Control of proteoglycan biosynthesis. Further studies on the effect of serum on cultured bovine articular cartilage.
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ABSTRACT:
SUBMITTER: McQuillan DJ
PROVIDER: S-EPMC1147052 | biostudies-other | 1986 Aug
REPOSITORIES: biostudies-other
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Project description:The addition of foetal calf serum to explant cultures of adult bovine articular cartilage is known to stimulate proteoglycan synthesis in a dose-dependent manner. We have now shown the activity in serum responsible for this effect to be heat- and acid-stable, to be associated with a high-Mr complex in normal serum but converted to a low-Mr form under acid conditions. The activity has an apparent Mr approximately 10,000 and isoelectric points similar to those reported for insulin-like growth factors (IGFs). Addition of a monoclonal antibody against insulin-like growth factor-I (IGF-I) prevented foetal calf serum from stimulating proteoglycan synthesis. Physiological concentrations of recombinant IGF-I or pharmacological levels of insulin when added to cartilage cultures mimicked the proteoglycan-stimulatory activity of serum. IGF-I appeared to act by increasing the rate of proteoglycan synthesis and did not change the nature of the proteoglycan synthesized nor the rate of proteoglycan catabolism by the tissue, suggesting that IGF-I may be important in the regulation of proteoglycan metabolism in adult articular cartilage. Furthermore, IGF-I can replace foetal calf serum in the culture medium, thereby allowing the use of a fully-defined medium which will maintain the synthesis and tissue levels of proteoglycan in adult articular cartilage explants for up to 5 days.
| S-EPMC1147434 | biostudies-other
Project description:The effects of mild or severe trypsin treatment of bovine articular-cartilage slices in tissue culture were studied by monitoring the incorporation of [35S]sulphate into proteoglycans. Moderate trypsin treatment caused a subsequent marked inhibition of proteoglycan biosynthesis, which was reversible with time. Analysis on Sepharose CL-2B of the proteoglycan species synthesized showed that, directly after trypsin treatment, there was a 30% increase in the synthesis of the low-Mr proteoglycan (Kav. 0.71), and the total decrease in proteoglycan biosynthesis was reflected in a decrease in the synthesis of the high-Mr proteoglycan species (Kav. 0.31). The small proteoglycan was partially characterized and shown to be a true biosynthetic product and not a breakdown product. Trypsin treatment (20 micrograms/ml per 100 mg of tissue) of cartilage slices also resulted in an increase in the glycosaminoglycan chain size of the large proteoglycan, but not of the small proteoglycan.
| S-EPMC1144862 | biostudies-other
Project description:The synthesis and distribution of hyaluronate and proteoglycan were studied in bovine articular cartilage in short-term explant culture with [3H]acetate and H2(35)SO4 as precursors. The incorporation of [3H]acetate into hyaluronate and sulphated glycosaminoglycans was linear with time, except that hyaluronate synthesis showed a marked lag at the beginning of the incubation. [3H]Hyaluronate represented 4-7% of the total [3H]glycosaminoglycans synthesized over a 6 h period. However, the distributions of [3H]hyaluronate and 3H-labelled sulphated glycosaminoglycans were different: about 50% of the newly synthesized [3H]hyaluronate appeared in the medium, compared with less than 5% of the 3H-labelled sulphated proteoglycans. A pulse-chase experiment revealed that the release of newly synthesized [3H]hyaluronate from cartilage was rapid. No difference was observed in the distribution of [3H]hyaluronate between medium and tissue by cartilage from either the superficial layer or the deep layer of articular cartilage. When articular cartilage was incubated with 0.4 mM-cycloheximide, proteoglycan synthesis was markedly inhibited, whereas the synthesis of hyaluronate was only partially inhibited and resulted in more of the newly synthesized hyaluronate being released into the medium. Analysis of the hydrodynamic size of [3H]hyaluronate isolated from cartilage on Sephacryl-1000 revealed one population that was eluted as a broad peak (Kav. less than 0.7), compared with two populations (Kav. greater than 0.5 and less than 0.5) appearing in the medium of cultures. These data suggest that hyaluronate is synthesized in excess of proteoglycan synthesis and that the hyaluronate that is not complexed with proteoglycans is rapidly lost from the tissue.
