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First published online March 23, 2006
doi: 10.1242/10.1242/dev.02308
1 Department of Biomedical Engineering and Orthopaedic Research Center, Lerner
Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue,
Cleveland, OH 44195, USA.
2 Laboratory of Connective Tissues Biology, Center of Biomedical Integrative
Genoproteomics, University of Liège, 4000 Sart Tilman, Belgium.
3 Department of Cell Biology, Thomas Jefferson University, Philadelphia, PA
19107, USA.
* Author for correspondence (e-mail: aptes{at}ccf.org)
Accepted 2 February 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: ADAMTS, Metalloprotease, Procollagen, Dermatosparaxis, Ehlers-Danlos syndrome
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). They form
organotypic matrices by tissue-specific expression and by assembly of the
collagen triple-helix into a variety of supramolecular aggregates
(Birk et al., 1991). Collagen I
is the major extracellular matrix (ECM) protein of bone, tendons, ligaments,
and the dermis of the skin. Collagen II is the major collagen of cartilage,
but it is also present in the vitreous of the eye and in the nucleus pulposus
of the intervertebral disc (Zhu et al.,
1999). Collagen III is a component of many stretchable tissues,
such as the vascular wall, dermis, lung, heart and gastrointestinal tract
(De Paepe and Malfait, 2004).
It can co-polymerize with collagen I to form heterotypic fibrils and it is
co-expressed in varying proportions with collagen I at many locations, with
the exception of bone (Niederreither et
al., 1995). Collagen I contains two 
1(I) chains and one
2(I) chain, whereas collagens II and III are homotrimers of
1(II) and 1(III) chains, respectively
(Olsen, 1991). Structural
mutations in collagen genes have serious consequences for tissues where single
collagen types are major constituents. Collagen I mutations cause osteogenesis
imperfecta (Rauch and Glorieux,
2004). Chondrodysplasias, sometimes with ocular anomalies, such as
Stickler syndrome, are caused by collagen II mutations
(Chan et al., 1995;
Cole, 1997;
Snead and Yates, 1999;
Tiller et al., 1995), and
collagen III mutations cause the vascular subtype of the Ehlers-Danlos
syndrome (type IV), which is characterized by aortic aneurysms and skin
fragility (De Paepe and Malfait,
2004).
Fibrillar collagens are made as a precursor (procollagen) and consist of a
long triple-helical (collagenous) domain flanked at each end by globular
non-collagenous propeptides (Olsen,
1991). The three major fibrillar procollagens share similar
biosynthetic mechanisms. Following intracellular assembly of the triple helix,
the procollagen molecules undergo proteolytic excision of each propeptide
either prior to or following secretion
(Canty et al., 2004;
Olsen, 1991). The C-propeptide
is excised by BMP1, an astacin-like protease
(Kessler et al., 1996).
ADAMTS2 (for A Disintegrin-like And Metalloprotease domain with Thrombospondin
type 1 motifs 2) can excise the N-propeptide of all three major fibrillar
collagens (Tuderman and Prockop,
1982; Wang et al.,
2003). The processed collagen monomers assemble into fibrils and
tissue-specific supramolecular aggregates. Because of a common biosynthetic
pathway, it was expected that defects in ADAMTS2 would have broader effects
than structural mutations involving individual procollagen chains. Instead,
ADAMTS2 mutations lead to a limited clinical syndrome -
dermatosparaxis (fragility of skin) in animals and the Ehlers-Danlos
syndrome-type VIIC (EDS-VIIC or the dermatosparactic type) in humans
(Colige et al., 2004;
Colige et al., 1999;
Lapiere and Nusgens, 1993;
Nusgens et al., 1992;
Petty et al., 1993;
Smith et al., 1992;
Wertelecki et al., 1992).
Collagen fibrils in the dermis of dermatosparactic calves, humans and mice are
thin, branched and `hieroglyphic' on cross-section, instead of being round and
of relatively uniform diameter (Li et al.,
2001; Nusgens et al.,
1992). Although this disorder is largely caused by anomalies in
skin (dermal) collagen, there is only a modest effect on other collagen I-rich
tissues, such as bone, tendon and muscle
(Lapiere and Nusgens, 1993).
Processing of procollagen II in the cartilage of dermatosparactic calves and
mice is only minimally compromised, or not at all
(Fernandes et al., 2001;
Li et al., 2001).
