Matrix (Stuttgart, Germany)最新文献

The structure, biosynthesis and catabolism of aggrecan has been studied in the bovine fetal rib growth plate. Comparative analyses were made on six 1-mm transverse slices which represent the resting zone (slice 6), proliferative zone (slices 5 and 4), upper hypertrophic zone (slice 3), middle hypertrophic zone (slice 2) and lower hypertrophic zone (slice 1). Aggrecan was abundant and exhibited very high aggregability in all zones. The aggrecan monomer was similar in structure in the resting and proliferative zones but showed a marked increase in hydrodynamic size in the lower hypertrophic zone; this was apparently due to an increase in the size of substituent glycosaminoglycans and an increase in core protein size as indicated by peptide analysis for G3 domain abundance. Biosynthetic studies with [³⁵S]-sulfate showed the rate of synthesis per cell to be highest in the upper hypertrophic zone, and the structure of the newly synthesised molecules to be similar to the resident population in all zones.

During explant culture in basal medium both aggregating and non-aggregating forms of aggrecan were released slowly from all zones. Addition of 10 nM retinoic acid to explants stimulated the release of both these forms of aggrecan whereas higher concentrations of retinoic acid (100 nM and 1000 nM) preferentially stimulated the release of the degraded forms. In this regard hypertrophic cells were the most responsive and resting cells were the least responsive. Analysis of the degraded fragments by polyacrylamide gel electrophoresis and by N-terminal sequencing indicated that aggrecan catabolism in all zones of the growth plate is due to the action of aggrecanase, a novel cartilage proteinase which is also active in normal and osteoarthritic articular cartilages (Sandy et al., 1992). These observations are discussed in terms of the role of aggrecan in the extensive matrix remodelling which accompanies chondrocyte hypertrophy in the growth plate.

The identification of mutations in cartilage-specific collagen genes in inherited forms of osteoarthritis (OA) and other heritable cartilage diseases has been hampered by the difficulty in obtaining sufficient tissue to isolate RNA and by the loss of the cartilage phenotype during in vitro chondrocyte culture. To overcome these limitations we employed RNA Polymerase Chain Reaction (PCR) to amplify cDNAs for the cartilage-specific collagens types II, IX and XI from mRNA obtained from small numbers of chondrocytes. We also amplified cDNAs for these collagens from “illegitimate transcripts” from Epstein-Barr virus (EBV)-transformed lymphocytes. Total RNA was obtained from freshly isolated human fetal and adult chondrocytes and from long-term (90 days) chondrocyte cultures. The RNA was reverse transcribed to cDNA and the cDNA amplified in the same tube with oligonucleotide primers specific for types II, IX and XI collagens. The amplified double-stranded products were cloned and sequenced. Successful amplification of the entire 4.4 kb of the type II pro collagen cDNA was obtained from as little as 6 ng of RNA from freshly isolated fetal human chondrocytes. Seven hundred and eighty base pairs of the α1(IX) collagen cDNA and the entire published sequence of α2(XI) collagen cDNA, were also amplified from these cells. We were also able to amplify cDNAs for the three cartilage-specific collagens from “illegitimate transcripts” from EBV-transformed lymphocytes. Thus, these methods will allow the identification of mutations in cartilage-specific collagen genes in patients with inherited OA and other heritable cartilage diseases from small amounts of cartilage or chondrocyte RNA or from non-cartilaginous sources.

Type IX collagen is a component of cartilage and vitreous humor. Its structure and matrix localization suggest it may serve to mediate interactions between fibrillar collagen, proteoglycan and other matrix components. Consequently, abnormalities in type IX collagen may result in chondrodysplasia. In this paper we describe the preparation and use of two monoclonal antibodies which recognize peptide sequences within the human cartilage α1(IX) collagen chain. Antibody 23-5D1 is highly sensitive and highly specific. It permits the immunoblot detection of type IX collagen extracted from milligram amounts of normal and chondrodysplastic cartilage; it also identifies the “short” form of the α1(IX) chain in human vitreous humor. Antibody 37-10H7 is highly specific, but of low sensitivity. It was used to make the new observation that an N-linked oligosaccharide is present in the amino-terminal globular domain of the a1(IX) chain. We anticipate that these antibodies may be valuable tools in the study of human and other mammalian chondrodysplasias.

