Cloning and expression of a type IX-like collagen in tissues of the ascidian Ciona intestinalis.
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Collagens are highly preserved proteins in invertebrates and vertebrates. To identify the collagens in urochordates, the total RNA extracted from the pharynx of the ascidian Ciona intestinalis was hybridized with a heterologous probe specific for the echinoderm Paracentrotus lividus fibrillar type I-like larval collagen. Using this probe, two main bands (i.e. 6 and 2.8 kb mRNA) were observed on Northern blot hybridization. The cDNA library prepared from poly(A)+RNA extracted from pharyngeal tissue was screened and a cDNA that specifies a type IX-like collagen was identified. This molecule presents a conceptual open reading frame for a protein containing 734 amino acids. In particular, we showed a 1 alpha chain type IX-like collagen characterized by three short triple-helical domains interspersed with four non-triple-helical sequences, with structural features of fibril-associated collagens with interrupted triple-helices (FACIT) collagens. Northern blot hybridizations indicate a 2.8 kb transcript size. Sequence comparison indicated homology (47.64%, 48.95%) between the type IX-like collagen of C. intestinalis and mouse and human type IX collagen. In situ hybridization of tunic and pharynx tissues shows the presence of transcripts in connective tissue cells. (+info)
Regulation of human COL9A1 gene expression. Activation of the proximal promoter region by SOX9.
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The COL9A1 gene contains two promoter regions, one driving expression of a long alpha1(IX) chain in cartilage (upstream) and one driving expression of a shorter chain in the cornea and vitreous (downstream). To determine how the chondrocyte-specific expression of the COL9A1 gene is regulated, we have begun to characterize the upstream chondrocyte-specific promoter region of the human COL9A1 gene. Transient-transfection analyses performed in rat chondrosarcoma (RCS) cells, human chondrosarcoma (HTB) cells, and NIH/3T3 cells showed that the COL9A1 promoter was active in RCS cells but not HTB or NIH/3T3 cells. Inclusion of the first intron had no effect on promoter activity. In transient-transfection analyses with promoter deletion constructs, it was found that full promoter activity in RCS cells depended on the region from -560 bp to +130 bp relative to the transcriptional start site (+1). Sequence analysis of the region from -890 bp to the transcriptional start predicted five putative SOX/Sry-binding sites. Mutation analysis revealed that two of three putative SOX/Sry binding sites within the -560 to +130 bp region are responsible for most of the COL9A1 promoter activity in RCS cells. Co-transfection experiments with a SOX9 expression plasmid revealed that a construct containing the five putative SOX/Sry-binding sites was transactivated 20- to 30-fold in both HTB and NIH/3T3 cells. Further co-transfection experiments showed that two of the SOX/Sry-binding sites located within the -560 to +130 bp region were required for full transactivation. However, mutation and deletion analyses indicated that a region from -560 to -357 bp, which does not contain any other conspicuous SOX9 sites, is also important for full promoter activity. DNA-protein binding assays and super-shift analysis revealed that SOX9 can form a specific complex with one of the SOX/Sry-binding sites with in the -560 to +130 region. (+info)
Intervertebral disc collagen. Usage of the short form of the alpha1(IX) chain in bovine nucleus pulposus.
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Nucleus pulposus, the central zone of the intervertebral disc, is gel-like and has a similar collagen phenotype to that of hyaline cartilage. Amino-terminal protein sequence analysis of the alpha1(IX)COL3 domain purified from bovine nucleus pulposus gave a different sequence to that of the long alpha1(IX) transcript expressed in hyaline cartilage and matched the predicted sequence of short alpha1(IX). The findings indicate that the matrix of bovine nucleus pulposus contains only the short form of alpha1(IX) that lacks the NC4 domain. The sequence encoded by exon 7, predicted from human COL9A1, is absent from both short and long forms of alpha1(IX) from bovine nucleus pulposus and articular cartilage. A structural analysis of the cross-linking sites occupied in type IX collagen from nucleus pulposus showed that usage of the short alpha1(IX) transcript in disc tissue had no apparent effect on cross-linking behavior. As in cartilage, type IX collagen of nucleus pulposus was heavily cross-linked to type II collagen and to other molecules of type IX collagen with a similar site occupancy. (+info)
Assembly of collagen types II, IX and XI into nascent hetero-fibrils by a rat chondrocyte cell line.
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The cell line, RCS-LTC (derived from the Swarm rat chondrosarcoma), deposits a copious extracellular matrix in which the collagen component is primarily a polymer of partially processed type II N-procollagen molecules. Transmission electron microscopy of the matrix shows no obvious fibrils, only a mass of thin unbanded filaments. We have used this cell system to show that the type II N-procollagen polymer nevertheless is stabilized by pyridinoline cross-links at molecular sites (mediated by N- and C-telopeptide domains) found in collagen II fibrils processed normally. Retention of the N-propeptide therefore does not appear to interfere with the interactions needed to form cross-links and mature them into trivalent pyridinoline residues. In addition, using antibodies that recognize specific cross-linking domains, it was shown that types IX and XI collagens, also abundantly deposited into the matrix by this cell line, become covalently cross-linked to the type II N-procollagen. The results indicate that the assembly and intertype cross-linking of the cartilage type II collagen heteropolymer is an integral, early process in fibril assembly and can occur efficiently prior to the removal of the collagen II N-propeptides. (+info)
Multilineage potential of cells from the artery wall.