It was hypothesized that enzymes closely related to ADAMTS2 may process
procollagen I in tissues other than skin
(Lapiere and Nusgens, 1993);
this is supported by the observation that in bovine dermatosparaxis, the
residual processed procollagen is cleaved at the expected site
(Colige et al., 2002). We
previously found that ADAMTS3, could process the amino-propeptide of
procollagen II (Fernandes et al.,
2001). Subsequently, it was shown that another homologous enzyme,
ADAMTS14, could process procollagen I
(Colige et al., 2002), and that
ADAMTS2 could process procollagen III
(Wang et al., 2003).
Nevertheless, a comprehensive understanding of how these enzymes participate
in collagen biosynthesis in vivo, particularly during embryogenesis, as the
first collagenous matrices are laid down, has not been achieved.
ADAMTS2, ADAMTS3 and ADAMTS14 constitute an evolutionarily related cluster
of ADAMTS proteases, the procollagen amino-propeptidases (PNPs). They have an
identical domain structure and a unique modular composition not present in any
other enzyme. The active site sequence in their catalytic domain
(HETGHVLGMEHD) differs from that of the other ADAMTS proteases. This, and
previous experimental evidence, suggested that their catalytic activities are
very similar (Colige et al.,
2002; Fernandes et al.,
2001; Wang et al.,
2003). Here, we demonstrate, through the temporal and spatial
analysis of mRNA encoding ADAMTS2, ADAMTS3 and ADAMTS14, as well as of that
encoding procollagen I, II and III, that there is highly coordinated
developmental expression of two of these enzymes and their procollagen
substrates during mouse embryogenesis. We demonstrate that ADAMTS3 can process
procollagen I. Together with its prominent expression in some procollagen I
rich tissues and in cartilage, ADAMTS3, and not ADAMTS2, may be the major
procollagen I and II aminopropeptidase from a developmental perspective.
ADAMTS2 is essential for procollagen III processing in mice, with possibly
important implications for human dermatosparaxis.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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1(I)
collagen (Col1a1) and mouse pro-1(III) collagen
(Col3a1) were provided by E. Vuorio (University of Turku, Finland)
(Niederreither et al., 1995).
The mouse pro-1(II) collagen (Col2a1) cDNA was provided by Dr
Naomi Fukai (Harvard Medical School). The Adamts2 probe was generated
by subcloning a 600 base pair (bp) NotI-PstI fragment from
IMAGE clone AA832579 into pBluescript II (Stratagene, La Jolla, CA). The
following primers were used to amplify Adamts3 and Adamts14
cDNA fragments by RT-PCR of mouse 17.5 days post-coitum (dpc) embryo cDNA. Adamts3 (amplifying a 528 bp fragment corresponding to the unique C-terminal domain of mouse Adamts3), 5'-CTGAAGAGCTGGTCTCAGTG-3' (forward) and 5'-TGGACTTGTCACCCAAACATG-3' (reverse); and
Adamts14 (producing a PCR product of 310 bp that corresponds to the unique C-terminal domain), 5'-AGGTGGGTGACAGAAGAGTGG-3' (forward) and 5'-AGGCTGCAGATCTGCACCATG-3' (reverse).
The PCR products were cloned into pGEMT-Easy (Promega, Madison, WI). Radioactive sense and antisense cRNA probes were generated by in vitro transcription using T3, T7 or SP6 RNA polymerases in the presence of [35S]-UTP (Perkin-Elmer).
In situ hybridization
In situ hybridization was performed on tissue sections of mouse embryos of
gestational age 7.5 to 17.5 dpc. Embryos, including the surrounding uterus at
7.5 dpc and 9.5 dpc, were fixed overnight in 4% paraformaldehyde, embedded in
paraffin wax, and sectioned at 6 µm. The sections were hybridized to
[35S]-UTP labeled riboprobes, as previously described
(Somerville et al., 2003).
