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PubMed2002

Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells.

Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast-related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase-pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow-derived mesenchymal progenitor cells. High-density pellet cultures of hOB maintained in chemically defined serum-free medium, supplemented with transforming growth factor-beta1, were composed of morphologically distinct, chondrocyte-like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high-density pellet cultures were surrounded by a sulfated proteoglycan-rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase-positive cells, that expressed osteoblast-related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, beta-glycerophosphate, and bone morphogenetic protein-2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte-specific genes, such as lipoprotein lipase and peroxisome proliferator-activated receptor gamma2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3-isobutyl-1-methylxanthine. Taken together, these results show that cells derived from collagenase-treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature.

Adipose Tissue↗
PubMed1988

Type X collagen alterations in rachitic chick epiphyseal growth cartilage.

We examined collagens of both normal and vitamin D-deficient chick epiphyseal growth cartilage. Special emphasis was placed on the study of Type X collagen, a recently described product of hypertrophic chondrocytes. Scanning electron microscopy of the epiphyseal growth cartilage of vitamin D-deficient chickens showed an enlarged growth cartilage with a disorganized extracellular matrix. The cartilage collagens were solubilized by proteolytic digestion and disulfide bond reduction of both normal and rachitic growth tissues. Sequential extraction with neutral salt and acetic acid buffers followed by pepsin digestion at 4 degrees C solubilized about 12% of normal tissues and about 7% of collagen from rachitic growth cartilage. Treatment of the pepsin-resistant collagens with neutral salt-dithiothreitol buffer under nondenaturing conditions and a subsequent pepsin digestion increased the yield of solubilized collagen to greater than 95% of the total tissue collagen. Results of the biochemical studies showed a marked increase in the relative proportion of Type X collagen (from 5.6 to 27.9%), a corresponding decrease in the proportions of Types II and IX collagens, and a moderate increase in Type XI collagen in rachitic cartilage. Amino acid analysis indicated that there were no differences in the Types II and X collagens of normal and rachitic cartilage. However, an abnormality in the relative proportions of the CNBr peptides of Type X collagen was detected in the rachitic cartilage. We suggest that the increase in collagen in the rachitic state may reflect increased levels of Type X collagen synthesis by cells in the hypertrophic region. It is likely that in rickets the overproduction of Type X collagen may be a compensatory mechanism by which the hypertrophic chondrocyte attempts to provide a maximum area of calcifiable matrix for the calcium-depleted serum.

Amino Acids↗
PubMed1995

Structural analysis of cross-linking domains in cartilage type XI collagen. Insights on polymeric assembly.

The collagen framework of hyaline cartilage is based on copolymers of types II, IX, and XI collagens. Previous studies have established specific covalent interactions between types II and IX collagens. The present study examined cross-linking sites in type XI collagen to define better the full heteropolymeric assembly. Pepsinsolubilized type XI collagen was purified from fetal bovine cartilage. The cross-linking amino acids in the preparation were primarily divalent, borohydride-reducible structures; pyridinoline residues were essentially absent. Individual alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains were resolved by high performance liquid chromatography. Telopeptides still attached by cross-links to helical sites were released by periodate oxidation and identified by microsequencing. Analysis of cross-linked peptides isolated from trypsin digest of each alpha-chain identified the attachment helical sites for the telopeptides. A high degree of interchain specificity was evident in the cross-linking between N-telopeptides and the COOH terminus of the triple-helix, consistent with a head-to-tail interaction of molecules staggered by 4D (D = 67 nm) periods. In addition, alpha 1(II) C-telopeptide was linked to the amino-terminal site of the alpha 1(XI) triple helix. In summary, the results show that type XI collagen molecules are primarily cross-linked to each other in cartilage, implying that a homopolymer is initially formed. Links to type II collagen are also indicated, consistent with an eventual cofibrillar assembly. Analysis of cartilage extracts showed that all three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), had at least in part retained their N-propeptides in cartilage matrix and that the alpha 3 (XI) chain was the IIB splicing variant product of the COL2A1 gene. Of particular note was the finding that the N-telopeptide cross-linking site in both alpha 1(XI) and alpha 2(XI) is located amino-terminal to the putative N-propeptidase cleavage site. This structural feature provides a potential mechanism for the proteolytic depolymerization of type XI collagen by proteases that can cleave between the cross-link and the triple helix (e.g. stromelysin).