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BACKGROUND: In diabetes or atherosclerosis, ectopic bone, fat, cartilage, and marrow often develop in arteries. However the mechanism is unknown. We have previously identified a subpopulation of vascular cells (calcifying vascular cells, CVC), derived by dilutional cloning of bovine aortic medial cells, and showed that they undergo osteoblastic differentiation and mineralization. We now show that CVC have the potential to differentiate along other mesenchymal lineages. METHODS AND RESULTS: To determine the multilineage potential of CVC, molecular and functional markers of multiple mesenchymal lineages were assessed. Chondrogenic potential of CVC was evidenced by expression of types II and IX collagen and cytochemical staining for Alcian blue. Leiomyogenic potential of CVC was evidenced by the expression of smooth muscle-alpha actin, calponin, caldesmon, and myosin heavy chain. Stromogenic potential of CVC was evidenced by the ability to support growth of colony-forming units of hematopoietic progenitor cells from human CD34+ umbilical cord blood cells for a period of 5 weeks. Adipogenic potential was not observed. CVC were immunopositive to antigens to CD29 and CD44 but not to CD14 or CD45, consistent with other mesenchymal stem cells. CVC retained multipotentiality despite passaging and expansion through more than 20 to 25 population triplings, indicating a capacity for self-renewal. CONCLUSIONS: These results suggest that the artery wall contains cells that have the potential for multiple lineages similar to mesenchymal stem cells but with a unique differentiation repertoire. (+info)
Covalent cross-linking of the NC1 domain of collagen type IX to collagen type II in cartilage.
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From a study to understand the mechanism of covalent interaction between collagen types II and IX, we present experimental evidence for a previously unrecognized molecular site of cross-linking. The location relative to previously defined cross-linking sites predicts a specific manner of interaction and folding of collagen IX on the surface of nascent collagen II fibrils. The initial evidence came from Western blot analysis of type IX collagen extracted by pepsin from fetal human cartilage, which showed a molecular species that had properties indicating an adduct between the alpha1(II) chain and the C-terminal domain (COL1) of type IX collagen. A similar component was isolated from bovine cartilage in sufficient quantity to confirm this identity by N-terminal sequence analysis. Using an antibody that recognized the putative cross-linking sequence at the C terminus of the alpha1(IX) chain, cross-linked peptides were isolated by immunoaffinity chromatography from proteolytic digests of human cartilage collagen. They were characterized by immunochemistry, N-terminal sequence analysis, and mass spectrometry. The results establish a link between a lysine near the C terminus (in the NC1 domain) of alpha1(IX) and the known cross-linking lysine at residue 930 of the alpha1(II) triple helix. This cross-link is speculated to form early in the process of interaction between collagen IX molecules and collagen II polymers. A model of molecular folding and further cross-linking is predicted that can spatially accommodate the formation of all six known cross-linking interactions to the collagen IX molecule on a fibril surface. Of particular biological significance, this model can accommodate potential interfibrillar as well as intrafibrillar links between the collagen IX molecules themselves, so providing a mechanism whereby collagen IX could stabilize a collagen fibril network. (+info)
Sequence variations in the collagen IX and XI genes are associated with degenerative lumbar spinal stenosis.
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BACKGROUND: Degenerative lumbar spinal stenosis (LSS) is usually caused by disc herniation or degeneration. Several genetic factors have been implicated in disc disease. Tryptophan alleles in COL9A2 and COL9A3 have been shown to be associated with lumbar disc disease in the Finnish population, and polymorphisms in the vitamin D receptor gene (VDR) (FokI and TaqI), the matrix metalloproteinase-3 gene (MMP-3) and an aggrecan gene (AGC1) VNTR have been reported to be associated with disc degeneration. In addition, an IVS6-4 a>t polymorphism in COL11A2 has been found in connection with stenosis caused by ossification of the posterior longitudinal ligament in the Japanese population. OBJECTIVE: To study the role of genetic factors in LSS. METHODS: 29 Finnish probands were analysed for mutations in the genes coding for intervertebral disc matrix proteins, COL1A1, COL1A2, COL2A1, COL9A1, COL9A2, COL9A3, COL11A1, COL11A2, and AGC1. VDR and MMP-3 polymorphisms were also analysed. Sequence variations were tested in 56 Finnish controls. RESULTS: Several disease associated alleles were identified. A splice site mutation in COL9A2 leading to a premature translation termination codon and the generation of a truncated protein was identified in one proband, another had the Trp2 allele, and four others the Trp3 allele. The frequency of the COL11A2 IVS6(-4) t allele was 93.1% in the probands and 72.3% in controls (p = 0.0016). The differences in genotype frequencies for this site were less significant (p = 0.0043). CONCLUSIONS: Genetic factors have an important role in the pathogenesis of LSS. (+info)
Identification of ciliary epithelial-specific genes using subtractive libraries and cDNA arrays in the avian eye.
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The ciliary epithelium of the ciliary body is derived from the anterior rim of the developing optic cup. Several recent studies have found that developmental abnormalities in this tissue can underlie congenital glaucoma. However, there is little known about the development of the ciliary epithelium. To better understand the developmental events responsible for the specification of this domain of the optic cup, we used a subtractive library, differential screening approach along with the construction of cDNA arrays to identify genes expressed in the ciliary epithelium of the chicken. We identified many genes specifically expressed in the ciliary epithelium, including a number that had been described previously as enriched in the ciliary epithelium of other species. By analyzing the expression of these genes during eye development, we were able to correlate the onset of ciliary epithelial gene expression with a reduction in mitotic activity in this region. We propose that the mechanisms that regulate the expression of ciliary epithelial genes are linked to the reduction in proliferation that results in the epithelial monolayer in this region. (+info)