Slides were dipped in NTB-2 emulsion (Kodak, Rochester, NY) and exposed at
4°C for 2 to 10 days. Nuclei were stained with Hoechst 33258 (Sigma, St
Louis, MO). Dark-field (autoradiographic signal) and fluorescence microscopy
(nuclear staining) images were captured on a Leica DMR upright microscope
(Leica Microsystems, Wetzlar GmbH), with a Retiga Exi Fast 1394 cooled CCD
camera with RGB liquid crystal color filter slider (Q Imaging, Burnaby, BC,
Canada). Images were viewed with ImagePro Plus software (Media Cybernetics,
Silver Spring, MD). Silver grains visualized on dark field microscopy were
given a red pseudocolor and superimposed on nuclear fluorescence (blue).
ADAMTS3 antibodies
A polyclonal antibody was generated in rabbits by immunization with a
C-terminal peptide from human ADAMTS3 (CKKDGKIIDNRRPTRSSTLERCOOH), coupled to
keyhole-limpet hemocyanin (Alpha Diagnostics International, Houston, TX). The
antiserum was affinity-purified against the peptide coupled to NHS-sulfolink
(Pierce).
Cloning of cells expressing ADAMTS3 and co-culture with dermatosparactic fibroblasts
HEK 293F cells (Invitrogen, Carslbad, CA) were maintained in growth medium
(DMEM supplemented with antibiotics and 10% fetal bovine serum). Clones stably
expressing the ADAMTS3 expression plasmid pSHTS3 were isolated as
previously described (Fernandes et al.,
2001). Culture media were analyzed by western blotting with
ADAMTS3 polyclonal antibody at a 1:1000 dilution, and clones expressing the
highest levels of ADAMTS3 were used in subsequent studies. Peptide
N-glycosidase F (PNGaseF, New England Biolabs, Beverly, MA) treatment of
conditioned medium was carried out as previously described
(Somerville et al., 2003).
Dermatosparactic calf fibroblasts were cultured alone or co-cultured with
equivalent numbers of HEK293 cells transfected with the empty plasmid vector
(a negative control), or with the plasmid vector expressing either ADAMTS3 or
ADAMTS2 (as a positive control) (Colige et
al., 2002). Co-culture experiments were performed in DMEM
supplemented with 2% FCS and ascorbic acid (50 µg/ml) in the absence of
G418. After 24 hours, the culture medium and the cell layer were collected
separately and denatured in Laemmli sample buffer with or without 0.1 M DTT.
The characterization of collagen polypeptides was performed by western
blotting using type I or type III collagen-specific antiserum and ECL. These
antisera were produced by repeated immunization of rabbit and guinea pig with
bovine collagen I or collagen III, respectively. These antisera, used at a
1:2000 dilution, were highly specific and did not cross-react with other
collagen types (data not shown).
Histology, transmission electron microscopy (TEM) and mCT of Adamts2-deficient (Adamts2-/-) mice
Adamts2-/- mice in the 129Sv strain were kindly
provided by D. Prockop (Tulane University) and were used under approved
institutional protocols. Genotype analysis was by PCR as previously described.
Two-month-old mice were killed by CO2 inhalation, and tissues and
organs were isolated for histological analysis and for transmission electron
microscopy. Lungs were fixed in 10% formalin under inflation as previously
described (Oblander et al.,
2005), followed by routine histology, including Hematoxylin and
Eosin stain, Hart's stain for elastin and the Mallory trichrome stain. The
skulls and hind limbs of the Adamts2-/- mice and wild-type
littermates were analyzed by micro-computerized tomography (mCT), followed by
three-dimensional reconstruction of the images. Samples for TEM were prepared
as previously described (Birk and Trelstad,
1986). Thick sections (1 µm) were cut and stained with
methylene blue-azure B. Thin sections were cut using a Leica UCT
ultramicrotome and a diamond knife. The lung was stained with 2.5% (w/v)
ethanolic uranyl acetate followed by 0.04% (w/v) bismuth subnitrate. The aorta
was stained with aqueous 2% (w/v) uranyl acetate followed by lead citrate.
Sections were examined and photographed at 80 kV using a Tecnai 12
transmission electron microscope equipped with a Gatan US1000 2K Ultrascan
digital camera.
Procollagen processing in Adamts2-/- mice
Individual organs were isolated from Adamts2-/- mice
and wild-type littermates. These were either ground in liquid nitrogen and
suspended in washing buffer [0.25 M sucrose, 20 mM EDTA, 2.5 mM NEM, 0.5 mM
PMSF, 50 mM Tris (pH 7.5)] or directly solubilized in Laemmli sample buffer
(aorta). After centrifugation (20000 g, 10 minutes),
supernatants were discarded and pellets washed again as described above.
