Mutations in the region encoding the von Willebrand factor A domain of matrilin-3 are associated with multiple epiphyseal dysplasia.

Multiple epiphyseal dysplasia (MED) is a relatively mild and clinically variable osteochondrodysplasia, primarily characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. Mutations in the genes encoding cartilage oligomeric matrix protein (COMP) and type IX collagen (COL9A2 and COL9A3) have previously been shown to cause different forms of MED (refs. 4-13). These dominant forms of MED (EDM1-3) are caused by mutations in the genes encoding structural proteins of the cartilage extracellular matrix (ECM); these proteins interact with high affinity in vitro. A recessive form of MED (EDM4) has also been reported; it is caused by a mutation in the diastrophic dysplasia sulfate transporter gene (SLC26A). A genomewide screen of family with autosomal-dominant MED not linked to the EDM1-3 genes provides significant genetic evidence for a MED locus on the short arm of chromosome 2 (2p24-p23), and a search for candidate genes identified MATN3 (ref. 18), encoding matrilin-3, within the critical region. Matrilin-3 is an oligomeric protein that is present in the cartilage ECM. We have identified two different missense mutations in the exon encoding the von Willebrand factor A (vWFA) domain of matrilin-3 in two unrelated families with MED (EDM5). These are the first mutations to be identified in any of the genes encoding the matrilin family of proteins and confirm a role for matrilin-3 in the development and homeostasis of cartilage and bone.

Effects of matrix macromolecules on chondrocyte gene expression: synthesis of a low molecular weight collagen species by cells cultured within collagen gels.

Chick-embryo sternal chondrocytes have been cultured within three-dimensional collagen gels as part of a study concerned with the effects of extracellular matrix macromolecules on chondrocyte gene expression. Data are presented indicating that chondrocytes cultured within such a collagenous environment synthesize significantly more of an hitherto unidentified, low molecular weight collagen species than do cells grown on plastic tissue-culture dishes in the conventional manner. This low molecular weight collagen species contains noncollagenous domains (as indicated by its decreased molecular size after mild pepsin digestion), is distinct from the known collagen types (as judged by CNBr peptide analysis), and forms part of the insoluble collagenous matrix produced by the chondrocytes. Cells growing within the gel tend to form colonies consisting of a linear array of cells reminiscent of the cellular organization in growth cartilage.

Type VI collagen microfibrils: evidence for a structural association with hyaluronan.

Type VI collagen, a widespread structural component of connective tissues, has been isolated in abundance from fetal bovine skin by a procedure involving bacterial collagenase digestion under nonreducing, nondenaturing conditions and gel filtration chromatography. Rotary shadowing electron microscopic analysis revealed that the collagen VI was predominantly in the form of extensive intact microfibrillar arrays. These microfibrils were seen in association with hyaluronan, which was identified by its ability to bind the G1 fragment of cartilage proteoglycan. Treatment with highly purified hyaluronidase largely disrupted the collagen VI microfibrils into component tetramers, double tetramers, and short microfibrillar sections. Subsequent incubation of disrupted collagen VI in the presence of hyaluronan facilitated a partial repolymerization of the microfibrils. In vitro binding studies have also demonstrated that type VI collagen binds hyaluronan with a relatively high affinity. These studies demonstrate that a specific structural relationship exists between type VI collagen and hyaluronan. This association is likely to be of primary importance in the growth and remodeling processes of connective tissues.

Macromolecular organization of chicken type X collagen in vitro.

The macromolecular structure of type X collagen in the matrices of primary cultures of chick hypertrophic chondrocytes was initially investigated using immunoelectron microscopy. Type X collagen was observed to assemble into a matlike structure with-in the matrix elaborated by hypertrophic chondrocytes. The process of self assembly was investigated at the molecular level using purified chick type X collagen and rotary-shadowing EM. It was shown that under neutral conditions at 34 degrees C, individual type X collagen molecules associate rapidly into multimeric clusters via their carboxy-terminal globular domains forming structures with a central nodule of carboxy-terminal domains and the triple helices radiating outwards. Prolonged incubation resulted in the formation of a regular hexagonal lattice by lateral association of the juxtaposed triple-helical domains from adjacent multimeric clusters. This extended lattice may play an important role in modifying the cartilage matrix for subsequent events occurring in endochondral bone formation.