Tropoelastin is composed of alternating hydrophobic and hydrophilic domains. A hydrophobic peptide, VGVAPG, has been shown to be a ligand for a 67-kDa elastin cell surface receptor expressed on fetal bovine auricular chondrocytes and ligamentum nuchae fibroblasts. To explore the possibility that tropoelastin contains additional peptide ligands for this elastin receptor, we have constructed two deletion proteins that are expressed in E. coli and lack the repeated VGVAPG sequence. These proteins supported bovine fibroblast attachment implying the presence of a receptor binding site. Experiments using synthetic peptides contained within these proteins identify a chemotactic peptide, PGAIPG, and a chemokinetic peptide, GAIPG, PGAIPG was identified as a ligand for the bovine elastin receptor.

Intact type VI collagen microfibrils and fibrillin-containing microfibrils were isolated from foetal bovine skin and investigated immunochemically and ultrastructurally. Substantial variations were detected in the abundance and macromolecular assembly of these structures at progressive stages of gestation. Microfibrils of collagen VI were increasingly abundant in skin through foetal development from late first trimester to term. The pattern of expression of fibrillin-containing microfibrils in foetal skin differed from that of collagen VI. Fibrillin-containing microfibrils were particularly sparse in first trimester skin, and present only as short assemblies. However, by early second trimester there had been a sharp increase in the abundance and length of these fibrillin-containing microfibrils. These observations are consistent with early second trimester being a key phase of fibrillin assembly. In the third trimester, fibrillin-containing microfibrils were frequently isolated in association with amorphous material. This information on the pattern of expression and assembly of collagen VI microfibrils and fibrillin-containing microfibrils in foetal skin implies temporally and functionally distinct contributions of these two components to the establishment of the fibrous dermal matrix.

Osteopontin (OPN) is a 34-kDa, highly-phosphorylated glycoprotein with cell attachment properties that is a prominent constituent of the bone matrix. To aid in elucidating the function of this protein we have studied the cellular expression of OPN mRNA during the formation, growth and maturation of rat calvarial (membranous) and tibial (endochondral) bone. From Northern hybridization analysis OPN expression was demonstrated in the kidney and gravid uterus as well as in bone tissues. Compared to collagen, the expression of OPN was low in early bone formation but increased subsequently and reached peak levels in 14-day-old bone. However, both the collagen and OPN mRNAs decreased markedly thereafter and remained low in young adult bone. From in situ hybridization studies using a [³⁵S]-labelled rat OPN cRNA probe, OPN mRNA was localized to osteoblastic cells in newly-forming calvariae, jaw bones, and in the metaphyseal and periosteal bone of the tibia. In contrast to bone sialoprotein (BSP), which is expressed almost exclusively by osteoblasts at sites of de novo bone formation, OPN transcripts were present in cells lining both endosteal and periosteal bone surfaces, and in osteocytes. Moreover, expression of OPN persisted during the subsequent growth and remodelling of both membranous and endochondral bone and was expressed at particularly high levels by bone cells and hypertrophic chondrocytes at sites of osteoclastic resorption. In the more mature bone of young adult rats OPN expression was significantly reduced but remained detectable in bone cells lining periosteal and endosteal surfaces and in the primary and secondary spongiosa of the tibia. These studies on the developmental expression of OPN support the concept of a multifunctional role for OPN in bone formation and remodelling. Thus, the expression of OPN by osteoblasts early in bone development is consistent with a role for this protein in the formation of bone matrix, whereas the peak expression of OPN later in bone development, together with high expression at sites of rapid remodelling, indicate that OPN deposited on the surface of mineralized connective tissues may provide a template for osteoclastic resorption.

When adult human articular cartilage was maintained in organ culture in the presence of interleukin 1β, increased destruction of the extracellular matrix was observed, as judged by increased type II collagen degradation in situ determined immunohistochemically and the increased release of proteoglycan into the culture medium. Concomitant with these changes was the increased release of latent metalloproteinases into the culture medium. Culture of cartilage in the presence of a peptidylhydroxamate metalloproteinase inhibitor indicated a key role for the active forms of these enzymes in situ, since it produced a marked reduction in both proteoglycan release and collagen degradation. This compound had no detectable cytotoxic effects in organ culture and did not reduce the secretion of the metalloproteinases. The results of this study provide direct evidence that the latent metalloproteinase precursors, whose release is greatly stimulated by interleukin 1, are indeed activated to some degree and participate in cartilage matrix degradation.