ARTICLE |
Correspondence to: Lynn Y. Sakai, Shriners Hospital for Children, 3101 SW Sam Jackson Park Rd., Portland, OR 97201.
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Summary |
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The molecular basis for Marfan's syndrome (MS), a heritable disorder of connective tissue, is now known to reside in mutations in FBN1, the gene for fibrillin-1. Classic phenotypic manifestations of MS include several skeletal abnormalities associated primarily with overgrowth of long bones. As a first step towards understanding how mutations in FBN1 result in skeletal abnormalities, the developmental expression of fibrillin-1 (Fib-1) in human skeletal tissues is documented using immunohistochemistry and monoclonal antibodies demonstrated here to be specific for Fib-1. At around 10-11 weeks of fetal gestation, Fib-1 is limited in tissue distribution to the loose connective tissue surrounding skeletal muscle and tendon in developing limbs. By 16 weeks, Fib-1 is widely expressed in developing limbs and digits, especially in the perichondrium, but it is apparently absent within cartilage matrix. Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage. However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage. Because these fibers can be extracted from cartilage using dissociative conditions, we postulate that they are laterally packed and crosslinked microfibrils. On the basis of these findings, we suggest that the growth-regulating function of Fib-1 may reside persistently within the perichondrium. In addition, the accumulation of special laterally crosslinked Fib-1 microfibrils around chondrocytes during late adolescence suggests that growth-regulating activities may also be performed by Fib-1 at these sites. (J Histochem Cytochem 45:1069-1082, 1997)
Key Words: fibrillin, cartilage, microfibrils, Marfan's syndrome, special banded fibers
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Introduction |
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Microfibrils are recognizable structural elements found throughout the connective tissue. They were first defined as small-diameter fibrils present near basement membranes (
The structural backbone of all microfibrils is believed to be composed of end-to-end polymers of fibrillin (Fib) molecules (
Because the fibrillins are highly homologous molecules, monoclonal antibodies (MAbs) that can distinguish between Fib-1 and Fib-2 are required to clarify the tissue distribution of the two fibrillins and to determine whether microfibrils are homopolymers or heteropolymers of the fibrillins. Here we report the characterization of two MAbs specific for Fib-1 and use them to demonstrate the distribution of Fib-1 in hyaline cartilage, the surrounding perichondrium and attachment sites for tendon, and in joint capsules. In addition, we identify a novel banded fiber unique to cartilage. This novel banded fiber contains Fib-1 and represents a special type of microfibril that appears to be laterally crosslinked.
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Materials and Methods |
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Characterization of Monoclonal Antibodies
MAbs 26, 69, and 201 have been previously described and demonstrated to bind specifically to epitopes present in fibrillin-1 (
Construction and Expression of Recombinant Fibrillin Subdomains.
To assemble expression vectors for recombinant production of Fib-1 and Fib-2 subdomains, FBN1 or FBN2 clones were PCR-amplified using Vent polymerase (New England Biolabs; Beverly, MA). To subclone the PCR products, the primers were designed to introduce an additional NheI restriction site at the 5' end and either a NotI or XhoI restriction site at the 3' end. In addition, the sequences for 6 histidine residues were introduced into the PCR product to facilitate purification of the recombinant peptide using chelating chromatography. Ligation of the PCR products into the episomal expression vector pCEP4/2III4, which contains the sequence for the BM40/SPARC signal peptide (
Fib-1 Recombinant Subdomains.
Expression vectors rF30 (coding for D330 to I489) and rF31 (coding for D288 to I489) were constructed from human FBN1 clone HFBN29 (2III4 to yield the plasmids pCEPSP-rF30 and pCEPSP-rF31. The construction, expression, and purification of rF18 and rF20 have been previously described in detail (
Fib-2 Recombinant Peptides.