Comparative studies of type X collagen expression in normal and rachitic chicken epiphyseal cartilage.

The levels of type X collagen in mineralizing normal chicken epiphyses and nonmineralizing rachitic chicken tibial epiphyses were measured and compared. Qualitative immunoperoxidase studies with anti-chick type X collagen monoclonal antibodies on sections from normal and rachitic cartilage demonstrated that the type X collagen levels in rachitic growth plates are reduced. Northern hybridization of mRNA and biosynthetic studies have confirmed that type X collagen synthesis in rickets is also decreased. In hypocalcemic rickets, the level of type X collagen mRNA is reduced by 80% whereas the level of type X collagen mRNA is only reduced by 50% in normocalcemic rickets. These observations provide additional evidence that type X collagen is involved in the process of cartilage mineralization and also suggest that the partial recovery of type X collagen synthesis in normocalcemic rickets may be related to the elevated plasma concentration of calcium. Calcium concentration may therefore play an important role in the control of type X collagen synthesis.

Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival.

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.

The interaction of adeninylalkylcobalamins with ribonucleotide reductase.

Several structural analogs of adenosylcobalamin, containing 2, 3, 4, 5 and 6 methylene carbons instead of the ribofuranose moiety, have been synthesized and their interaction with ribonucleotide reductase from Lactobacillus leichmannii has been investigated. Kinetic studies of the inhibition of the reductase by these analogs showed that the adeninylalkylcobalamins with 4, 5 and 6 carbons interposed between the adenine moiety and the cobalt atom are potent inhibitors of ribonucleotide reduction. The stronger interaction between adeninylpentylcobalamin and the enzyme than that between adenosylcobalamin and the enzyme suggests that the more flexible acyclic analog of adenosine requires fewer adjustments of the protein upon binding.

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.

Vascular pericytes express osteogenic potential in vitro and in vivo.

At postconfluence, cultured bovine pericytes isolated from retinal capillaries form three-dimensional nodule-like structures that mineralize. Using a combination of Northern and Southern blotting, in situ hybridization, and immunofluorescence we have demonstrated that this process is associated with the stage-specific expression of markers of primitive clonogenic marrow stromal cells (STRO-1) and markers of cells of the osteoblast lineage (bone sialoprotein, osteocalcin, osteonectin, and osteopontin). To demonstrate that the formation of nodules and the expression of these proteins were indicative of true osteogenic potential, vascular pericytes were also inoculated into diffusion chambers and implanted into athymic mice. When recovered from the host, chambers containing pericytes were found reproducibly to contain a tissue comprised of cartilage and bone, as well as soft fibrous connective tissue and cells resembling adipocytes. This is the first study to provide direct evidence of the osteogenic potential of microvascular pericytes in vivo. Our results are also consistent with the possibility that the pericyte population in situ serves as a reservoir of primitive precursor cells capable of giving rise to cells of multiple lineages including osteoblasts, chondrocytes, adipocytes, and fibroblasts.

A novel cell culture model of chondrocyte differentiation during mammalian endochondral ossification.

Endochondral ossification (EO) occurs in the growth plate where chondrocytes pass through discrete stages of proliferation, maturation, hypertrophy, and calcification. We have developed and characterized a novel bovine cell culture model of EO that mirrors these events and will facilitate in vitro studies on factors controlling chondrocyte differentiation. Chondrocytes derived from the epiphyses of long bones of fetal calves were treated with 5-azacytidine (aza-C) for 48 h. Cultures were maintained subsequently without aza-C and harvested at selected time points for analyses of growth and differentiation status. A chondrocytic phenotype associated with an extensive extracellular matrix rich in proteoglycans and collagen types II and VI was observed in aza-C-treated and -untreated cultures. aza-C-treated cultures were characterized by studying the expression of several markers of chondrocyte differentiation. Parathyroid hormone-related protein (PTHrP) and its receptor, both markers of maturation, were expressed at days 5-9. Type X collagen, which is restricted to the stage of hypertrophy, was expressed from day 11 onward. Hypertrophy was confirmed by a 14-fold increase in cell size by day 15 and an increased synthesis of alkaline phosphatase during the hypertrophic period (days 14-28). The addition of PTHrP to aza-C-treated cultures at day 14 led to the down-regulation of type X collagen by 6-fold, showing type X collagen expression is under the control of PTHrP as in vivo. These findings show that aza-C can induce fetal bovine epiphyseal chondrocytes to differentiate in culture in a manner consistent with that which occurs during the EO process in vivo.