Pellets were then suspended in 0.15 M extraction buffer [0.15 M NaCl, 50 mM
Tris (pH 7.5)], rotated for 2 hours at 4°C and centrifuged (20000
g, 10 minutes). Extracts were collected and pellets
sequentially extracted in 1 M extraction buffer [1 M NaCl, 50 mM Tris (pH
7.5)] and 0.1 M acetic acid. Adequate amounts of the various samples were then
dialyzed at 4°C in 0.1 M acetic acid, lyophilized, denatured in Laemmli
sample buffer with or without 0.1 DTT, and analyzed by SDS-PAGE. Sponge
granulomas were induced by the subcutaneous insertion of circular PVA sponges
(10 mm diameter, clinical PVA sponges grade 3, M-PACT Worldwide Management,
Eudora, KS) rehydrated in PBS. After 4 weeks, mice were sacrificed and sponges
were collected. Histology demonstrated the presence of abundant granulation
tissue containing fibroblasts and blood vessels (data not shown). The sponges
were ground, washed as described above, and then proteins were extracted in
Laemmli sample buffer, separated by SDS-PAGE and western blotted with
anti-collagen III as described above.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Thus, Col1a1 was primarily expressed in bone,
skin, perichondrium, periosteum, tendons and the adventitia (outermost layer)
of blood vessels. Col2a1 was expressed almost exclusively in
cartilage. Col3a1 expression was prominent in the vascular wall, lung
mesenchyme, peritoneal lining and the wall of visceral organs. Col3a1
was co-expressed with Col1a1 in skin, but not in bone. None of the
sense probes for any of the collagen or ADAMTS genes gave a signal above
background levels.
Adamts2 and Adamts3 are expressed in the maternal tissues at day 7.5 of gestation
Northern analysis of commercial embryo RNA blots had shown the highest
expression of both Adamts2 and Adamts3 at 7 dpc
(Fernandes et al., 2001).
However, in situ hybridization clarified that there was no expression in the
embryo itself, whereas there was strong expression in maternal tissues,
specifically in the decidual reaction and the myometrium of the uterus.
Prominent Col1a1 expression was seen in these tissues.
Col1a1 and Adamts3 expression overlapped significantly in
the decidual reaction (Fig. 1).
The Adamts2 expression pattern was different, but also overlapped
with Col1a1 (Fig. 1).
The prominent co-expression of Col1a1 with these biosynthetic enzymes
reflects the intense remodeling of the uterus that occurs during pregnancy.
These data suggest that the previously described northern analysis findings
resulted from the inclusion of a significant amount of maternal tissue.
Adamts3, but not Adamts2 or Adamts14, is co-expressed with Col2a1 in cartilage
Most of the skeleton is formed from cartilage models that undergo
endochondral ossification. Cartilage is first formed around 12 dpc in
mesenchymal condensations that begin to express procollagen II and aggrecan.
We have previously shown that ADAMTS3 processed procollagen II in vitro, and
that ADAMTS3 mRNA was present at higher levels than ADAMTS2
mRNA in cultured chondrocytes by RT-PCR
(Fernandes et al., 2001).
However, in that study, the spatial and temporal association of
Adamts3 and Col2a1 was not determined. Adamts3 mRNA
was associated with Col2a1 mRNA expression from the initiation of
chondrogenesis (Fig. 2A) and,
subsequently, throughout skeletal development in all cartilage models, whether
in the appendicular, axial or craniofacial skeleton
(Fig. 2A-G). Chondrocytes in
developing growth plates differentiate into hypertrophic chondrocytes and, as
they do so, they suppress collagen II production and produce an abundance of a
short-chain collagen, type X (LuValle et
al., 1989), whose N-propeptides are not processed. Interestingly,
Adamts3 was downregulated as chondrocytes became hypertrophic
(Fig. 2C,E-G). After 12 dpc,
pre-cartilaginous mesenchymal tissue is located in the perichondrium around
cartilage (Zhu et al., 1999);
The Adamts3 expression domain extended beyond the limits of the
Col2a1 expression domain into the perichondrium
(Fig. 2B-G). Neither
Adamts2 nor Adamts14 were detectable by in situ
hybridization in cartilage at any developmental stage examined.