Expression vectors rF33 (coding for D317 to I533) and rF37 (coding for S145 to L995) were constructed from human FBN2 cDNA clones obtained by screening a gt11 unamplified placenta library (Clontech; Palo Alto, CA) with FBN1-specific PCR products, as described previously (
22-3, was used to amplify sequences for rF33 and rF37. For rF33, primers DR 67 (5'-CGTAGCTAGCAGACATTGATGAGTGCAGCATC-3') and DR 70 (5'-ATAGTTTAGCGGCCGCTAGTGATGGTGATGGTGATGTATACAATC-TCCATTTGCATCCTGC-3') were used. A 679-BP NheI-NotI fragment was then subcloned into pCEP4/
2III4 and designated pCEPSP-rF33. For rF37, primers DR 77 (5'-CCCGGTGTATTGACGCAGATTCC-3') and DR 86 (5'-ATA- AGAATGCGGCCGCGCTAGCATCAATTCAGCAGTGCA- GTGTGAG-3') were used, which introduced an additional NheI and EagI restriction site 5' to the FBN2 sequence (nucleotides 432-3404). A 2893-BP EagI-XbaI fragment was then subcloned into EagI-XbaI restricted pBluescript II SK (+) (Stratagene; La Jolla, CA). The resulting plasmid was designated pBS-UP
22-3. To modify the 3' end, pBS-UP
22-3 was PCR-amplified with primer F5.306S (5'-TGAATGTGAAAGCAACCCATG-3') and DR 89 (5'-ACCGCTC- GAGCTAGTGATGGTGATGGTGATGCAAACATACAC-GGCCAGTCCC-3'). The MunI-XhoI fragment from pBS-UP
22-3 was then replaced by the 391-BP MunI-XhoI PCR fragment to yield plasmid pBS-rF37. The 2581-BP NheI-XhoI fragment from pBS-rF37 was subcloned into pCEP4/
2III4 and designated pCEPSP-rF37.
The complete sequences of subcloned PCR amplified products were confirmed by automated DNA sequencing (Applied Biosystems; Foster City, CA). The sequences obtained from pCEPSP-rF30, pCEPSP-rF31, and pCEPSP-rF33 corresponded with the expected sequences. Some differences were observed for FBN2 sequences from pBS-UP22-3 and pBS-rF37: 574insGA and 582insC leading to an amino acid exchange from residues 192-194 (AQP to GPNR) [a sequencing error also previously reported (
Immunoblotting
Recombinant subdomains of Fib-1 (rF18, rF20 (
Other Analytical Methods
Molecular masses of recombinant proteins were determined by comparison of mobilities in SDS-PAGE with globular marker proteins.
N-terminal sequence analyses were performed using a gas phase sequencer (Applied Biosystems 475). Amino acid compositions and protein concentrations were determined after hydrolysis of peptides in 6 M HCl (24 hr at 110C) on an amino acid analyzer (Beckman 6300; Palo Alto, CA).
Light Microscopy
AMEX immunostaining was performed on fetal human tissues (10-11 and 16 weeks' gestation) fixed in acetone (
Immunofluorescence was performed on acetone-fixed frozen sections as previously described (
Confocal Microscopy
A specimen of 14-year-old human rib cartilage was frozen in hexanes and sectioned to a thickness of 40 µm using a Leitz cryostat. Sections were collected on poly-L-lysine-coated slides, dried, then fixed in ice-cold acetone for 10 min. Sections were then removed from the slides by immersion in PBS and submersed in chondroitinase ABC (Sigma) (290 U/ml PBS) for 90 min at 37C. The sections were rinsed in PBS, immersed overnight at 4C in MAb 69 diluted 1:50 in PBS, washed, then immersed in goat anti-mouse TRITC conjugate (Jackson ImmunoResearch Laboratories; West Grove, PA) diluted 1:50 in PBS for 30 min at RT. The sections were rinsed, mounted on slides using Aquamount (Lerner Laboratories; Pittsburgh, PA), and viewed with a Leica confocal microscope equipped with an argon/krypton laser. Images from 2-µm-thick optical sections were collected and recorded.