Amino Acid Sequence↗
PubMed1997

Skeletal development in transgenic mice expressing a mutation at Gly574Ser of type II collagen.

Skeletal development of transgenic mice with a type II collagen mutation was analyzed and compared with wild-type littermates. The single base substitution in Col2a1 resulted in a glycine to serine mutation within the helical domain and corresponded to one previously identified in a patient with the lethal human chondrodysplasia, hypochondrogenesis (Horton et al. [1992] Proc. Natl. Acad. Sci. U.S.A. 89:4583-4587). Skeletal staining of embryos from 14.5 through 18.5 days of gestation demonstrated a dwarf phenotype in the transgenic embryos, most notably short limb bones and vertebral column that was first detected at 15.5 days post-coitus. In addition to the reduced length, the extent of ossification was less in the transgenic mice. The architecture of the long bone growth plate was abnormal in the transgenic tissue, in particular there was no discernible proliferative zone. There were few stacks of characteristically flattened cells and the overall length of the growth plate in the mutant embryos was reduced. At the ultrastructural level, there were fewer collagen fibrils present in the transgenic mouse cartilage compared to that of wild-type littermates. Ultrastructural localization of collagen types II, IX and XI revealed a similar pattern between the transgenic and wild-type pups, suggesting that the collagen fibrils present in the matrix of littermates with both phenotypes had a similar composition. Skeletal analysis and cartilage histochemistry indicated that effect of the type II collagen mutation was to reduce the density of the collagen fibrils within the cartilage matrix which was associated with delayed bone formation and resulted in a short-limbed phenotype.

Animals↗
PubMed1995

Use of a new rat chondrosarcoma cell line to delineate a 119-base pair chondrocyte-specific enhancer element and to define active promoter segments in the mouse pro-alpha 1(II) collagen gene.

We show that a new rat chondrosarcoma (RCS) cell line established in long-term culture from the Swarm tumor displayed a stable differentiated chondrocyte-like phenotype. Indeed, these cells produced the collagen types II, IX, and XI and alcian blue-stainable cartilage-specific proteoglycans, but no type I or type III collagen. To functionally characterize their chondrocytic nature, the cells were stably transfected with a type II collagen/beta geo chimeric gene which confers essentially perfect chondrocyte-specific expression in transgenic mice. RCS cells expressed both beta-galactosidase and G418 resistance, in comparison with similarly transfected 10T1/2 and NIH/3T3 fibroblasts which did not. These cells were then used to perform a systematic deletion analysis of the first intron of the mouse type II collagen gene (Col2a1) using transient expression experiments to determine which segments stimulated expression of a luciferase reporter gene in RCS cells but not in 10T1/2 fibroblasts. Cloning of two tandem copies of a 156-base pair (bp) intron 1 fragment (+2188 to +2343) in a construction containing a 314-bp Col2a1 promoter caused an almost 200-fold increase in promoter activity in RCS cells but no increase in 10T1/2 cells. DNase I footprint analysis over this 156-bp fragment revealed two adjacent protected regions, FP1 and FP2, located in the 3'-half of this segment, but no differences were seen with nuclear extracts of RCS cells and 10T1/2 fibroblasts. Deletion of FP2 to leave a 119-bp segment decreased enhancer activity by severalfold, but RCS cell specificity was maintained. Further deletions indicated that sequences both in the 5' part of the 119-bp fragment and in FP1 were needed simultaneously for RCS cell-specific enhancer activity. A series of deletions in the promoter region of the mouse Col2a1 gene progressively reduced activity when these promoters were tested by themselves in transient expression experiments. However, these promoter deletions were all activated to a similar level in RCS cells by a 231-bp intron 1 fragment that included the 156-bp enhancer. The RCS cell-specific activity persisted even if the Col2a1 promoter was replaced by a minimal adenovirus major late promoter. This 231-bp intron 1 fragment also had strong enhancing activity in transiently transfected mouse primary chondrocytes. Our experiments establish the usefulness of RCS cells as an experimental system for studies of the control of chondrocyte-specific genes, provide an extensive delineation of segments in the Col2a1 first intron involved in chondrocyte-specific activity, and show that promoter sequences are dispensable for chondrocyte specificity.