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Adamts2-/- mice have a normal skeleton and teeth
mCT analysis of two-month-old mice in whom skeletal maturity was attained,
demonstrated no consistent anomalies in either the appendicular, axial or
craniofacial skeleton. The overall morphology of the skull, as well as the
surface features such as vascular foramina and the dimensions of its component
bones, appeared no different from that of wild-type littermates
(Fig. 4A). Severe abnormalities
of the secondary dentition have been documented in EDS-VIIC patients
(De Coster et al., 2003;
Malfait et al., 2004).
Incisors from Adamts2-/- mice appeared normal, although
molar teeth from Adamts2-/- mice showed a subtle loss of
surface contour when compared with the wild-type littermates
(Fig. 4A). Although
Adamts2 expression was not detectable during tooth development,
Adamts3, but not Adamts14, was highly expressed in the
dental papilla and at lower levels in ameloblasts
(Fig. 4B).
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130-150 kDa protein that was
reactive with the affinity-purified ADAMTS3 antibodies. The reactive band
migrated faster after digestion with PNGase F as expected, since ADAMTS3 is
predicted to have a N-linked carbohydrate
(Fig. 4C). Type I collagen
produced by dermatosparactic calf skin fibroblasts (DS) in culture consists of
pro 1(I) and pro 2(I) in the cell layer, and pro 1(I),
pro 2(I) and pN1(I) in the medium, demonstrating the absence of
significant type I aminoprocollagen peptidase activity
(Fig. 4D). In co-culture with
control HEK293F transfected cells (Ct), pro 1(I) and pro 2(I)
are also the most abundant products, but pC1(I), pN1(I),
1(I) and pN2(I) are also detected, especially in the conditioned
medium. These data illustrate the presence of low amino- and
carboxy-procollagen peptidase activity. This is probably related to the
synthesis of low levels of BMP1, ADAMTS3 and/or ADAMTS14 by DS, enzymes that
may be more active in the high-confluence conditions observed in co-cultures
than in monocultures. HEK293F cells expressing ADAMTS2 were used as a positive
control. When co-cultured with DS, fully processed 1(I) and
2(I), and a significant amount of pC1(I), were found associated
with the cell layer. In the conditioned medium, only collagen molecules fully
processed at the N terminus [1(I), 2(I), pC1(I) and
pC2(I)] were detected, illustrating a high aminoprocollagen peptidase
activity. In DS/ADAMTS3-HEK293F cell co-culture, analysis of the collagen
pattern demonstrates a high aminoprocollagen peptidase activity, although it
is lower than that observed in presence of ADAMTS2.
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1(III) was observed in the
presence of ADAMTS2. Procollagen III processing was also detected when DS were
co-cultured with cells expressing ADAMTS3.
Adamts3 is expressed at extraskeletal locations
Col2a1 expression has been previously reported at locations that
are not associated with cartilage formation
(Cheah et al., 1991). We found
Adamts3 to be precisely co-expressed with Col2a1 at one such
location in the hindbrain at 12.5 dpc (Fig.
5A). In addition, Adamts3 was highly expressed at other
locations where Col2a1, Col1a1 or Col3a1 gene expression was
absent. At 15.5 dpc, Adamts3 was highly expressed in the developing
cerebral cortex (Fig. 5B), and
at 17.5 dpc it was strongly expressed in the subcommisural organ, a
specialized ependymal zone between the third and fourth ventricles
(Fig. 5C), and in dorsal root
ganglia (Fig. 5D). We have not
investigated Adamts3 expression in the vitreous humor, another
extraskeletal site of Col2a1 expression. Adamts3 expression
was seen in the wall of the urinary bladder
(Fig. 5E).
Adamts2, but not Adamts3 or Adamts14, is co-expressed with Col3a1
Adamts2 was co-expressed with Col3a1 in the wall of large
arteries, such as the aorta, carotid and pulmonary arteries, throughout mouse
embryogenesis (Fig. 6A,B). By
contrast, Col1a1 was expressed only in the adventitial (outermost)
layer of arteries (data not shown). Expression of both Col3a1 and
Adamts2 was highest in the lung mesenchyme
(Fig. 6B,C). Adamts2
was also co-expressed with Col3a1 at other sites, such as the palate
(Fig. 6D), the intestinal wall,
peritoneum and mesentery (Fig.