Electron Microscopy
An overview of the methods used in this study has been described previously (
Extracts of 14-year-old rib cartilage were prepared by first trimming away all exterior surfaces of the rib (eliminating perichondrium), mincing the remaining tissue with a sharp blade, then homogenizing the tissue in ice-cold 0.2 M ammonium bicarbonate, pH 7.4, using the 1/4'' blade of a polytron homogenizer until no visible tissue fragments remained. The resulting homogenate was then either exposed to MAb 69 overnight at 4C, followed by washing and an additional overnight exposure to a 1:1 mixture of 5- and 10-nm gold particulates conjugated to goat anti-mouse IgG (Amersham; Arlington Heights, IL) or to 20 U/ml collagenase (Worthington; Freehold, NJ) in 0.05 M Tris-HCl, pH 7.5, 5 mM CaCl2 for 4 hr at RT, followed by exposure to 6 M guanidine-HCl (Amresco; Solon, OH) in water. All homogenates were allowed to settle onto the surface of carbon-coated grids made hydrophilic by glow-discharge, then stained in 3% phosphotungstic acid, pH 7.0.
Immunolocalization of fibrillin within 13-year-old humeral head articular cartilage and also in 13-, 14-, and 31-year-old rib was accomplished by first digesting the tissue with chondroitinase ABC (Sigma) (290 U/ml PBS) for a minimum of 2 hr at RT, followed by en bloc incubation overnight at 4C in either MAb 69, MAb 26, or MAb 201, followed by washing and immersion overnight at 4C in goat anti-mouse IgG 5-nm colloidal gold conjugate (Amersham; Arlington Heights, IL). Control antibodies included those specific for collagen Types II, VI, IX, XI, and XII. Immunolabeled tissues were then fixed, dehydrated, and embedded as described above.
In addition, one sample of 20-year-old cartilage was fixed in ice-cold 4.0% paraformaldehyde-0.1% glutaraldehyde, rinsed in cacodylate buffer and then in 0.15 M Tris-HCl overnight, then dehydrated in a graded alcohol series at progressively lower tempertures (30% EtOH -10C; 50% EtOH -20C; 70% EtOH -20C), infiltrated in LR White embedding Resin (Electron Microscopy Sciences; Fort Washington, PA), and polymerized at 60C for several days. Ultrathin sections were mounted on formvar-coated single-hole slot grids, which were floated section side down at RT in the following solutions: Tris-HCl with 0.1% Tween-20 for 15 min; Tris-HCl containing 0.05M glycine for 60 min; Tris-HCl with 0.1% Tween-20, 0.5% ovalbumin, 0.5% fish gelatin, and 2% nonfat dry milk for 15 min; Tris-HCl containing 0.1% Tween-20 for 5 min; MAb 26 diluted 1:10 in Tris-HCl with 0.1% Tween-20, 0.5% ovalbumin, 0.5% fish gelatin for 120 min; rinsed for 30 min in several changes of Tris-HCl with 0.1% Tween-20; goat anti-mouse 10-nm gold conjugate (Amersham) diluted 1:15 in Tris-HCl with 0.1% Tween, 0.05% ovalbumin, 0.05% fish gelatin; rinsed in Tris-HCl with 0.1% Tween-20; and finally rinsed in distilled water for 30 min.
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Results |
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Characterization of Fib-1-specific Antibodies
Previously published results positioned the epitopes recognized by MAb 26, MAb 201, and MAb 69 to distinct regions in Fib-1 (
The apparent molecular masses of rF30 (27 kD), rF31 (32 kD), rF33 (30 kD), and rF37 (110 kD) corresponded well with their calculated masses, and Edman degradation resulted in the expected N-terminal sequences (Figure 1). Amino acid compositions also agreed with calculated values. Rotary shadowing of the large Fib-2 peptide rF37 (not shown) demonstrated an extended thread-like shape 38.1 ± 3.6 nm long, similar to shapes of recombinant Fib-1 peptides (
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MAb 26 reacted positively with both rF30 (not shown) and rF31 (Figure 2A), locating the epitope to a position between amino acid residues 330 and 489 of Fib-1. rF20, which begins with the EGF-like motif (residues 450-489) adjacent to the proline-rich region, does not react with MAb 26 (
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Fib-1 Expression in Fetal Tissues
Immunohistochemical staining of tissues from a developing human fetus (10-11 weeks' gestation) showed a limited distribution of Fib-1. In Figure 3, MAb 26 (Figure 3B and Figure 3E) and MAb 201 (Figure 3C and Figure 3F) demonstrate similar staining patterns, which are restricted to the loose connective tissue surrounding skeletal muscle and tendon. Endomysium and perimysium, as well as dermis, vasculature, nerves, perichondrium, joint capsules, and cartilage, are not stained. A positive control antibody specific for tenascin-C, which stains the perichondrium, is shown in Figure 3D. Other control antibodies specific to components known to be present in fetal cartilage, trabecular bone, and skin were also positive in sections similar to these (not shown).
