the primers
5'-CCGGATCCATGAAGGCAACTCTCATCTTC and
5'-GGGGTACCTTGTAGTTTCCAAGTTGAATG (42) and the DNA polymerase Dynazyme (Finnzymes Oy, Espoo, Finland). Amplification conditions were
30 cycles of 95 °C for 30 s, 55 °C for 45 s, and
72 °C for 2 min. The polymerase chain reaction product was ligated
in the BamHI/KpnI site of pQE 30 (Qiagen, Hilden,
Germany), expressed as a His-tagged protein in bacteria and purified on
nickel-agarose.
Mouse fibromodulin antigen was also produced as a His-tagged protein.
Single-stranded cDNA derived from mouse tail mRNA (see above
under "RNA and Protein Transfer Blot Analysis") was amplified using
the primers 5'-TCCCCCGGGGATGCAGTGGGCCTCCGTC and
5'-CCCAAGCTTCAGATCCGATGAGGTTGG (GenBankTM accession number
X94998) and polymerase chain reaction conditions as described above for
the production of mouse decorin. A BglII/HindIII fragment, representing a 189-amino acid-residue C-terminal fragment was
ligated in pQE30 (Qiagen). Recombinant protein was expressed in
bacteria and purified as described above.
Protein extracts from tails or tail tendons were prepared as described
(39), precipitated with ethanol and redissolved in 0.14 M
NaCl, 8 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4,
3 mM NaN3, pH 7.4, containing 0.8% SDS.
Protein concentrations in extracts were determined according to
Bradford (43) using a Coomassie protein assay reagent kit (Pierce).
Immunolocalization of proteins in mouse tissues was performed on
paraffin sections of mouse tails and Achilles tendon as described (44).
Tails were fixed in 95% ethanol, 1% acetic acid and decalcified in
6% EDTA in phosphate-buffered saline for 1 week prior to dehydration and embedding. Achilles tendons were fixed in 4% paraformaldehyde in
phosphate-buffered saline. For histological analysis, paraffin sections
of tail, sternum, knee-joint, muscle, and inner organs were stained
with hematoxylin and Chromotrope 2R (Sigma).
Quantitation and Localization of mRNA--
Ribonuclease
protection assays were used to determine amounts of mRNA. Total RNA
was isolated from tails or tail tendons after extraction with guanidine
isothiocyanate and centrifugation through a CsCl cushion as described
(6). Poly(A)+ RNA was isolated from the total RNA using an
oligo(dT) matrix kit (Qiagen). Antisense mouse lumican mRNA was
prepared using a 258-bp MscI/XbaI fragment
(GenBankTM accession number AFO13262) of the mouse lumican
gene ligated in pBluescript KSII, using T7 RNA polymerase and a RNA
transcription kit (Ambion, Austin, TX). Antisense mouse actin RNA was
transcribed from pTRI-
-actin-mouse (Ambion). The protection assay
was performed using a kit according to the manufacturer's
recommendations (Ambion). Protected double-stranded RNA was separated
on a 6% polyacrylamide sequencing gel, and the radioactivity was
determined using a Bio Imaging Analyzer (Fuji Photo Film Co., Japan).
Fibromodulin, lumican and decorin mRNA were localized by in
situ hybridization on longitudinally cut paraffin sections of tail
as described (45) after fixation in 4% paraformaldehyde and
decalcification in 6% EDTA, 0.5% paraformaldehyde in
phosphate-buffered saline. RNA probes were labeled with digoxigenin-UTP
by in vitro transcription with SP6, T7, or T3 RNA
polymerases using a digoxigenin RNA labeling kit (Boehringer Mannheim)
according to the manufacturer's recommendations. Antisense mouse
fibromodulin probe was transcribed from a 267-bp
BamHI/BglII cDNA fragment in pBluescript KSII
using T7 RNA polymerase. Mouse lumican probes were transcribed from a
258-bp MscI/XbaI fragment ligated in pBluescript,
where T7 RNA polymerase was used to produce an antisense probe and T3
RNA polymerase was used to produce a sense probe. A 324-bp
BamHI/HincII mouse decorin cDNA fragment was
ligated in pGEM7Z (Promega, Madison, Wisconsin), and T7 polymerase was
used to produce an antisense probe, whereas SP6 was used to produce a
sense probe.
Electron Microscopy--
Tissues from littermates representing
fibromodulin-null, heterozygous, and wild type mice of the same sex at
7 and 20 weeks of age were subjected to electron microscopy.