6E), the wall of the urinary bladder
(Fig. 6F) and the skin (see
Fig. 8A). Adamts14
showed no co-expression with Col3a1, although expression of
Adamts3 in the wall of the urinary bladder partially overlapped with
the expression of both Adamts2 and Col3a1
(Fig. 5E,
Fig. 6F). With this exception,
Adamts3 was not otherwise co-expressed with Col3a1.
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Adamts2-/- mice have distal airspace distension
The lung was the most prominent site of developmental expression of
Adamts2, where its distribution corresponded with that of
Col3a1 (Fig. 6B,C).
Lungs from two-month-old Adamts2-/- mice and wild-type
littermates were evaluated histologically after fixation under inflation to
optimize parenchymal distension. Adamts2-/- lungs had a
consistent decrease in parenchymal density at both 2 months
(Fig. 7A) and 2 weeks (data not
shown). This emphysema-like appearance was unaccompanied by inflammatory cells
or obvious fibrosis. Histochemical detection of elastin and collagen (data not
shown) did not reveal any abnormalities and the ultrastructure showed the
presence of normal basement membranes (Fig.
7B), elastic fibers and surfactant-producing pneumocytes (data not
shown). By contrast, the histology (Fig.
7C) and electron microscopy of the aorta from
Adamts2-/- mice did not show consistent changes from that
of the wild-type littermates. In both the lung and aorta
(Fig. 7D) of
Adamts2-/- mice, the shape and diameter of collagen
fibrils appeared to be unaffected.
We investigated whether the processing of both procollagen I and procollagen III were impaired in the aorta and lungs from Adamts2-/- mice. Although it is not possible to comment from this data on the relative proportion of the two collagens in these tissues, it is clear that both newborn and adult Adamts2-/- mice demonstrated significantly less processing of both collagens than did their wild-type littermates (Fig. 7E,F).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Halila et al.,
1986; Peltonen et al.,
1985; Tuderman and Prockop,
1982). Wang et al. recently demonstrated that ADAMTS2 could
process procollagen III in vitro, but the in vivo relevance was unclear
(Wang et al., 2003). The
overlapping expression of Adamts2 and Col3a1, and the
substantial reduction in procollagen III processing in
Adamts2-/- mice provides solid proof that ADAMTS2 is the
major procollagen III-processing enzyme.
The role of ADAMTS2 in processing procollagen III may further contribute to
the loss of mechanical integrity of dermatosparactic skin, as collagen I and
III are both present in the dermis. In situ hybridization revealed prominent
Col3a1 expression during lung development, in accordance with a
previous study suggesting that, in mice, collagen III is a crucial structural
component of the lung (Shiomi et al.,
2003). The pseudo-emphysematous appearance of the lungs and the
near-total lack of lung procollagen III processing in
Adamts2-/- mice strongly suggests a vital requirement for
collagen III in the lung. Lung abnormalities may result from a fragility of
the pulmonary walls, similar to skin fragility in dermatosparaxis. Neither
Adamts3 nor Adamts14 were expressed in the lung and,
therefore, there was no compensating processing enzyme. However, in the aorta,
which is severely affected by structural collagen III mutations, the
near-total lack of procollagen III processing had no apparent effect on
histology and collagen ultrastructure at 2 months of age. It is possible that
changes may be seen in older mice, or by the induction of stress on the aortic
wall in appropriate in vivo models.
The data suggest that ADAMTS3 probably makes a significant contribution to procollagen I processing during musculoskeletal development in mice. Expression of Adamts3 in many Col1a1-expressing tissues, such as bone and tendon, may explain why the most severe deficiencies in procollagen I processing in mouse dermatosparaxis are in the dermis. Together with our previous analysis of bovine dermatosparactic cartilage that demonstrated complete procollagen II processing, the data suggest that ADAMTS3 is probably the major procollagen II-processing enzyme, and predict that Adamts3 mutations will lead to a chondrodysplasia or osteochondrodysplasia. Although Col2a1 and Col1a1 expression are generally mutually exclusive in tissues, Adamts3 expression overlapped with that of both collagen genes. Although we detected Adamts14 mRNA by RT-PCR of total embryo RNA (data not shown), in situ hybridization with three non-overlapping riboprobes showed no discernible developmental expression.
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). It is possible that ADAMTS3 might also be a
SCO-spondin-processing enzyme.