3T3 Cells↗
PubMed2001

Chondrocyte phenotype and cell survival are regulated by culture conditions and by specific cytokines through the expression of Sox-9 transcription factor.

OBJECTIVE: To investigate the effects of culture conditions, serum and specific cytokines such as insulin-like growth factor (IGF) 1 and interleukin (IL) 1alpha on phenotype and cell survival in cultures of Syrian hamster embryonic chondrocyte-like cells (DES4(+).2). METHODS: Proteins and RNA extracted from subconfluent and confluent early- and late-passage DES4(+).2 cells cultured in the presence or absence of serum and IL-1alpha or IGF-1 or both cytokines together were analysed for the expression of chondrocyte-specific genes and for the chondrogenic transcription factor Sox-9 by Western and Northern blotting. Apoptosis was assessed by agarose gel electrophoresis of labelled low-molecular weight DNA extracted from DES4(+).2 cells and another Syrian hamster embryonic chondrocyte-like cell line, 10W(+).1, cultured under the different conditions and treatments. RESULTS: Early passage DES4(+).2 cells expressed chondrocyte-specific molecules such as collagen types alpha1(II) and alpha1(IX), aggrecan, biglycan and link protein and collagen types alpha1(I) and alpha1(X) mRNAs, suggesting a prehypertrophic chondrocyte-like phenotype. The expression of all genes investigated was cell density- and serum-dependent and was low to undetectable in cell populations from later passages. Early-passage DES4(+).2 and 10W(+).1 cells survived when cultured at low cell density, but died by apoptosis when cultured at high cell density in the absence of serum or IGF-1. IGF-1 and IL-1alpha had opposite and antagonistic effects on the chondrocyte phenotype and survival. Whereas IL-1alpha acting alone suppressed cartilage-specific gene expression without significantly affecting cell survival, IGF-1 increased the steady-state mRNA levels and relieved the IL-1alpha-induced suppression of all the chondrocyte-specific genes investigated; it also enhanced chondrocyte survival. Suppression of the chondrocyte phenotype by the inflammatory cytokine IL-1alpha correlated with marked down-regulation of the transcription factor Sox-9, which was relieved by IGF-1. The expression of the Sox9 gene was closely correlated with the expression of the chondrocyte-specific genes under all conditions and treatments. CONCLUSIONS: The results suggest that the effects of cartilage anabolic and catabolic cytokines IGF-1 and IL-1alpha on the expression of the chondrocyte phenotype are mediated by Sox-9. As Sox-9 appears to be essential for matrix production, the potent effect of IL-1alpha in suppressing Sox-9 expression may limit the ability of cartilage to repair during inflammatory joint diseases.

Aggrecans↗
PubMed1991

Loss of a tumor suppressor gene function is correlated with downregulation of chondrocyte-specific collagen expression in Syrian hamster embryo cells.

We previously described the isolation of closely related, preneoplastic Syrian hamster cell lines that have retained (supB+) or lost (supB-) the ability to suppress the anchorage-independent growth and tumorigenicity of a sarcoma cell line (BP6T) in cell hybrids. In this report, we have used differential cDNA screening to clone several genes that are expressed in supB+ cells and downregulated in supB- cells. The nontumorigenic supB+ and supB- variants are advantageous for differential cDNA cloning because multiple independent cell lines differing in their tumor suppressor activity have been isolated. Differentially expressed cDNAs were isolated and placed into one of four groups based on DNA cross-hybridization. Representative cDNAs from Groups I and II, which were expressed at relatively high levels in two independently derived supB+ cell lines (DES4 and 10W) and downregulated in the supB- and tumor cell lines, were sequenced. The DNA and predicted amino acid sequences of these genes were found to be highly homologous to the chondrocyte-specific collagens type II and type IX. In contrast to the chondrocyte-specific collagens, another collagen isoform, collagen type I, was expressed at similar levels in both supB+ and supB- cells. These results suggest that carcinogen-induced immortalization selected for chondrocyte-like cell lines from the mixed embryo cell population. As these cells progressed toward tumorigenicity, the ability to express the chondrocyte differentiation markers was lost concomitantly with the ability to suppress the tumorigenicity of the BP6T sarcoma cell line. These results are consistent with the hypothesis that the supB+ tumor suppressor gene is involved in the regulation of differentiation. The identification of genes regulated by this suppressor gene may aid in its isolation.