Immediately after sacrifice, Achilles tendon samples were fixed by
immersion in 0.1 M sodium cacodylate-buffered 2%
glutaraldehyde containing 0.1 M sucrose. The specimens were
postfixed in 0.1 M s-collidine-buffered 2%
osmium, dehydrated in graded ethanol, and embedded in an epoxy resin
according to standard procedure (46). Ultrathin sections were
contrasted with uranyl acetate and lead citrate. Samples cut
longitudinally or transversely with respect to the main axis of
collagen fibers were studied. Micrographs from transverse sections of
the tendon were sampled by systematic random sampling and subjected to
morphometry. Thus, from each animal the diameter of each of the
transversely cut collagen fibrils of the tendon was measured on 20 micrographs from two tissue blocks with an interactive optomechanical particle size analyzer and presented in a histogram.
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RESULTS |
Generation of Fibromodulin-null Mice--
The fibromodulin gene
was inactivated by homologous recombination in embryonic stem cells
with a targeting vector interrupting the coding sequence in exon 2 with
a neomycin resistance cassette (Fig. 1).
After transformation and selection with G418 and FIAU, we identified
three correctly targeted clones out of 150 screened. Two of these were
used to produce chimeric mice, which were mated with C57/B6 females.
Chimeric males with germ line transmission were mated with 129/sv
females to generate an inbred strain. Breeding of heterozygous mice
resulted in the recovery of homozygous and heterozygous mice in the
proportions expected for a single copy gene mutation.

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Fig. 1.
Targeted disruption of the fibromodulin
gene. A, restriction enzyme maps showing the relevant
sites in the FM gene (top), the structure of the targeting
vector (middle) and the structure of the targeted gene
(bottom). E, EcoRI; H,
HindIII; B, BamHI; S,
SalI. B, Southern blot analysis of DNA isolated
from a mouse homozygous for the wild type allele (lane 1), a
heterozygous mouse (lane 2), and a mouse homozygous for the
mutated allele (lane 3). The wild type and targeted alleles
gave 9.8- and 3.4-kilobase pair fragments, respectively, after
digestion with EcoRI and hybridization with the indicated
300-bp EcoRI/SalI probe.
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The fibromodulin-null mice did not exhibit any gross anatomical
abnormalities, grew to normal size, were fertile, and had a normal life
span. Light microscopic investigations of heart, liver, lung, kidney,
skin, and cartilage did not reveal any abnormalities. Transfer blot
analysis of proteins extracted from tails showed the absence of
fibromodulin in the null mice (Fig.
2A). Northern blot analysis
showed an absence of fibromodulin mRNA in fibromodulin-null mice
and about half the amount in heterozygous mice as compared with wild
type mice (Fig. 2B).

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Fig. 2.
Transfer blot analysis of fibromodulin and
fibromodulin mRNA. A, transfer blot analysis of
proteins extracted from the tail of a mouse homozygous for the wild
type allele (lane 1), a heterozygous mouse (lane
2), and a mouse homozygous for the mutated allele (lane
3). Proteins were separated on a 10% polyacrylamide gel in SDS
and stained with Coomassie (left). Identical samples
electrophoresed on the same gel were transferred to nitrocellulose
filters, and fibromodulin was detected using an antiserum followed by
chemoluminiscence (right). B, Northern blot
analysis of RNA isolated from the tail of a mouse homozygous for the
wild type allele (lane 1), a heterozygous mouse (lane
2), and a mouse homozygous for the mutated allele (lane
3). The filter was hybridized with a fibromodulin cDNA probe
and also with a glyceraldehyde phosphodehydrogenase (GAPDH)
probe as control.
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Connective Tissue Histology and
Immunohistochemistry--
Hematoxylin/chromotrope stained
cross-sections of tails revealed abnormal tendon collagen fiber bundles
in fibromodulin-null and heterozygous mice. In comparison the tendon
fiber bundles in wild type animals are highly organized with evenly
distributed cells (Fig. 3,
A-C). In fibromodulin-null animals most fiber bundles have
a different appearance and show an abnormal morphology (Fig. 3,
G-I), as compared with those in wild type mice. Also in
heterozygous animals, 10-20% of the collagen fiber bundles are
similar to the abnormal bundles in fibromodulin-null mice (Fig. 3,
D-F). The abnormal fiber bundles appear less organized with
unevenly distributed cells. The transverse sections suggest fewer cells
in fibromodulin-null as compared with wild type tendon. However,
longitudinal sections indicate that a similar number of fibroblasts are
present in fibromodulin-null and wild type tendon fibers. The abnormal
fiber bundles also appear to have reduced endotenon tissue, which is
composed of cells and loose connective tissue surrounding these bundles
(47). In association the fibromodulin-null collagen bundles appear to
shrink more during the fixation and also detach from the surrounding
tissues. Furthermore, the number of fiber bundles, surrounded by
epitenon (47), is reduced in fibromodulin-null and to a lesser degree
also in heterozygous animals. Counting fiber bundles in four groups of
littermates (1, 1.5, 3.5, and 20 months old) shows a 25-55% reduction
in fibromodulin-null as compared with wild type littermates.