The complete cDNA derived sequence of the rat prolyl 4-hydroxylase alpha subunit.

A rat cDNA encoding the prolyl 4-hydroxylase alpha subunit (P4H alpha) was isolated and sequenced. The primary aa sequence deduced from the nucleotide sequence reveals a 534-aa protein that shows extensive aa identity with the human (88%) and chick (77%) P4H alpha.

Sequence comparison of three mammalian type-X collagen promoters and preliminary functional analysis of the human promoter.

The mechanism(s) controlling the specific expression of the type-X collagen (COL10A1)-encoding gene in the growth plate of developing long bones is not known. In preparation for identifying and characterizing the 5'-regulatory sequences and transcription factors which control mammalian Col10a1 gene expression, we have isolated and sequenced the first exon and 5' flanking promoter regions of bovine Col10a1. Sequence comparisons, including those previously published for mouse Col10a1, highlighted a number of conserved domains within the promoter and upstream elements. Reporter cat gene (encoding chloramphenicol acetyltransferase, CAT) constructs containing 5'-regulatory sequences of human COL10a1 (hCOL10a1) were transfected into primary cultures of foetal bovine growth plate chondrocytes producing COL10A1 and non-producing epiphyseal cartilage chondrocytes. Constructs containing up to 900 bp of promoter sequence exhibited low levels of CAT production in expressing cells and non-expressing cells. Addition of a further 1.5 kb of upstream sequence resulted in a dramatic increase in CAT production in expressing cells only. The results demonstrate the presence of enhancer-like elements between 900 bp and 2.4 kb upstream of the transcription start point(s) of hCOL10a1, which is distinctly different from that reported for the chick.

Partial characterization of type X collagen from bovine growth-plate cartilage. Evidence that type X collagen is processed in vivo.

Sequential extraction of bovine growth-plate cartilage with 4 M guanidinium chloride and pepsin was used to identify the intact and pepsinized forms respectively of type X collagen. This collagen occurs predominantly as the processed [alpha 1(X)]3 form in vivo, although the procollagen [pro alpha 1(X)]3 form can also be detected. The bovine pro alpha 1(X) and alpha 1(X) chains have Mr values identical to the corresponding chick species (Mr 59,000 and 49,000). However, the pepsinized alpha 1(X)p chains (Mr 47,000) are larger than those of the chick (Mr 45,000), and the bovine collagen type X is further distinguished by being disulphide-bonded within the triple-helical domain.

Identification of disulphide-bonded type X procollagen polypeptides in embryonic chick chondrocyte cultures.

A high-Mr (Mr 120,000), disulphide-bonded collagenous polypeptide was observed to co-purify with the prox1(X) chain during isolation of cartilage collagens from culture medium of embryonic chick tibiotarsal chondrocytes. This high Mr polypeptide was subsequently shown by two-dimensions l SDS-PAGE and peptide mapping to represent a dimer of the prox1(X) chain of type X collagen linked by disulphide bonding in the non-collagenous domains.

The fibrillar collagens, collagen VIII, collagen X and the C1q complement proteins share a similar domain in their C-terminal non-collagenous regions.

A sequence comparison of the C-termini of collagens X, VIII, the collagen-like complement factor C1q, and the fibrillar collagens showed a conserved cluster of aromatic residues. This conserved cluster was in a domain of approximately 130 amino acids that exhibited marked similarities in hydrophilicity profiles between the different collagens, despite a low level of sequence similarity. These data suggest that the 'collagen X-like family' and the fibrillar collagens contain a domain within their C-termini that adopts a common tertiary structure, and that a conserved cluster of aromatic residues in this domain may be involved in C-terminal trimerization.

Embryonic chick cartilage collagens. Differences in the low-Mr species present in sternal cartilage and tibiotarsal articular cartilage.

