Altered proteoglycan synthesis by micromelial limbs induced by 6-aminonicotinamide. Appearance of abnormal forms of cartilage-characteristic proteoglycan (PG-H).

Since administration of 6-aminonicotinamide (10 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs, biosynthesis of cartilage-characteristic proteoglycan-H (PG-H) as an important index of limb chondrogenesis was examined in day-7 normal and micromelial hind limbs by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]glucosamine and either [35S]sulphate or [35S]methionine, followed by analyses of labelled PG-H by glycerol density-gradient centrifugation under dissociative conditions, showed a marked reduction in the PG-H synthesis. (2) PG-H synthesized by the micromelial limbs was much lower than that synthesized by the normal limbs in the biosynthetic ratio of chondroitin sulphate to keratan sulphate and glycoprotein-type oligosaccharide, although no significant difference was observed in the immunological properties of these proteoglycans. (3) The degree of sulphation of chondroitin sulphates of PG-H was lowered in the micromelial limbs as judged by the increase of unsulphated disaccharide (delta Di-OS) released by chrondroitinase ABC digestion, although there were no significant differences between the normal and the micromelial limbs in the average molecular size (Mr = 38,000) of labelled chondroitin sulphates of PG-H. (4) Addition of beta-D-xyloside, an artificial initiator for chondroitin sulphate synthesis, to the micromelial limbs in culture recovered the incorporation of labelled glucosamine into chondroitin sulphate to that comparable with the normal control with beta-D-xyloside, although the incorporation of [35S]sulphate was lower in the micromelia than in the control with beta-D-xyloside. These results suggest that the reduction in the biosynthesis of the PG-H as well as the production of altered forms of PG-H induced by 6-aminonicotinamide during a critical period of limb morphogenesis may be an important factor for the micromelia.     

The role of proteoglycan in the development of sea urchins : I. Abnormal development of sea urchin embryos caused by the disturbance of proteoglycan synthesis

The development of the sea urchins, Hemicentrotus pulcherrimus and Clypeaster japonicus, was examined under conditions which inhibit the synthesis of proteoglycan in various ways. The changes in the developmental fate were compared with the degree of inhibition of proteoglycan synthesis as measured by incorporation of labeled precursors. The inhibition of proteoglycan synthesis was achieved by treating the embryos with (1) aryl-β- -xyloside (inhibition of coupling between protein and polysaccharide moieties); (2) sodium selenate (inhibition of sulfation of polysaccharide); and (3) 2-deoxy- -glucose (inhibition of polysaccharide chain elongation). It was found that the development of embryos ceased at the blastula stage in all cases tested, irrespective of the step of the inhibition. The cessation of development became apparent when the incorporation of the labeled precursors was decreased to a level below 50% compared with the normal level. From these facts, it appears that the synthesis of proteoglycan is necessary for the realization of post-gastrular development.     

Aggregated proteoglycan synthesis in organ cultures of human nucleus pulposus.

Proteoglycans isolated under associative conditions in the presence of protease inhibitors from human nucleus pulposus contained 17% aggregate and 83% non-aggregating monomer (Kav = 0.5 on Sepharose CL-2B). Isolated aggregate after reduction and alkylation was resolved into two components (Kav = 0.15 and 0.43) on Sepharose CL-2B. Labeled proteoglycans isolated from parallel samples pulsed with [35S]sulfate and chased for up to 18 h were present largely as aggregated material (up to 78%). Reduction and alkylation of the labeled samples gave a labeled proteoglycan monomer with Kav = 0.15. Both the labeled and unlabeled chondroitin sulfate chains had the same distribution on Sepharose CL-6B and equivalent molecular weights (Mr = 2.0 x 10(3)). After chondroitinase ABC digestion, the unlabeled keratan sulfate-protein core was polydisperse with a Kav = 0.38 on Sepharose CL-4B while the labeled keratan sulfate-protein core had a Kav = 0.05. This indicates that the newly synthesized proteoglycan had a large core protein and suggests that the proteoglycans present in nucleus pulposus are originally synthesized as large molecular weight, aggregating proteoglycans.     