Heterozygous animals had 0-32% less bundles than wild type.

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Fig. 3.
Histology of connective tissue in tails.
Hematoxylin/chromotrope stained sections of tails from wild type
(A-C), heterozygous (D-F), and
fibromodulin-null (G-I) littermates. A,
B, D, E, G, and
H, transverse sections. C, F, and
I, longitudinal sections. Dermis (d), muscle
(m), and tendon (t) are indicated.
Photomicrographs A, D, and G show an
overview (magnification, ×31). Micrographs B, C,
E, F, H, and I show tendon
bundles at higher magnifications (×78). Arrows indicate
abnormal fiber bundle in heterozygous tail.
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The distribution of fibromodulin, lumican, and decorin was investigated
in cross-sections of tails using immunohistochemistry. In wild type
animals fibromodulin is preferentially present in the collagen bundles
of tendons, but also a weaker staining of the dermis was observed (Fig.
4A). Lumican was
preferentially detected in dermis and in peritenon tissues immediately
surrounding tendons in wild type mice (Fig. 4B), whereas the
decorin antiserum showed staining in tendons and skin (Fig.
4C). The fibromodulin, lumican, and decorin staining
patterns in heterozygous and wild type tail samples showed differences
in the collagen fiber bundles. Fiber bundles with an abnormal
morphology stained weakly with the lumican antiserum (Fig.
4E) and showed an uneven, patchy staining pattern with
fibromodulin (Fig. 4D) and decorin (Fig. 4F)
antisera. Fibromodulin was absent in fibromodulin-null mice (Fig.
4G), where we observed a change in the amount and
distribution of lumican. The anti-lumican staining in fibromodulin-null
mice was increased relative to the staining in wild type animals, with
staining of most tendon collagen bundles (Fig. 4H). The
morphologically abnormal collagen fiber bundles showed a uneven
staining pattern with both lumican and decorin antisera (Fig.
4I).

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Fig. 4.
Immunostaining of transverse sections of
tails. Shown are transverse sections of tails from wild type
(A-C), heterozygous (D-F), and
fibromodulin-null (G-I) littermates. Serial sections were
incubated with antibodies against fibromodulin (FM;
A, D, and G), lumican (LUM;
B, E, and H), and decorin
(DEC; C, F, and I) followed
by a peroxidase-conjugated second antiserum and incubation with
3,3'-diaminobenzidine. Arrows indicate abnormal fiber
bundles in heterozygous tail, which stain with the lumican antiserum.
Magnification, ×31.
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Similar staining patterns were seen in Achilles tendons. The collagen
fiber bundles in wild type Achilles tendon were stained with the
fibromodulin (Fig. 5A) and
decorin (Fig. 5C) antisera. The lumican antiserum stained
connective tissues surrounding the tendon but not the tendon collagen
fibers (Fig. 5B). The staining patterns of wild type and
heterozygous tendon (Fig. 5, D-F) were similar. In
fibromodulin-null tendon no fibromodulin was detected (Fig.
5G), whereas the lumican antiserum stained the tendon
collagen bundles (Fig. 5H). The decorin antiserum stained
wild type, heterozygous, and fibromodulin-null Achilles tendon in a
similar way, with staining in both tendon collagen bundles and
surrounding tissues (Fig. 5, C, F, and
I).

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Fig. 5.
Immunostaining of Achilles tendon.
Serial transverse sections of Achilles tendons from wild type
(A-C), heterozygous (D-F), and
fibromodulin-null (G-I) littermates were stained with
antibodies against fibromodulin (FM; A,
D, and G), lumican (LUM; B,
E, and H), and decorin (DEC;
C, F, and I). Magnification,
×31.
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Ultrastructural Analysis of Tendon--
Electron microscopic
analysis of Achilles tendon from 7- and 20-week-old littermates showed
abnormalities in the structure of collagen fibrils. Longitudinal
sections show a higher abundance of thin fibrils in the
fibromodulin-null mouse (Fig.