Animals↗
PubMed2005

Establishment and characterization of the permanent human cell line C3842 derived from a secondary chondrosarcoma in Ollier's disease.

The permanent human cell line C3842 was established from a secondary chondrosarcoma in a typical case of Ollier's disease. In the present study, we analyzed the morphological, cytogenetic and molecular biological characteristics of the cultured cells in comparison with the original tumor and investigated the invasion properties of the tumor model using functional imaging of proteolysis, matrigel assay and chick chorioallantoic membrane assay. C3842 cells exhibit the typical features of malignant cartilage tumor cells in vitro, including the expression of collagen types II, IX, XI and aggrecan. The proteolytic ability of C3842 cells is attributed to the expression of several proteases, such as cathepsin B, urokinase plasminogen activator and matrix-metalloproteinase-2, which enable the cells to degrade collagen type I and to permeate matrigel matrix. In accordance with the biological features in vivo, C3842 cells are not able to invade through the epithelium of the chick chorioallantoic membrane. In conclusion, the cell line C3842 provides the first model of a secondary chondrosarcoma in Ollier's disease in vitro, which is characterized by distinct features of such malignant cartilage tumors.

Adult↗
PubMed1991

Serum IgE anti-cartilage collagen antibodies in rheumatoid patients.

The presence of circulating IgG, IgA and IgM antibodies to native cartilage collagens in some patients with rheumatoid arthritis (RA) suggests that an autoimmune response to cartilage collagens may be involved in the pathogenesis of RA. However, the relevance of such antibodies to the pathological process remains unclear, and it is likely that many humoral and cellular derived factors combined to trigger events leading to the chronicity of the rheumatoid lesion. Since histological and biochemical studies have suggested the involvement of mast cells in the rheumatoid joint, we have studied the frequency of IgE antibodies directed against the cartilage collagens type II, IX and XI in patients with active rheumatoid disease. Of the 91 patients' sera tested, 32 had significant levels of IgE anti-cartilage collagen antibodies when compared with non-arthritic controls. Total serum IgE levels did not correlate with the presence of IgE anti-collagen antibodies, nor were any patients positive for IgE antibodies to fibronectin, a widely distributed extracellular matrix component. These results are consistent with an allergic type I hypersensitivity reaction to cartilage antigens in RA involving mast cell and basophil degranulation.

Arthritis, Rheumatoid↗
PubMed1999

Mice lacking matrilin-1 (cartilage matrix protein) have alterations in type II collagen fibrillogenesis and fibril organization.

Matrilin-1 (cartilage matrix protein) is a homotrimeric protein that forms collagen-dependent and collagen-independent fibrils in the extracellular matrix of cartilage. In the growth plate of developing long bones, the gene for matrilin-1 is transcribed exclusively by the chondrocytes of the zone of maturation which is situated between the zones of proliferation and hypertrophy. When associated with the cartilage collagen fibril, which consists of collagens type II, IX, and XI, matrilin-1 displays a periodicity of 59.3 nm. Matrilin-1 also interacts with the proteoglycan, aggrecan. Because of its association with the collagen fibril, we tested the hypothesis that matrilin-1 may play a role in collagen fibril formation and cartilage matrix assembly by generating mice with targeted mutations in the matrilin-1 gene. Ultrastructural studies of the cartilage of growth plates of matrilin-1 null mice reveal an abnormal type II collagen fibrillogenesis and fibril organization in the matrix of the zone of maturation. These results represent the first report on the regulation of the heterotypic type II collagen fibril by a non-collagenous protein. The abnormal fibrillogenesis had no obvious effects on skeletal development, on the organization of chondrocytes in the growth plate and on the deposition of aggrecan and the hypertrophic-specific type X collagen in the cartilaginous matrix.

Animals↗
PubMed1992

Purification and characterization of native type XIV collagen.