Somite chondrogenesis in vitro: 1. Alterations in proteoglycan synthesis

During embryonic development, somites undergo chondrogenic differentiation when stimulated by notochord or spinal cord. The present study shows that, when cultured in suitable medium, explanted somites incorporated radioactive sulfate into cartilage-specific proteoglycans and the synthetic rate increased when notochord was included with somites. With increased culture time, explanted somites also synthesized proteoglycan monomers which were larger in size along with a larger proportion that were capable of interacting with exogenous hyaluronic acid. Interaction with notochord also resulted in increased synthesis of chondroitin 4-sulfate. Gel electrophoretic analysis showed that proteoglycans from unstimulated somites did not contain link protein (required for stable aggregate formation), even on day 9, while notochord-induced somites showed link protein as early as day 3, increasing 3-fold by day 9.     

Effect of beta-xylosides on synthesis of cartilage-specific proteoglycan in chondrocyte cultures.

Monolayer cultures of embryonic chick chondrocytes were incubated with ³⁵SO₄ in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free chondroitin sulfate chains were measured following gel filtration on Sephadex G-200. Synthesis of β-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. The amount of core protein was determined from equivalent numbers of β-xyloside-treated and untreated cells by a radioimmune assay. Similar amounts of core protein were found in both types of cultures, indicating that decreased synthesis of cartilage-specific core protein is not responsible for the observed decrease in overall chondroitin sulfate proteoglycan production.     

Collagen synthesis and deposition in cartilage during disrupted proteoglycan production.

A simple system was developed to investigate in vitro the possible relationship between collagen and proteoglycan synthesis in cartilage. When production of complete proteoglycan molecules was effectively inhibited with 4-methylumbelliferyl beta-D-xyloside collagen synthesis and distribution were virtually unaffected.     

Abnormal hemoglobin synthesis in some leukemic patients.

Hemoglobin chain synthesis during leukemic processes has been studied on patients having fetal hemoglobin. All cases showed the following abnormalities : (1) a relatively increased synthesis of the beta chain ; (2) an important increase of the free dimeric precursors pool, with, most of the time, a predominance of alpha chain. If the first point suggests an alpha-thalassemia feature, the presence of free alpha chains shows evidence for a more complex mechanism not only due to a decrease of messenger RNA. The hypothesis of a clonal disorder could neither be demonstrated nor ruled out. The observed abnormalities could be due to a defect in a alpha chain depending regulation mechanism.     

Extracellular matrix metabolism by chondrocytes. III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. Benzyl-beta-D-xyloside, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.     

Compression loading in vitro regulates proteoglycan synthesis by tendon fibrocartilage.

The regulation of proteoglycan synthesis in a fibrocartilaginous tissue by mechanical loading was assessed in vitro. Discs of bovine tendon fibrocartilage were loaded daily with unconfined, cyclic, uniaxial compression (5 s/min, 20 min/day) and the synthesis of large and small proteoglycans was measured by incorporation of [35S]sulfate. All discs synthesized predominantly large proteoglycan when first placed in culture. After 2 weeks in culture nonloaded discs synthesized predominantly small proteoglycans whereas loaded discs continued to produce predominantly large proteoglycan. The turnover of 35S-labeled proteoglycan was not significantly altered by the compression regime. Increased synthesis of large proteoglycans was induced by a 4-day compression regime following 21 days of culture without compression. Inclusion of cytochalasin B during compression mimicked this induction. Autoradiography demonstrated that cell proliferation was minimal and confined to the disc edges whereas 35S-labeled proteoglycan synthesis occurred throughout the discs. These experiments demonstrate that mechanical compression can regulate synthesis of distinct proteoglycan types in fibrocartilage.     

Changes in mouse intervertebral-disc proteoglycan synthesis with age. Hereditary kyphoscoliosis is associated with elevated synthesis.

Mice with hereditary kyphoscoliosis (ky/ky) develop intervertebral-disc degeneration at the cervico-thoracic junction. Disc proteoglycans were investigated to determine whether changes in synthesis or structure were associated with this. Elevated 35S-proteoglycan synthesis was found in one or more cervico-thoracic discs in 80-day-old ky/ky mice. The hydrodynamic size and aggregation properties of ky/ky-mouse disc 35S-proteoglycans extracted with 4 M-guanidinium chloride were normal. Increased proportions of small 35S-proteoglycans were extracted with 0.5 M-guanidinium chloride from discs of normal and ky/ky mice with increasing age.     