6A), as compared with the wild
type littermate (Fig. 6D). Some of the thin fibrils in the
fibromodulin-null tendon appear more flexible and show variation in
their orientation. The transverse sections demonstrate more thin
fibrils in fibromodulin-null (Fig. 6, B and C) as
compared with wild type littermates (Fig. 6, E and
F). Many fibrils in the fibromodulin-null tendon were
irregular and rough in cross-section, suggesting abnormalities in the
lateral fusion of fibrils or incorporation of more collagen molecules
into fibril precursors. In comparison, the wild type tendon had a
normal appearance with rounded and smooth fibrils. Electron micrographs
of the heterozygous tendons were similar to the wild type (not
shown).

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Fig. 6.
Transmission electron micrographs of Achilles
tendons. Shown are longitudinal sections of tendons from
fibromodulin-null (A) and wild type (D)
7-week-old littermates (magnification, ×25,000), transverse sections
of tendon from fibromodulin-null (B) and wild type
(E) 20-week-old littermates (magnification, ×57,000), and
transverse sections of tendons from fibromodulin-null (C) and wild type
(F) 20-week-old littermates (magnification,
×122,000).
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Morphometric analysis of fibrils from two groups of littermates
demonstrate that fibromodulin-null tendons have on the average thinner
fibrils than wild type. The Achilles tendons appear to contain two main
populations of fibrils. The thinner fibrils are 50-80 nm, and the
thicker fibrils are 150-250 nm in diameter. The decrease in
fibromodulin-null fibril diameter appears to be the result of a
relative increase in the thinner fibril population with an overall
similar range of fibril diameters in wild type and fibromodulin-null
animals. The fibrils in the wild type tendons (Fig.
7, A and D) were on
the average 152 and 143 nm in diameter in the 7- and 20-week-old mice,
respectively. The fibrils in the heterozygous mice (Fig. 7,
B and E) had an average diameter of 154 and 157 nm in the 7- and 20-week-old mice, respectively. The fibrils in the 7- and 20-week-old fibromodulin-null mice (Fig. 7, C and
F) showed an average diameter of 128 and 122 nm,
respectively.

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Fig. 7.
Morphometric analysis of Achilles tendon
fibrils. Shown are the diameters of collagen fibrils from 7 weeks
(A-C) and 20 weeks (D-F) old littermates. Wild
type (A and D), heterozygous (B and
E), and fibromodulin-null (C and F)
fibril diameters were measured and presented in a histogram. The
average fibril diameter and the total number of fibrils measured
(n) are presented. The smallest size class includes fibrils
18-50 nm in diameter.
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LRR Glycoprotein/Proteoglycan Composition--
Transfer blot
analysis of proteins extracted from the tails of fibromodulin-null and
wild type mice suggested at least a 2-fold increase in the amount of
lumican in the fibromodulin-null mice as compared with the wild type
animals (Fig. 8). Analysis of filters from the same transfer with a decorin antiserum indicated that the
amount of decorin was slightly decreased in the fibromodulin-null mice.
To confirm these semiquantitative results, proteins were extracted from
the tails of wild type, heterozygous, and fibromodulin-null littermates. The extracts were subjected to ELISA using lumican, decorin, and fibromodulin antisera. Littermates of the same sex between
1 and 14 months of age showed a 2.6-3.7-fold increase in the lumican
content in the fibromodulin-null mice as compared with wild type
animals (Table I). The amounts of decorin
in the fibromodulin-null mice were virtually unchanged as compared with wild type littermates. ELISA of the tail extracts from heterozygous animals did not detect any changes in lumican or decorin contents as compared with wild type mice (results not shown). The
fibromodulin content, determined by ELISA in three wild type animals,
was 6-8-fold higher than the lumican content (Table I).

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Fig. 8.
Protein transfer blot analysis of wild type
and fibromodulin-null littermates. Proteins extracted from the
tails of wild type (lanes 1-3) and fibromodulin-null
(lanes 4-6) littermates were electrophoresed on a 10%
polyacrylamide gel in SDS and transferred to nitrocellulose. The gel
was loaded with 90 µg of protein (lanes 1 and
4), 45 µg of protein (lanes 2 and
5), and 22.5 µg of protein (lanes 3 and
6). Lumican (top) and decorin (bottom)
were detected with rabbit antisera followed by 125I goat
anti-rabbit IgG.
