Fibromodulin-null Mice Have Abnormal Collagen Fibrils, Tissue Organization, and Altered Lumican Deposition in Tendon*

Liz SvenssonDagger , Attila Aszódi§, Finn P. Reinholt, Reinhard Fässler§, Dick HeinegårdDagger , and Åke OldbergDagger parallel

From the Dagger  Department of Cell and Molecular Biology, University of Lund, P.O. Box 94, S-221 00 Lund, Sweden, the § Department of Experimental Pathology, University Hospital, S-221 85 Lund, Sweden, and the  Laboratory for Electron microscopy, Department of Pathology, National Hospital, 0027 Oslo, Norway

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fibromodulin is a member of a family of connective tissue glycoproteins/proteoglycans containing leucine-rich repeat motifs. Several members of this gene family bind to fibrillar collagens and are believed to function in the assembly of the collagen network in connective tissues. Here we show that mice lacking a functional fibromodulin gene exhibit an altered morphological phenotype in tail tendon with fewer and abnormal collagen fiber bundles. In fibromodulin-null animals virtually all collagen fiber bundles are disorganized and have an abnormal morphology. Also 10-20% of the bundles in heterozygous mice are similar to the abnormal bundles in fibromodulin-null tail tendon. Ultrastructural analysis of Achilles tendon from fibromodulin-null mice show collagen fibrils with irregular and rough outlines in cross-section. Morphometric analysis show that fibromodulin-null mice have on the average thinner fibrils than wild type animals as a result of a larger preponderance of very thin fibrils in an overall similar range of fibril diameters. Protein and RNA analyses show an approximately 4-fold increase in the content of lumican in fibromodulin-null as compared with wild type tail tendon, despite a decrease in lumican mRNA. These results demonstrate a role for fibromodulin in collagen fibrillogenesis and suggest that the orchestrated action of several leucine-rich repeat glycoproteins/proteoglycans influence the architecture of collagen matrices.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -helices and beta -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.

    MATERIALS AND METHODS
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ABSTRACT
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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-beta -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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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.

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.

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.

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.

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.

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).

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.

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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Table I
Protein and mRNA levels in wild type and fibromodulin-null littermates

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 (open circle ), 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.

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 beta -actin using lumican and beta -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.

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 beta -actin antisense probes (Fig. 11). The level of lumican mRNA in fibromodulin-null tendon fibroblasts relative to beta -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 beta -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 beta -actin probe (probe) and control RNase digestion of the beta -actin probe in the absence of RNA (probe + RNase) are shown. A 250-bp RNase-protected RNA was generated with the beta -actin probe in the presence of approximately 2 ng of mRNA from wild type (+/+), heterozygous (+/-), and fibromodulin-null (-/-) littermates.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* This work was supported by the Swedish Medical Research Council, Anna-Greta Crafoord's Stiftelse, Greta och Johan Kock's Stiftelse, Gustav V 80-års Fond, and Alfred Österlund's Stiftelse.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.

parallel To whom correspondence should be addressed. Tel.: 46-46-2228577; Fax: 46-46-2223128; E-mail: ake.oldberg{at}medkem.lu.se.

    ABBREVIATIONS

The abbreviations used are: LRR, leucine-rich repeat; bp, base pair(s); ELISA, enzyme-linked immunosorbent assays.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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