Cell culture of rabbit meniscal fibrochondrocytes II. Sulfated proteoglycan synthesis

Rabbit meniscal fibrochondrocytes were grown in vitro under culture conditions previously shown to foster growth of this cell type. Regardless of the culture regimen employed, the cells synthesized sulfated proteoglycans which could be differentiated by their solubility when dialyzed against water. The water soluble proteoglycans (WSPG) were monomeric in nature and could be separated into sub-types based on their hydrodynamic size when analyzed by gel-filtration chromatography. The water insoluble proteoglycans (WIPG) appeared to represent hyaluronic acid-dependent aggregates of the larger of the two WSPG. The proteoglycans contained approximately 87% chondroitin sulfate and 5% dermatan sulfate. Keratan sulfate could not be detected. Addition of ascorbate to the culture medium did not change the amount or the hydrodynamic size of the proteoglycan aggregates but did alter the quantity of the larger WSPG monomer synthesized depending upon the culture regimen used. Thus, these cells are capable of expressing their differentiated phenotype in short-term monolayer cell culture.     

Extracellular matrix metabolism by chondrocytes III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. $\text{Benzyl}\text{-β-}\text{D}\text{-}\text{xyloside}$, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.     

Ultrasound stimulates proteoglycan synthesis in bovine primary chondrocytes

Mechanical forces can stimulate the production of extracellular matrix molecules. We tested the efficacy of ultrasound to increase proteoglycan synthesis in bovine primary chondrocytes. The ultrasound-induced temperature rise was measured and its contribution to the synthesis was investigated using bare heat stimulus. Chondrocytes from five cellular isolations were exposed in triplicate to ultrasound (1 MHz, duty cycle 20%, pulse repetition frequency 1 kHz) at average intensity of 580 mW/cm2 for 10 minutes daily for 1-5 days. Temperature evolution was recorded during the sonication and corresponding temperature history was created using a controllable water bath. This exposure profile was used in 10-minute-long heat treatments of chondrocytes. Heat shock protein 70 (Hsp70) levels after one-time treatment to ultrasound and heat was analyzed by Western blotting, and proteoglycan synthesis was evaluated by 35S-sulfate incorporation. Ultrasound treatment did not induce Hsp70, while heat treatment caused a slight heat stress response. Proteoglycan synthesis was increased approximately 2-fold after 3-4 daily ultrasound stimulations, and remained at that level until day 5 in responsive cell isolates. However, chondrocytes from one donor cell isolation out of five remained non-responsive. Heat treatment alone did not increase proteoglycan synthesis. In conclusion, our study confirms that pulsed ultrasound stimulation can induce proteoglycan synthesis in chondrocytes.     

The role of proteoglycan synthesis in the development of sea urchins : II. The effect of administration of exogenous proteoglycan

The role of proteoglycan synthesis in the early development of sea urchins was studied by treating the embryos with a variety of inhibitors of proteoglycan synthesis and also with proteoglycan of exogenous origin. Developmental arrest at the blastula stage caused by p-nitrophenyl-β- -xyloside (p-NP-xyl) was cancelled by the administration of proteoglycan of exogenous origin. Proteoglycan of post-gastrular origin was effective for cancellation, but proteoglycan of blastular origin was not effective. This suggests that the hindrance of development by p-NP-xyl was due to the lack of proteoglycan synthesis at the late blastula stage. On the other hand, developmental arrest caused by 2-deoxy- -glucose (deoxy-glucose) and Na-selenate was not cancelled by the administration of proteoglycan under any condition tested. Apart from the cancelling action of proteoglycan, solely administered proteoglycan caused a blockage in development at a stage corresponding to the stage from which it was extracted, indicating that proteoglycan may be characterized by a kind of stage specificity.     

Control of proteoglycan synthesis. Studies on the activation of synthesis observed during culture of articular cartilages.

When slices of adult rabbit articular cartilage were incubated in culture medium, the rate of incorporation of [35S]sulphate or [3H]acetate into glycosaminoglycans increased 4-8 fold during the first 5 days of incubation. Similar changes in biosynthetic activity were observed during culture of adult bovine cartilage. The activation of synthesis was not serum-dependent, but appeared to be a result of the depletion of tissue proteoglycan that occurs under these incubation conditions [Sandy, Brown & Lowther (1978) Biochim. Biophys. Acta 543, 536--544]. Thus, although complete activation was observed in serum-free medium, it was not observed if the cartilage was cultured inside dialysis tubing or in medium containing added proteoglycan subunit. The average molecular size of the proteoglycans synthesized by activated tissue was slightly larger than normal, as determined by chromatography on Sepharose CL-2B, and the average molecular size of the glycosaminoglycans synthesized by activated tissue was markedly increased over the normal. The increase in chain size was accompanied by an increase in the proportion of the chains degraded by chondroitinase ABC; these results are consistent with the preferential synthesis by activated chondrocytes of chondroitin sulphate-rich proteoglycans. The increase in glycosaminoglycan chain size was observed whether the chains were formed on endogenous core protein or on exogenous benzyl-beta-D-zyloside. An approximate 4-fold activation in culture of glycosaminoglycan synthesis on protein core was accompanied by a 1.54-fold increase in the rate of incorporation of [3H]serine into the chondroitin sulphate-linkage region of the proteoglycans. A 2.8-fold activation in culture of glycosaminoglycan synthesis on benzyl-beta-D-zyloside was accompanied by a 1.7-fold increase in the rate of incorporation of [3H]benzyl-beta-D-zyloside into glycosaminoglycans. The activation of glycosaminoglycan synthesis was, however, accompanied by no detectable change in the activity of xylosyltransferase (EC 2.4.2.26) in cell-free extracts. These results are discussed in relation to current ideas on the control of proteoglycan synthesis in cartilage.     