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The tail extracts contain components derived from many connective
tissues, e.g. tendon, dermis, bone, and cartilage. To
specifically study the composition in tendon, we dissected tail tendons
from three groups of littermates. Tendon extracts were subjected to ELISA using fibromodulin, lumican, and decorin antisera (Fig. 9). Fibromodulin was expectedly absent in
the fibromodulin-null animals. Tendons from the heterozygous animals
contained 0-50% less fibromodulin than the wild type littermates. The
lumican content was 3-5-fold higher in the fibromodulin-null animals
as compared with wild type littermates, and the heterozygous animals contained 30-60% more lumican than the wild type littermates. The
decorin contents were similar in all genotypes. We estimated a 1:3:4
molar ratio of lumican:fibromodulin:decorin in wild type tendon.

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Fig. 9.
ELISA of tail tendon extracts. Proteins
extracted from the tail tendons of wild type ( ), heterozygous ( ),
and fibromodulin-null mice (×) were diluted as indicated and used to
inhibit the binding of antibodies to mouse recombinant fibromodulin
(FM), lumican (LUM), and decorin
(DEC). The protein extracts were from three groups of
littermates (4, 6, and 20 weeks old) and diluted as indicated from an
initial concentration of 0.3 mg/ml.
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Lumican mRNA Analysis--
To investigate whether the increase
in the amount of lumican was reflected by an increase in the steady
state level of lumican mRNA, we analyzed poly(A)+ RNA
isolated from tails using ribonuclease protection assays. The amount of
lumican mRNA was determined relative to the level of
-actin
using lumican and
-actin antisense RNA probes. Surprisingly, the
assays showed 21-49% lower levels of lumican mRNA relative to
actin mRNA in fibromodulin-null mice, as compared with wild type
littermates (Table I). Studies by in situ hybridizations of
longitudinal sections of tails using fibromodulin, lumican, and decorin
antisense RNA probes corroborate the ribonuclease protection results.
In wild type animals the antisense fibromodulin probe hybridized with
RNA in tendon fibroblasts (Fig.
10A). The lumican probe
hybridized with mRNA in skin and tendon (Fig. 10B). Similar results were obtained with a decorin probe, staining cells in
skin and tendon (Fig. 10C). In connective tissues from
heterozygous mice the staining pattern with fibromodulin (Fig.
10E), lumican (Fig. 10F), and decorin (Fig.
10G) antisense probes were similar to those observed in wild
type tissues. In fibromodulin-null tissues no hybridization with the
fibromodulin probe was detected (Fig. 10H). The staining
pattern with the lumican antisense probe showed a decrease in lumican
mRNA in tendon fibroblasts, whereas the staining of cells in skin
was unchanged as compared with wild type tissues (Fig. 10I).
The vast majority of tendon fibroblasts in fibromodulin-null mice
lacked expression of lumican mRNA, and large segments of tendon
showed no staining with the antisense lumican probe. The decorin probe
showed an unaltered staining pattern as compared with wild type tissues
(Fig. 10J). Lumican (Fig. 10D) and decorin (not
shown) control sense probes showed no hybridization.

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Fig. 10.
In situ hybridizations of
longitudinal sections of tails. Shown are serial sections from
wild type (A-D), heterozygous (E-G), and
fibromodulin-null (H-J) littermates. Skin (upper
panel) and tendon (lower panel) are shown. The sections
were hybridized with a fibromodulin antisense RNA probe (FM;
A, E, and H), a lumican antisense
probe (LUM; B, F, and I),
and a decorin antisense probe (DEC; C,
G, and J). Panel D shows control
lumican sense probe (SENSE). After the hybridization with
digoxigenin-labeled RNA and incubation with a anti-digoxigenin alkaline
phosphatase antibody, sections were developed with nitro blue
tetrazolium, 5-bromo-4-chloro-3-indolylphosphate, and
levamisole. Arrows indicate staining resulting from
hybridization with probe. A brown precipitate in skin, which was
also present in the control, is indicated (*).
Magnification, ×78.
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The lumican mRNA content in tendon was further studied by
ribonuclease protection assays. Poly(A)+ RNA was purified
from tail tendons dissected from 10-month-old littermates and was
analyzed using lumican and
-actin antisense probes (Fig.
11). The level of lumican mRNA in
fibromodulin-null tendon fibroblasts relative to
-actin mRNA was
only 2% of the relative lumican mRNA content in wild type cells.
The lumican mRNA level in heterozygous tendon was 89% of the level
in wild type tendon. Similar results were obtained using
poly(A)+ RNA from 5-month-old littermates (results not
shown).

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Fig. 11.