A new molecule, type XIV collagen, with domains homologous to type IX and XII collagens has been recently discovered in pepsin extracts of fetal bovine tissues (Dublet, B., and van der Rest, M. (1991) J. Biol. Chem. 266, 6853-6858). In the present study, we describe the purification and the characterization of the intact native form of this newly discovered collagen. By using only two chromatographic steps we were able to obtain pure type XIV collagen. Furthermore, minor modifications of the protocol allowed us to perform the simultaneous large scale purification of type XII and type XIV collagens from the same tissue. Intact type XIV collagen migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as two bands of 220 and 290 kDa (reducing conditions). After collagenase treatment, a single band of 190 kDa is observed, which represents the large non-collagenous domain of the molecule (NC3). Rotary shadowing electron micrographs of intact type XIV collagen show a cross-shaped structure formed by a thin tail attached through a central globule to three identical "fingers." These properties are similar to those previously described for intact chicken type XII collagen (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156), but the two molecules are different gene products and have charge and glycosylation differences. Finally, we show that the three chains of purified type XIV collagen have an apparent molecular mass of approximately 220 kDa and are not cross-linked to each other by bonds other than disulfide bridges. The same observation was made for type XII collagen. In both cases, the 290-kDa migrating band in SDS-PAGE is due to incomplete denaturation in electrophoresis sample buffer in the absence of urea.

Animals↗
PubMed2003

Expression patterns of cartilage collagens and Sox9 during mouse heart development.

A majority of congenital heart defects are due to abnormal development of the valves and membranous septa, i.e., connective tissue components of the heart. During development, an interesting feature of cardiac connective tissue is transient expression of collagens typical for cartilage. To better understand the role of these collagens in the heart, we have performed a systematic study on the temporospatial expression of type II and IX collagen isoforms during mouse heart development employing northern hybridization and RNase protection assay. The mRNAs for alpha1(II) and alpha1(IX) collagens were expressed transiently between embryonic days 10.5 and 14.5 in embryonic mouse heart. RNase protection assays revealed that for both transcripts the embryonic ("prechondrogenic") variants of the alternatively spliced mRNA isoforms dominated. Immunohistochemistry demonstrated that type IIA collagen and Sox9, its key transcriptional regulator, were expressed in the epithelial-mesenchymal areas of the developing heart, with partially overlapping patterns particularly in valvular and septal regions. In addition, Sox9 expression was detected widely in the developing heart. These observations support the hypothesis that cartilage collagens, especially the long isoform of type II collagen, participate in the morphogenesis of cardiac valves and septa.

Animals↗
PubMed1996

Type VI collagen is present in human and bovine vitreous.

BACKGROUND: Heterotypic (mixed) collagen fibrils that contain collagen types II, IX and V/XI have previously been identified in vitreous gel. The purpose of this study was to determine whether vitreous gel also contains type VI collagen microfibrils, as they are widely distributed in other tissues where they may play an important structural role. METHODS: Bovine and human vitreous gel was analysed by rotary shadowing electron microscopy after bacterial collagenase digestion and gel filtration chromatography. Bovine vitreous was extracted in 4 M guanidine hydrochloride, and following isopycnic centrifugation fractions were analysed by western blotting. RESULTS: Type VI collagen microfibrils were identified by electron microscopy in human and bovine vitreous. Type VI collagen was identified by western blot analysis. CONCLUSION: Type VI collagen is present in vitreous and may participate in the structural assembly of the vitreous gel.

Animals↗
PubMed1991

1991 Volvo Award in basic sciences. Collagen types around the cells of the intervertebral disc and cartilage end plate: an immunolocalization study.

Several types of collagen are known to exist in the intervertebral disc in addition to the fibrillar collagens, Types I and II. Although they constitute only a small percentage of the total collagen content, these minor collagens may have important functions. This study was designed to investigate the presence of Types I, II, III, IV, VI, and IX collagens in the intervertebral disc and cartilage end plate by immunohistochemistry, thereby establishing their location within the tissues. Types III and VI collagen have a pericellular distribution in animal and human tissue. No staining for Type IX collagen was present in normal human disc, but in rat and bovine intervertebral disc, it was also located pericellularly. These results show that cells of the intervertebral disc and cartilage end plate sit in fibrous capsules, forming chondrons similar to those described in articular cartilage. In pathologic tissue the amount and distribution of the collagen types, and the organization of the pericellular capsule, differ from that seen in control material.

Adult↗
PubMed1991

Degradation of cartilage matrix components by the cysteine proteinases, cathepsins B and L.

Rat chondrosarcoma chondrocytes are able to secrete both the precursor and mature forms of cathepsins B and L. When these two cysteine proteinases were added to a native cartilaginous matrix near neutral pH they were found to release and degrade both the proteoglycan and collagen components. One cleavage site in the proteoglycan monomer was found to be near the amino terminal globular domain. Collagen types II, IX, and XI in the matrix were also released and partially degraded. Ultimately these proteinases act as matrix depolymerases as they attacked the regions which are involved in collagen crosslinking. We conclude that these enzymes may play a role in matrix destruction as seen in arthritis.