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By 16 weeks' gestation, Fib-1 is widely expressed in the fetus. Figure 4 shows cross-sections of a finger (Figure 4A and Figure 4D) demonstrating intense Fib-1 staining throughout the connective tissue (Figure 4D), except within the matrix of cartilage. Increased staining is apparent in the loose connective tissue, around nerves as well as muscle (Figure 4B). By this developmental stage, there is abundant and classical staining of Fib-1 in both skin (Figure 4E) and perichondrium (Figure 4C and Figure 4F). High-magnification views suggested that Fib-1 staining entered the periphery of the cartilage matrix but was largely absent from the interior of the cartilage (Figure 4F). The abundant Fib-1 staining of the perichondrium seen at this time persists through maturity (data not shown).
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Fib-1 staining was found within the matrix of cartilage from the arm after 20 weeks of fetal gestation (Figure 5). A large mesh network of fine fibrils was present. This network of fibrils became more flattened closer to the peripheries of the cartilage (not shown).
Special Banded Fib-1 Fibers in Aging Human Cartilage
Immunofluorescence confocal microscopy of cartilage from adolescents showed Fib staining primarily pooled around chondrocytes within resting and proliferative zones (data not shown). Serial immunofluorescence images demonstrated bright baskets around chondrocytes. Figure 6 is one of a series of sections of Fib staining around a single chondrocyte. Yellow immunofluorescence indicates the Fib network encapsulating the cell. Hypertrophic zones were relatively negative for Fib-1 (data not shown).
Electron microscopy revealed typical microfibrils (Figure 7A and Figure 7B), fibrils that were more densely packed (Figure 7C), as well as very densely packed banded fibers (Figure 8). All these fibrils labeled with Fib-1-specific antibodies (Figure 7A, Figure 8E) but did not label with antibodies specific for collagens II, VI, IX, XI, and XII. These banded Fib-1 fibers most commonly demonstrated a periodicity of approximately 50 nm (with a range up to 65 nm) and could be distinguished ultrastructurally from thin collagen fibers as well as from thick "amianthoid" fibers (
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Of 44 cartilage samples scored for the presence or absence of the banded Fib-1 fibers, 19 came from individuals under the age of 8, and 17 of these samples did not appear to contain the banded fibers. Of 25 samples from individuals ranging in age from 9 to 71 years, all but one (a sample from an 18-year-old) contained banded fibers. These data are shown in Table 1. Athough most of these samples were rib cartilage and articular cartilage, samples of elastic cartilage, fibrous cartilage, and vertebral cartilage were also included. Samples were not scored for their apparent abundance.
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Because banded Fib-1 fibers were always detected in the matrix of aged cartilage (Figure 9A), other comparably aged tissues were also examined. Laterally packed and banded Fib-1 fibers were not found in any other tissue. In skin, Fib-1 microfibrils appeared typically loosely bundled (Figure 9B). In cornea, laterally packed and banded fibers were seen (Figure 9C). However, the periodicity of the banding pattern of the corneal fiber was was much longer (115 nm compared to
50 nm), and these corneal fibrils were not recognized by Fib-specific antibodies (data not shown).
To test whether the banded Fib-1 fibers might be a peculiar artifact of preparing cartilage for microscopy, cartilage homogenates were extracted with collagenase and guanidine, and were stained with phosphotungstic acid. Even after exposure to these dissociative conditions, large banded Fib-1 fibers were visualized (Figure 10A and Figure 10B) and could be immunolabeled with some periodicity using an antibody specific to fibrillin (Figure 10C). The variable thickness, tapered ends (Figure 10A), and the banding pattern (Figure 10B) of the extracted fibers probably represent their authentic morphologies because these features were comparable to those noted in fixed, dehydrated, and embedded tissues (e.g., Figure 8C).