Ribonuclease protection assay of RNA from
dissected tail tendon. Poly(A)+ RNA was isolated from
the tail tendon fibroblasts of 10-month-old wild type, heterozygous,
and fibromodulin-null littermates and subjected to RNase protection
assays using a lumican probe (left) and a -actin probe
(right). The 320-bp lumican probe (probe) and
control RNase incubation of the lumican probe in the absence of RNA
(probe + RNase) are shown. A 258-bp RNase-protected RNA was
generated with the lumican probe in the presence of approximately 10 ng
of mRNA from wild type (+/+), heterozygous (+/ ), and
fibromodulin-null ( / ) mice. The 304-bp -actin probe
(probe) and control RNase digestion of the -actin probe
in the absence of RNA (probe + RNase) are shown. A 250-bp
RNase-protected RNA was generated with the -actin probe in the
presence of approximately 2 ng of mRNA from wild type (+/+),
heterozygous (+/ ), and fibromodulin-null ( / ) littermates.
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DISCUSSION |
Fibrillar collagens provide the basis for the tensile
properties as well as the biomechanical scaffold for anchorage of
macromolecules and cells in connective tissues. Collagen fibers are
composite structures of several collagens and a number of
noncollagenous molecules bound to their surface. Thereby these
molecules provide sites for potential interactions with neighboring
fibers and other matrix constituents. The LRR
proteoglycans/glycoproteins fibromodulin, lumican, and decorin bind to
the surface of collagen fibrils and have been implicated as regulators
of collagen fibrillogenesis (24, 27). It is likely that also other
members of this protein family have this property, but this has not yet
been verified. Information provided by decorin-null (32) and
lumican-null mice (33) suggested that these LRR
glycoproteins/proteoglycans indeed have fundamental roles in
determining the architecture and mechanical properties of collagen
fibers in connective tissues in vivo.
In this report we demonstrate that mice lacking a functional
fibromodulin gene have a phenotype involving thinner collagen fibrils
and abnormal fibers in tendon. This phenotype is associated with an
increased deposition of lumican, suggesting that lumican and
fibromodulin share the same binding site on tendon collagen fibrils.
This further suggests that the orchestrated action of several collagen
binding LRR glycoproteins/proteoglycans, with different effects on
collagen fibrillogenesis, influence the architecture and mechanical
properties of the collagen matrix.
The phenotype resulting from the inactivation of the fibromodulin gene
is rather subtle, and mice lacking fibromodulin did not show any gross
anatomical defects, grew to normal size, were fertile, and had a normal
life span. Fibromodulin is comparatively prominent in tendon and
histological investigations of wild type, heterozygous, and
fibromodulin-null mice show morphological changes in tail tendon
collagen fiber bundles. In transverse sections most fiber bundles in
the fibromodulin-null mice had an abnormal morphology and also 10-20%
of the collagen fiber bundles in the heterozygous mice had a similar
abnormal appearance. In comparison with wild type collagen bundles, the
fibromodulin-null bundles appear differently organized with fewer and
unevenly distributed cells. However, longitudinal sections of tendons
indicate that a similar number of cells are present in wild type and
fibromodulin-null tendons. The reason for this discrepancy is unknown,
but an uneven and disorganized distribution of cells may give the
impression of fewer cells along the collagen fibers in transverse
sections. In addition, the fibromodulin-null and to a lesser degree the heterozygous mice have reduced endotenon tissue and also a reduced number of fiber bundles. The heterozygous and fibromodulin-null animals
had an average of 15 and 38% fewer collagen bundles, respectively, in
their tails than wild type littermates.
The location of fibromodulin, lumican, and decorin in tail was studied
by immunohistochemistry. The collagen fiber bundles of wild type tendon
stained with the fibromodulin and decorin antisera but not with the
lumican antiserum. In contrast, the morphologically abnormal fiber
bundles in heterozygous and fibromodulin-null mice stained to varying
degrees with the lumican antiserum. The stainings with fibromodulin,
lumican, and decorin antisera of heterozygous and fibromodulin-null
tail tendon showed an uneven distribution among the bundles and also
within a single bundle. These uneven staining patterns of collagen
fiber bundles suggest the presence of fibroblasts expressing varying
amounts LRR glycoproteins/proteoglycans. Ultrastructural analysis of
chick tendon fibroblasts have suggested that fibril segments are
assembled by lateral and axial fusion into progressively thicker
fibrils, while still in clefts of the fibroblast plasma membrane (48).
The observed uneven staining patterns of collagen fibers may be the
result of fusion of fibril bundles synthesized by subpopulations of
fibroblasts or by fibroblasts at different times. The morphological
abnormalities in heterozygous tail tendons also suggest that the
collagen fiber morphology depends on the synthesis and matrix
deposition of different LRR proteoglycans/glycoproteins in a
dose-dependent fashion.