Can the major fibrillar procollagen amino-propeptides be processed by other
proteases? The highly conserved PNP modular organization (except for the
variable C-terminal module) is different from all other proteases and is
probably highly specialized for binding to triple-helical collagens.
Heat-denatured collagen is not efficiently processed by ADAMTS2
(Tuderman et al., 1978),
suggesting a need for spatially stringent interactions between the ancillary
domain and the triple helix. Because residual procollagen processing in
dermatosparactic skin occurs at the same cleavage site as in normal skin
(Colige et al., 2002), it
supports processing by an enzyme(s) closely related to ADAMTS2. It is clear
from the Adamts2-/- mice that absence of ADAMTS2 leads to
a significant loss of processing of procollagen III in many tissues. At these
locations, neither Adamts3 nor Adamts14 are expressed, and,
thus, do not compensate for its absence. Thus other enzymes do not appear to
compensate significantly. A splice variant of collagen II, termed IIA is
expressed in pre-cartilaginous mesenchyme and in perichondrium
(Ryan and Sandell, 1990;
Ryan et al., 1990).
Ultrastructural and biochemical studies suggest its N-propeptide is not
excised during development, suggesting perhaps that it is resistant to the
action of these proteases (Zhu et al.,
1999). By contrast, matrix metalloproteinases (MMPs) have been
shown to cleave the propeptide from collagen IIA at numerous sites other than
the PNP bond (Fukui et al.,
2002).
ADAMTS2 mutations in humans lead to characteristic craniofacial
changes and decreased growth; features that are absent in
Adamts2-/- mice. As individuals with EDS-VIIC age, the
skin fragility is somewhat ameliorated and there is increasing joint laxity,
which is not a prominent feature in early childhood.
Adamts2-/- mice also have fragile skin, but they are not
significantly growth retarded and have no craniofacial dysmorphism
(Li et al., 2001). These
differences between EDS-VIIC and mouse dermatosparaxis are compatible with the
different deployment of functionally equivalent enzymes in humans and mice
through the transcriptional regulation of their genes. The expression of
ADAMTS3 and ADAMTS14 may change with age in humans, perhaps
explaining how the properties of dermatosparactic skin change as affected
individuals get older.
In addition to skin fragility, humans with EDS-VIIC manifest with a variety
of other anomalies. Easy bruising, a universal sign in patients with EDS-VIIC,
can be partly attributed to the ability of ADAMTS2 to process procollagen III,
and to our observation, albeit in mice, that it is the only PNP expressed at
significantly high levels in the walls of blood vessels. The procollagen
III-processing defect in the absence of ADAMTS2 appears to be dramatic in most
mouse tissues. We were particularly concerned that procollagen III processing
in the mouse aorta was defective. Although histology and electron microscopy
did not reveal specific anomalies in the aortic wall, and no aortic or other
vascular aneurysms have been hitherto noted in EDS-VIIC patients, our findings
suggest a need for the monitoring of EDS-VIIC patients as they age. Bladder
rupture and intestinal perforations have been reported in individuals with
EDS-VIIC. Our studies demonstrate co-expression of procollagen III and ADAMTS2
in the developing mouse at these locations. A recent clinical study emphasized
the abnormal permanent dentition of individuals with EDS-VIIC, including
hypodontia (De Coster et al.,
2003). Adamts2-/- mice have mild dental
changes and compensation by Adamts3 may account for this.
Most of the 19 ADAMTS proteases can be assigned to phylogenetically
distinct clades (Apte, 2004;
Huxley-Jones et al., 2005;
Nicholson et al., 2005).
Recent analysis of ADAMTS gene evolution suggests that genes within mammalian
ADAMTS clades arose by duplication of their primitive ancestors
(Huxley-Jones et al., 2005;
Nicholson et al., 2005). We
speculated that, following duplication, closely related genes, such as
ADAMTS2, ADAMTS3 and ADAMTS14, were highly conserved to provide
subfunctionalization (retention of molecular function but with diversification
of expression patterns) rather than neofunctionalization (acquisition of new
functions) (Huxley-Jones et al.,
2005). The ability of all three PNPs to process procollagen I, but
specialization in this regard as a result of differential gene regulation,
supports this hypothesis. Other ADAMTS clades may exhibit the same
phenomenon.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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