Animals↗
PubMed1993

Type XIV collagen is encoded by alternative transcripts with distinct 5' regions and is a multidomain protein with homologies to von Willebrand's factor, fibronectin, and other matrix proteins.

The combined nucleotide sequences of several overlapping cDNAs provide the first complete amino acid sequence of type XIV collagen. Independent confirmation of the deduced sequence is provided by amino acid sequencing of several tryptic peptides isolated from purified chicken skin type XIV collagen. Comparative analyses show that the amino-terminal non-triple-helical region of alpha 1(XIV) chains contains sequence motifs that are similar to alpha 1(IX) collagen, fibronectin type III repeats, and von Willebrand's factor A-domains. The results also strongly suggest that the alpha 1(XIV) collagen gene is identical to the gene encoding the matrix component previously named undulin. cDNAs covering the 5' region of alpha 1(XIV) mRNA fall into two classes with distinct sequences in their 5'-untranslated regions. We believe the two alternative sequences result from differential splicing of the primary transcript. Interestingly, one of the untranslated sequences shows a high degree of identity with the cis-regulatory translational control sequence in the 5'-untranslated region of a Drosophila ribosomal protein mRNA. We hypothesize therefore that the sequence in alpha 1(XIV) collagen may play a role in the control of alpha 1(XIV) protein synthesis.

Alternative Splicing↗
PubMed1995

Biosynthesis and characterization of type X collagen in human fetal epiphyseal growth plate cartilage.

We examined in vitro collagen biosynthesis by organ cultures from human fetal epiphyseal growth plate cartilage. The biosynthetic products were characterized by NaCl fractional precipitation, limited proteolytic digestion, and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Organ cultures of human fetal epiphyseal growth plate cartilage synthesized large amounts of type X collagen in addition to type II, type IX, and type XI collagens. The individual polypeptide chains of human type X collagen migrated with an apparent M(r) of 45 kDa after proteolytic digestion with pepsin. The migration pattern of these molecules did not change when examined under reducing and nonreducing conditions, indicating that they did not contain intrahelical disfulfide bonds. Comparison of the rates at type X collagen biosynthesis at weeks 20 and 24 of human fetal development showed a marked increase of 24 weeks. Northern hybridization analysis of total RNA from freshly isolated epiphyseal growth plate chondrocytes with a cDNA corresponding to the carboxyl terminus of human type X collagen indicated that the developmental increase of type X collagen production is determined by pre-translational mechanisms.

Base Sequence↗
PubMed1992

Primary structure and function of stromelysin/transin in cartilage matrix turnover.

Stromelysin/Transin is a member of the matrix metalloprotease gene family. This metalloprotease is synthesized as a preproenzyme with a predicted size of 53,977 Da including a 17 amino acid signal peptide. Prostromelysin is secreted from normal and transformed cells in two forms with apparent molecular masses on NaDodSO4 gels of 60 and 58-kDa. The minor 60-kDa species contains N-linked oligosaccharide(s). Stromelysin consists of three domains the amino terminal propeptide(s) domain contains the tribasic amino acid sequence RRK which is important in the proteolytic activation of this zymogen by trypsin-like serine proteases. The second domain consists of the catalytic domain which contains the zinc binding site. The carboxyl-terminal hemopexin domain has no known function and can be removed without a loss of enzymatic activity. Stromelysin has a broad range of substrate specificity including proteoglycans, casein, fibronectin, laminin, native type IV and IX collagen and gelatin but not type I collagen. In the presence of trypsin or plasmin, catalytic amounts of this enzyme can also fully activate interstitial fibroblast collagenase. We have developed a panel of monoclonal antibodies against stromelysin which will be useful for the tissue localization of the various species of this enzyme in tissues. In addition, we have demonstrated that either human rIL-1 (alpha) or rTNF (alpha) can stimulate the expression of this enzyme in cultured bovine articular cartilage at least 10-fold. Based on western blot analysis, the zymogen form of the enzyme was the major enzyme species detected in either the media or cartilage matrix compartments of cytokine treated cultures.

Amino Acid Sequence↗