Immunohistochemical analysis of Achilles tendon showed a similar
distribution of fibromodulin, lumican, and decorin as in tail tendon.
The Achilles tendon fiber bundles from wild type mice stained with the
anti-fibromodulin serum but not with the lumican antiserum. In analogy
with the tail tendon, the lumican antiserum showed an intense staining
of the collagen fiber bundles in fibromodulin-null Achilles tendon.
This indicates that tail and Achilles tendons are biochemically
similar. Presumably tendons have similar properties, because they are
primarily exposed to tensile forces, regardless of location. We did not
observe an uneven distribution of LRR glycoproteins/proteoglycans
between fiber bundles in heterozygous Achilles tendon, as was seen in heterozygous tail tendon. The Achilles tendon is composed of two fiber
bundles, which are some 10-fold larger in diameter than the bundles in
tail. As discussed above, the tail tendon fiber bundles may be
synthesized by subpopulations of fibroblasts or by fibroblasts at
different times. Possibly, the fewer and larger bundles in Achilles
tendon are produced by less diversified fibroblasts leading to a more
homogeneous tendon tissue.
Ultrastructural analysis of Achilles tendons from fibromodulin-null
mice show collagen fibrils with somewhat irregular outlines in
cross-section, suggesting an altered fusion of fibrils as was previously observed in decorin-null mice (32). The tendons appear to
contain fibrils centered around two main populations. Thinner fibrils
are centered around 50-80 nm in diameter, and the thicker fibrils are
centered around 150-250 nm. Morphometric analysis of tail tendons from
7- and 20-week-old wild type mice show an average fibril diameter of
152 and 143 nm, respectively. This difference in average diameter is
due to more fibrils belonging to the thin fibril population in the
20-week-old wild type mouse. Omitting the three smallest size classes
(18-82 nm) leads to an identical average fibril diameter (176 nm) in
the two wild type mice. In fibromodulin-null animals a much larger
proportion of the fibrils have a thinner diameter, although the range
is the same as in wild type. The 7- and 20-week-old mice showed fibril diameter of 128 and 122 nm, respectively. As a consequence, the fibromodulin-null mice have some 60% more fibrils than wild type animals per unit area. Collagen fibrils in the 7-week-old heterozygous animal had an average diameter and size distribution, which is almost
identical to that seen in the wild type. However, for unknown reasons,
the 20-week-old heterozygous tendon has a broader size distribution and
a 10% larger average diameter as compared with the wild type
littermate. It is possible that there is a key level concentration of
the LRR proteins for their effect on collagen fibrillogenesis. Because
the heterozygous Achilles tendon presumably has similar alterations in
LRR protein composition as tail tendon, it appears that the
fibrillogenesis is not very sensitive to smaller changes in the
concentration of these proteins. Interestingly, the irregularity and
larger abundance of thin collagen fibrils in the fibromodulin-null mice
may be taken to indicate that fiber growth occurs in steps regulated
and possibly catalyzed by changes in the pattern of LRR protein
synthesis. As is discussed below, this may result from an increased
content of lumican that apparently fulfills some of the roles of
fibromodulin but has a somewhat different overall function in the
assembly process. It remains to be determined whether there is a
progression through stages, such that initially primarily one of the
LRR glycoproteins/proteoglycans populate the fibril only to be
substituted by another as the fibril grow. In light of the present
data, it appears possible that lumican precedes fibromodulin. It has
been shown that collagen fibril growth is a rapid process leading to a
4-5-fold increase in fibril length between days 16 and 18 of chicken
metatarsal tendon development (49). This rapid increase in length is
due to an extracellular association of preformed fibril segments.
Hypothetically these segments are stabilized or activated by
interacting with other extracellular matrix components, e.g.
fibromodulin, lumican, and decorin. Possibly lumican stabilizes the
fibril segments and is not replaced by fibromodulin in the
fibromodulin-null mice. Alternatively, the segments are stabilized by
e.g. decorin and is replaced by lumican, due to lack of
fibromodulin, when the fibrils grow in length. To shed more light on
the sequence of events leading to mature tendon, the developmental
expression of collagen-binding LRR glycoproteins/proteoglycans in wild
type and fibromodulin-null mice is of key interest for future studies.