| S-EPMC1133497 | biostudies-other
Project description:BackgroundRheumatoid arthritis (RA) is an autoimmune disease of the synovial joints. The autoimmune character of RA is underscored by prominent production of autoantibodies such as those against IgG (rheumatoid factor), and a broad array of joint tissue-specific and other endogenous citrullinated proteins. Anti-citrullinated protein antibodies (ACPA) can be detected in the sera and synovial fluids of RA patients and ACPA seropositivity is one of the diagnostic criteria of RA. Studies have demonstrated that RA T cells respond to citrullinated peptides (epitopes) of proteoglycan (PG) aggrecan, which is one of the most abundant macromolecules of articular cartilage. However, it is not known if the PG molecule is citrullinated in vivo in human cartilage, and if so, whether citrulline-containing neoepitopes of PG (CitPG) can contribute to autoimmunity in RA.MethodsCitPG was detected in human cartilage extracts using ACPA+ RA sera in dot blot and Western blot. Citrullination status of in vitro citrullinated recombinant G1 domain of human PG (rhG1) was confirmed by antibody-based and chemical methods, and potential sites of citrullination in rhG1 were explored by molecular modeling. CitPG-specific serum autoantibodies were quantified by enzyme-linked immunosorbent assays, and CitPG was localized in osteoarthritic (OA) and RA cartilage using immunohistochemistry.FindingsSera from ACPA+ RA patients reacted with PG purified from normal human cartilage specimens. PG fragments (mainly those containing the G1 domain) from OA or RA cartilage extracts were recognized by ACPA+ sera but not by serum from ACPA- individuals. ACPA+ sera also reacted with in vitro citrullinated rhG1 and G3 domain-containing fragment(s) of PG. Molecular modeling suggested multiple sites of potential citrullination within the G1 domain. The immunohistochemical localization of CitPG was different in OA and RA cartilage.ConclusionsCitPG is a new member of citrullinated proteins identified in human joints. CitPG could be found in both normal and diseased cartilage specimens. Antibodies against CitPG may trigger or augment arthritis by forming immune complexes with this autoantigen in the joints of ACPA+ RA patients.
| S-EPMC4778950 | biostudies-literature
Project description:The negatively charged proteoglycans (PG) provide compressive resistance to articular cartilage by means of their fixed charge density (FCD) and high osmotic pressure (?(PG)), and the collagen network (CN) provides the restraining forces to counterbalance ?(PG). Our objectives in this work were to: 1), account for collagen intrafibrillar water when transforming biochemical measurements into a FCD-?(PG) relationship; 2), compute ?(PG) and CN contributions to the compressive behavior of full-thickness cartilage during bovine growth (fetal, calf, and adult) and human adult aging (young and old); and 3), predict the effect of depth from the articular surface on ?(PG) in human aging. Extrafibrillar FCD (FCD(EF)) and ?(PG) increased with bovine growth due to an increase in CN concentration, whereas PG concentration was steady. This maturation-related increase was amplified by compression. With normal human aging, FCD(EF) and ?(PG) decreased. The ?(PG)-values were close to equilibrium stress (?(EQ)) in all bovine and young human cartilage, but were only approximately half of ?(EQ) in old human cartilage. Depth-related variations in the strain, FCD(EF), ?(PG), and CN stress profiles in human cartilage suggested a functional deterioration of the superficial layer with aging. These results suggest the utility of the FCD-?(PG) relationship for elucidating the contribution of matrix macromolecules to the biomechanical properties of cartilage.
| S-EPMC3175069 | biostudies-literature
Project description:Terahertz-spectroscopy probes dynamics and spectral response of collective vibrational modes in condensed phase, which can yield insight into composition and topology. However, due to the long wavelengths employed (λ = 300 μm at 1THz), diffraction limited imaging is typically restricted to spatial resolutions around a millimeter. Here, we demonstrate a new form of subwavelength hyperspectral, polarization-resolved THz imaging which employs an optical pattern projected onto a 6 μm-thin silicon wafer to achieve near-field modulation of a co-incident THz pulse. By placing near-field scatterers, one can measure the interaction of object with the evanescent THz fields. Further, by measuring the temporal evolution of the THz field a sample's permittivity can be extracted with 65 μm spatial resolution due to the presence of evanescent fields. Here, we present the first application of this new approach to articular cartilage. We show that the THz permittivity in this material varies progressively from the superficial zone to the deep layer, and that this correlates with a change in orientation of the collagen fibrils that compose the extracellular matrix (ECM) of the tissue. Our approach enables direct interrogation of the sample's biophysical properties, in this case concerning the structure and permittivity of collagen fibrils and their anisotropic organisation in connective tissue.