beta-D-xyloside-mediated alteration in the synthesis of basement membrane proteoglycan

The effect of nitrophenyl-beta-D-xyloside (xyloside), a synthetic initiator of glycosaminoglycan synthesis, on proteoglycan and glycosaminoglycan synthesis by a basement membrane producing tumor was studied. While xyloside markedly stimulated the formation of chondroitin sulfate chains, it depressed the formation of a basement membrane heparan sulfate proteoglycan and caused only little formation of free heparan sulfate chains. However, when the synthesis of the core protein of the proteoglycan was inhibited by cycloheximide, heparan sulfate chains were produced by xyloside treatment. These heparan sulfate chains had a sulfate content higher than that of heparan sulfate found on the proteoglycan. The data indicate that xyloside can substitute for the heparan sulfate initiation site on the core protein of the proteoglycan and that this initiation is enhanced in the absence of core protein. This suggests that under normal conditions the formation of heparan sulfate chains may be tightly linked to the production of the core protein.     

Extracellular matrix metabolism by chondrocytes. VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[3H]proline into hydroxyproline and [3H]acetate into glycosaminoglycans, was shown to be depressed by 58% and 39%, respectively, by the addition of exogenous proteoglycan at a concentration of 10 mg/ml growth media. The incorporation of L-[3H]proline into acid-insoluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity on the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-translational modification of collagen and proteoglycan.     

Separation of a proteoglycan fraction from Kurloff cells stimulating protein synthesis in macrophages.

Proteoglycan from Kurloff cells, when present in the medium in low concentrations, increased the incorporation of (3-H)leucine into trichloracetic acid-precipitable material by macrophages from peritoneal exudates, in addition to inhibiting their migration from capillary tubes, as observed previously. After treatment with 0.5 M-NaOH, followed by dialysis or ultrafiltration, material with the distinctive u.v. and i.r. spectra of the whole proteoglycan appeared in the diffusate, and biological activity was lost from the proteoglycan which remained in the dialysis residue. The diffusible material absorbed near 260 nm and had i.r. bands at 805 cm-minus-1 and 1260 cm-minus-1, but did not display the i.r. bands characteristic of chondrotin 4-sulphate. It contained little sulphate, no hexosamine and less than 1% of the uronic acid present in the whole proteoglycan, and there were only trace amounts of amino acids, xylose and galactose. However, significant amounts of ribose and organic phosphate were present, each representing about 1% of the whole proteoglycan. After proteolysis and chondroitanase digestion of the proteoglycan, a fraction with absorbance at 260 mn was eluted from Dowex 1 with water which stimulated the incorporation of (3H)leucine by macrophages and inhibited their migration from capillary tubes.     

Inhibition of cartilage proteoglycan synthesis by interleukin I

We have investigated the mechanism of inhibition of cartilage proteoglycan by interleukin 1. Proteoglycan synthesis was inhibited using lower doses of interleukin 1 than those required to cause cartilage resorption. There was no significant effect on DNA or total protein synthesis. Gel electrophoresis showed a direct inhibitory effect on core protein synthesis while pulse-chase experiments using radiolabelled sulphate showed no alteration in the rate of intracellular transport and secretion of completed proteoglycan. Chondrocytes incubated with cycloheximide showed a first-order decrease in rate of uptake of radiolabelled sulphate (t1/2 = 25 mins) but interleukin 1 induced inhibition showed a delay of at least 1 hr, consistent with a requirement to deplete intracellular pools of protein before effects on post-translational events could be observed. Foetal and neonatal cartilage responded to the cytokine in a similar way to adult cartilage.