Further information pertinent to the role of the LRR
glycoproteins/proteoglycans was obtained by ELISA to show the relative abundance of fibromodulin, lumican, and decorin. Analysis showed that
fibromodulin-null tail tendon contained approximately 4-fold more
lumican than the wild type. In the heterozygous tail tendon the lumican
content was increased by some 45%, and fibromodulin decreased by
approximately 25%. The amount of decorin remained unchanged. Previous
studies have shown that decorin and fibromodulin bind to separate sites
on collagen fibrils (27), which may explain the unchanged decorin
content in the fibromodulin-null mice. On the other hand it can
be anticipated that lumican and fibromodulin share the same binding
site on collagen fibrils, because they are more closely related,
showing 48% protein sequence identity. Possibly the loss of
fibromodulin leads to collagen fibrils, which instead bind lumican.
However, it remains to be shown whether fibromodulin and lumican
share binding sites on collagen fibrils and also to compare the
fibromodulin and lumican collagen binding properties. Based on a 1:3:4
molar ratio of lumican:fibromodulin:decorin in wild type tail tendon,
we estimate that the fibromodulin molecules are putatively replaced by
lumican in the heterozygous and fibromodulin-null tendon. Therefore,
the altered collagen fibril morphology could be the consequence of an
increase in the amount of collagen-bound lumican. It is interesting
that the overall size distribution of collagen fibrils is similar in
wild type and fibromodulin-null mice, whereas the latter show a greater
preponderance of thin fibrils. One question that arises is whether
there are indeed two different fibril-forming systems. If so, it is
possible that the relative balance of these two systems is influenced
by the LRR glycoproteins/proteoglycans. It is of interest to note that cornea has a rather monodisperse population of thin fibrils and is rich
in lumican (33).
The increase in lumican after targeted disruption of the fibromodulin
gene does not appear to be primarily the result of an increase in the
steady state level of lumican mRNA. Ribonuclease protection assays
rather indicated that the level of lumican mRNA in tail extracts
was decreased by 20-50% in the fibromodulin-null mice as compared
with wild type littermates. In situ hybridizations and
ribonuclease protection assays showed lumican mRNA expression in
wild type tendon fibroblasts and drastically reduced expression in
fibromodulin-null tendon fibroblasts. Interestingly, the in situ hybridizations suggest that the decrease in lumican mRNA is restricted to tendon, and no change in expression was observed in
skin fibroblasts. Thus, the down-regulation of lumican mRNA is
preferentially observed in tendon fibroblasts, where the increase in
lumican deposition is most pronounced. These results suggest that the
amount of lumican is influenced by the availability of binding sites in
the extra cellular matrix and that the expression of lumican mRNA
is regulated by the extra cellular matrix composition. The lack of
fibromodulin in fibromodulin-null tendons allows the incorporation of
lumican, and the extracellular matrix content of lumican is probably
monitored and regulated by the fibroblasts. The dramatic increase in
lumican content in fibromodulin-null tendons may signal to cells to
decrease the expression of lumican mRNA. A possible feedback
mechanism could involve cellular receptors for LRR proteins. Indeed, it
has previously been shown that cultured fibroblasts degrade about 30%
of newly synthesized decorin (50) via efficient receptor-mediated
endocytosis (51). In analogy, it is possible that lumican is also
endocytosed and degraded, providing a sensing mechanism. Alternatively,
the connective tissue cells may somehow sense the structure of collagen
fibrils, perhaps via alterations in the mechanical properties of the
matrix. They may then respond by regulating the synthesis of LRR
glycoproteins/proteoglycans to adjust the properties of the collagen scaffold.
In addition to the collagen-binding LRR glycoproteins/proteoglycans,
processing of procollagen (52) and fibril associated collagens have
been implicated in the regulation of fibril formation. Targeted
disruption of collagen type V, which associates with collagen type I,
leads to abnormalities caused by disorganized type I collagen fibrils
including thicker than normal collagen type I fibrils in cornea (53).
Recently thrombospondin 2 has also been shown to affect collagen
fibrillogenesis (54). Mice lacking thrombospondin 2 contain abnormal
collagen type I fibrils in tendons having greater than normal diameter
and irregular contours in cross-section, similar to the decorin-null
mice. The thrombospondin 2 effect on fibril formation is suggested to
depend on altered cell adhesion properties, because trombospondin 2 is
not known to associate with collagen in connective tissues. Collagen
fibrillogenesis is clearly a complex process involving the action of
many factors influencing lateral and axial growth, fibril diameters,
and interfibrillar spacing. These factors, which include the LRR
glycoproteins/proteoglycans, are crucial in determining the precise
supramolecular architecture of collagen matrices in connective tissues.