| S-EPMC5932036 | biostudies-literature
Project description:1. The effect of different batches of fetal bovine serum and of growth factors on [35S]sulphate incorporation into glycosaminoglycans and on UDP-sugar pools in explant cultures of bovine articular cartilage was investigated. 2. [35S]Sulphate incorporation was variably stimulated between 1.2- and 3.5-fold by four different batches of serum. The UDP-glucuronate pool size expanded 4.3-6.5-fold in the presence of serum, even in those cultures in which little stimulation of [35S]sulphate incorporation occurred. The UDP-N-acetylhexosamine and UDP-hexose pools expanded by about 1.5- and 2.0-fold respectively in the presence of serum. UDP-xylose was not detected. 3. Equilibrium-labelling and pulse-chase experiments with D-[1-3H]glucose indicated that the rate of flux through the UDP-sugar pools was unaffected by serum. UDP-hexose, UDP-N-acetylhexosamine and UDP-glucuronate have approximate half-lives (t1/2) of 7, 12 and 3-4 min respectively. At equilibrium, the 3H specific activities of UDP-hexose and UDP-N-acetylhexosamine were very similar but that for the UDP-glucuronate pool was much higher, especially in serum-supplemented cultures. The results suggest that UDP-glucuronate synthesis occurs via a pathway which is independent of the main UDP-hexose pathway. 4. Supplementing cultures with heat-treated serum had no effect on the serum-induced expansion of UDP-sugar pools but stimulation of [35S]sulphate incorporation into glycosaminoglycans was 50% lower than for native serum. Acid-treated serum promoted a 2-fold expansion of the UDP-glucuronate and UDP-N-acetylhexosamine pool over that obtained with native serum but was 20% less effective in stimulating [35S]sulphate incorporation than the latter. Prior dialysis of serum had no effect on its modulatory action on either [35S]sulphate incorporation or on the size of UDP-sugar pools. 5. Insulin-like growth factor 1 (IGF-1), transforming growth factor beta-1 (TGF beta-1), platelet-derived growth factor (PDGF) (BB homodimer) and epidermal growth factor (EGF) all stimulated [35S]sulphate incorporation into glycosaminoglycans as expected. The UDP-glucuronate pool expanded by 1.5- and 2.0-fold in the presence of IGF-1 and TGF beta-1 respectively, and by about 1.8-fold in the presence of PDGF or EGF. None of the factors investigated, or combinations of IGF-1 and TGF beta-1 or IGF-1 and EGF, stimulated expansion of the UDP-glucuronate pool to the same extent as native serum.(ABSTRACT TRUNCATED AT 400 WORDS)
| S-EPMC1137605 | biostudies-other
Project description:A homogeneous preparation of catabolin from pig leucocytes caused a reversible dose-dependent (0.01-1 nM) decrease in the synthesis of proteoglycan in slices of pig articular cartilage cultured in serum-free medium. The monomers that were synthesized and secreted in the presence of catabolin had the same average hydrodynamic size and ability to aggregate as the controls, and the core protein was substituted with the same number of glycosaminoglycan chains. The chains were the same average length and charge as normal and were sulphated to the same extent as the controls. Newly synthesized extracellular proteoglycan was not preferentially degraded. A 2-3-fold increase in glycosaminoglycan synthesis occurred in control and catabolin-treated cartilage in the presence of beta-D-xyloside (1 mM), more than 80% being secreted into the medium as free chains. Decreased incorporation of sulphate was not reversed in the presence of lysosomal-enzyme inhibitors, and there was no evidence in pulse-chase experiments of increased intracellular degradation of glycosaminoglycan chains before secretion. It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules.
| S-EPMC1144916 | biostudies-other
Project description:Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.
| S-EPMC1138657 | biostudies-other
Project description:Proteoglycans (A1D1) extracted from bovine femoral-head cartilage were examined by electron microscopy using benzyldimethylammonium chloride as a spreading agent. The preparation contained a mixture of particles, some with a 'beaded' structure and a contiguous filamentous 'tail' at one end and others which appeared as round 'blobs', some of which also had filamentous tails. Previous electron-microscopic studies of proteoglycan monomers have indicated that their length distributions were apparently unimodal, a finding that contrasted with agarose/polyacrylamide-gel-electrophoresis results, which generally indicated two bands. In the present study proteoglycans isolated from the slowly migrating electrophoretic band were shown to be predominantly the larger molecules of beaded appearance, whereas the rapidly migrating proteoglycans were predominantly molecules with the 'blob-like' appearance. Gel-filtration, isopycnic-density-gradient-centrifugation and rate-zonal-centrifugation techniques were evaluated as means of proteoglycan fractionation by electron microscopy and agarose-gel electrophoresis. Rate-zonal centrifugation in mixed-salt gradients of caesium chloride/4 M-guanidinium chloride yielded the most effective fractionation.
| S-EPMC1147373 | biostudies-other