The collagenous polypeptides present in embryonic chick sternal and tibiotarsal cartilages have been solubilised by digestion with pepsin and separated by salt fractionation. Type II collagen, 1 alpha 2 alpha 3 alpha collagen, and two polypeptides (apparent molecular mass 150 and 42 kDa), which were reducible to a number of smaller peptides, were extracted from both tissues. However, also present in the peptic digests of tibiotarsal cartilages was a major non-reducible highly-soluble polypeptide of 45 kDa. This short-chain collagen is apparently identical to the pepsinized product of G collagen (Mr 59 000), a major low-Mr procollagen-like species previously detected in chick chondrocyte cultures.

Matrix Gla protein is differentially expressed during the deposition of a calcified matrix by vascular pericytes.

PCR-based subtractive hybridisation was used to identify genes up-regulated when pericytes undergo osteogenic differentiation and deposit a calcified matrix. cDNA pools were generated from confluent pericytes and from pericyte cultures containing calcified nodules. A pericyte cDNA library was screened with the product of the subtraction procedure (calcified minus confluent cDNA) and the majority of the positive clones were identified as matrix Gla protein (MGP). Northern analysis and immunohistochemistry demonstrated that MGP was only expressed by pericytes in calcified nodules. Antibodies to MGP inhibited the deposition of a calcified matrix by pericytes, suggesting that MGP regulates both cell differentiation and calcification.

Single-dose subcutaneous iodine-131-iodohippurate for determination of renal plasma flow.

Subcutaneous administration of a single dose of 131I-iodohippurate was used for determination of renal plasma flow (RPF) in 20 subjects during water diuresis. Slow release of tracer (200 microCi) permitted serial clearance measurements over 5 hr that were compared to standard, constant infusion, PAH clearance (mean 379.5 +/- 34.9 ml/min/1.73 m2, range 50.9 to 696.3 ml/min/1.73 m2). RPF(Isotope) was 424.9 +/- 30.3 ml/min/1.73 m2 (range 144.4 to 746.5 ml/min/1.73 m2) and highly correlated with RPFPAH (r = 0.883, p less than 0.0001). This technique permits prolonged studies of renal plasma flow under steady-state conditions without constant infusion.

Distribution of the matrix metalloproteinases stromelysin, gelatinases A and B, and collagenase in Crohn's disease and normal intestine.

AIMS: To investigate the role of the matrix metalloproteinases (MMPs) in the connective tissue changes seen in the intestine in Crohn's disease. METHODS: Indirect immunofluorescence microscopy using specific antibodies to the MMPs (collagenase, gelatinase A and B, and stromelysin) were used to assess the distribution of these enzymes in normal and diseased intestine. RESULTS: In normal intestine the matrix metalloproteinases were confined to a few isolated inflammatory cells, but in Crohn's disease, the inflammatory infiltrate was associated with increased numbers of polymorphonuclear leucocytes which stained positive for gelatinase B. Stromelysin was also detected extracellularly on the connective tissue matrix in regions of smooth muscle cell proliferation and mucosal degradation. Interestingly, in ulcerative colitis, another inflammatory bowel disease, stromelysin was localised in the lamina propria in regions of mucosal loss. CONCLUSIONS: The increased numbers of inflammatory cells containing gelatinase B, and the localisation of extracellular stromelysin in regions of fibrosis and mucosal degradation, suggest that these enzymes have a role in the pathological changes seen in Crohn's disease. In cases of ulcerative colitis stromelysin was also detected on the lamina propria in regions of mucosal loss, and seems to be associated with the connective tissue changes that precede mucosal loss.

Cartilage proteoglycan aggregate and fibronectin can modulate the expression of type X collagen by embryonic chick chondrocytes cultured in collagen gels.

Chick embryo sternal chondrocytes from the caudal and cephalic regions were cultured within type I collagen gels and type I collagen/proteoglycan aggregate composite gels in normal serum. Caudal region chondrocytes were also cultured within type I collagen gels in the presence of fibronectin-depleted serum. There was a marked stimulation of type X collagen synthesis by the caudal region chondrocytes after 9 days in the presence of fibronectin-depleted serum and after 14 days in the presence of proteoglycan aggregate. These results provide evidence for the ability of chondrocytes from a zone of permanent cartilage to synthesise type X collagen and for the involvement of extracellular matrix components in the control of type X collagen gene expression.