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Because en bloc incubation with antibodies results in labeling of fiber surfaces but not interiors, techniques for labeling section surfaces were employed to investigate the presence of Fib-1 within the dense banded fibers. After exposure of fiber interiors to MAbs 69 (not shown), 15 (not shown), and 26 (Figure 11), gold labeling was found throughout the interior of the fiber rather than only at the surface.
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Discussion |
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Using MAbs (26 and 201) that bind to specific epitopes in Fib-1 but do not recognize similar domains present in Fib-2, we have documented the developmental expression of Fib-1 in human fetal limbs and digits. Early in fetal gestation (10-11 weeks), Fib-1 is first expressed in the loose connective tissue around skeletal muscle and tendon. However, by 16 weeks' gestation, abundant and classical immunohistochemical staining is found in all tissues, with the exception of cartilage matrix. At this time, and continuing through adulthood, perichondrium contains abundant Fib-1 microfibrils, which are also endowed with elastin. By 20 weeks of fetal gestation, a loose meshwork of immunofluorescent Fib-1 fibers is detected within cartilage matrix. These results are consistent with the general pattern of expression of fbn1 in fetal mouse tissues examined by in situ hybridization techniques (
Between these fetal times and adolescence, immunoelectron microscopy reveals sparse but otherwise classic loose bundles of Fib-1 microfibrils within cartilage matrix. The thin meshwork of Fib-1 immunofluorescence in fetal cartilage matrix corresponds to these loose bundles of microfibrils. By late adolescence, densely packed and laterally cross-striated fibers containing Fib-1 accumulate around the periphery of resting chondrocytes, but hypertrophic chondrocytes appear not to be encapsulated by these fibers. Because fetal expression of Fib-1 by hypertrophic chondrocytes has been reported (
Fib-1 broad banded fibers can be distinguished morphologically and immunochemically. They have not been described previously and appear to be unique to cartilage. To investigate whether the appearance of these special banded fibers might be due to artifacts associated with dehydration and preparation of cartilage for microscopic examination, cartilage was extracted using dissociative conditions and extracts were examined by electron microscopy. Identical banded fibers were found in these extracts, suggesting that these fibers represent densely packed and laterally crosslinked Fib-1 microfibrils. Additional immunolabeling of the interiors of these fibers by Fib-1-specific antibodies further supports this suggestion. How Fib-1 microfibrils become packed and crosslinked in cartilage and determination of other molecules that might be present in these banded fibers require further investigation. Collagens II, VI, IX, XI, and XII, however, do not appear to be components of these fibers.
Mutations in FBN1 result in Marfan's syndrome, a heritable disorder of connective tissue affecting 1:10,000 individuals. A single mutation in FBN1 can cause multiple and variable phenotypic manifestations (
In the perichondrium/periosteum, Fib-1 microfibrils are highly organized into long elastic fibers uniformly aligned parallel to the long axis of the bone (data not shown) (
Alternatively, or in addition, growth-regulating activities of Fib-1 may be performed specifically at the time when growth ceases. We have documented the presence of special banded Fib-1 fibers that accumulate around chondrocytes at the time when growth ceases (late adolescence) and which are abundant in adult cartilage. This new finding suggests the possibility that Fib-1 fibers may exert a special influence on chondrocytes to cease proliferation and differentiation. Because fibrillins are highly homologous to the family of latent transforming growth factor-ß-binding proteins (
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Acknowledgments |
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Supported by grants from the Shriners Hospitals for Children (DRK, LYS) and by the Deutsche Forschungsgemeinschaft (DPR).
We thank Maudine Waterman (Dartmouth Medical Center) and Noe Charbonneau (Shriners) for excellent technical assistance. Expertise provided by Kenneth Fish at the Laboratory for Confocal Microscopy, Oregon Health Sciences University, is sincerely appreciated. Electron microscopy facilities were provided in part by the Fred Meyer and R. Blaine Bramble Charitable Trust Foundations.
Received for publication October 3, 1996; accepted January 21, 1997.
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