Total Glycosaminoglycan Synthesis Research Articles

β-d-Xylosides Stimulate GAG Synthesis in Chondrocyte Cultures Due to Elevation of the Extracellular GAG Domains, Accompanied by the Depletion of the Intra–pericellular GAG Pools, with Alterations in the GAG Profiles

The familial disease of hereditary multiple exostoses is characterized by abnormal skeletal deformities requiring extensive surgical procedures. In hereditary multiple exostoses patients there is a shortage in the pericellular glycosaminoglycan (GAG) of heparan sulfate (HS), related to defective activity of HS glycosyltransferases, mainly in the pericellular regions of chondrocytes. This study searched for a novel approach employing xylosides with different aglycone groups priming a variety of GAG chains, in attempting to alter the GAG compositional profile. Cell cultures of patients with osteochondroma responded to p-nitrophenyl β-d-xyloside by a significant increase in total GAG synthesis, expressed mainly in the extracellular domains, limited to chondroitin sulfate). The different β-d-xylosides, in addition to increasing the synthesis of extracellular GAGs, led to a significant depletion of the intracellular GAG domains. In mouse chondrocyte cultures, β-d-xylosides with different aglycones created a unique distribution of the GAG pools. Of special interest was the finding that the naphthalene methanol β-d-xyloside showed the highest absolute levels of HS-GAGs in both extracellular and intra–pericellular moieties compared with other β-d-xylosides and with controls without xyloside. In summary, β-d-xylosides can be utilized in chondrocyte cultures to modify the distribution of GAGs between the extracellular and intracellular compartments. In addition, xylosides may alter the profile of specific GAG chains in each moiety.

Effect of Extracellular pH on Matrix Synthesis by Chondrocytes in 3D Agarose Gel

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.

Age-related Changes in the Response of Human Articular Cartilage to IL-1α and Transforming Growth Factor-β (TGF-β): CHONDROCYTES EXHIBIT A DIMINISHED SENSITIVITY TO TGF-β

Cartilage glycosaminoglycan (GAG) synthesis and composition, upon which its structural integrity depends, varies with age, is modified by anabolic and catabolic stimuli, and is regulated by UDP-glucuronate availability. However, how such stimuli, prototypically represented by transforming growth factor-beta1 (TGF-beta1) and IL-1alpha, modify GAG synthesis during aging of normal human articular cartilage is not known. Using explants, we show that chondroitin sulfate (CS):total GAG ratios decrease, whereas C6S:C4S ratios increase with cartilage maturation, and that chondrocytes in the cartilage mid-zone, but not the superficial or deep zones, exhibit uridine 5'-diphosphoglucose dehydrogenase (UDPGD) activity, which is also increased in mature cartilage. We also show that IL-1alpha treatment reduces both total GAG and CS synthesis, decreases C6S:C4S ratios (less C6S), but fails to modify chondrocyte UDPGD activity at all ages. On the other hand, TGF-beta1 increases total GAG synthesis in immature, but not mature, cartilage (stimulates CS but not non-CS), age-independently decreases C6S:C4S (more C4S), and increases chondrocyte UDPGD activity in a manner inversely correlated with age. Our findings show that TGF-beta1, but not IL-1alpha, modifies matrix synthesis such that its composition more closely resembles "less mature" articular cartilage. These effects of TGF-beta1, which appear to be restricted to periods of skeletal immaturity, are closely associated although not necessarily mechanistically linked with increases in chondrocyte UDPGD activity. The antianabolic effects of IL-1alpha are, on the other hand, likely to be independent of any direct modification in UDPGD activity and manifest equally in human cartilage of all ages.

Hypoxia differentially enhances the effects of transforming growth factor-beta isoforms on the synthesis and secretion of glycosaminoglycans by human lung fibroblasts.

Interstitial lung diseases associated with hypoxia, such as lung fibrosis, are characterized by enhanced production of transforming growth factor-beta (TGF-beta) and increased deposition of extracellular matrix (ECM) molecules, including glycosaminoglycans (GAGs). In this study, we investigated the effect of hypoxia (3% O(2)) on TGF-beta-induced GAG synthesis by primary human pulmonary fibroblasts, established from lung biopsies. Total GAG synthesis was assessed by the incorporation of [(3)H]glucosamine into GAGs associated with the cell layer (cells and ECM) or secreted in the medium. GAGs were isolated and purified by gel filtration, fractionated by electrophoresis on cellulose acetate membranes, and characterized using GAG-degrading enzymes. GAG molecules identified in the cell layer and the medium were: hyaluronic acid, and chondroitin, dermatan, and heparan sulfates. All TGF-beta isoforms time dependently induced [(3)H]glucosamine incorporation into GAGs of the cell layer or the medium. Characterization of individual GAG molecules indicated that this was attributed to dermatan and heparan sulfates in the cell layer and to hyaluronic acid and chondroitin and dermatan sulfates in the medium. Hypoxia enhanced the effect of all TGF-beta isoforms, particularly that of TGF-beta3, on the secretion of hyaluronic acid and chondroitin and dermatan sulfates. In the cell layer, hypoxia stimulated only the effect of TGF-beta2-induced [(3)H]glucosamine incorporation into GAGs. Our data indicate that hypoxia differentially enhances the effect of TGF-beta isoforms on the secretion and deposition of GAGs and may hasten ECM remodeling associated with the pathogenesis of lung fibrosis.

Myofibroblast Involvement in Glycosaminoglycan Synthesis and Lipid Retention during Coronary Repair

Myofibroblasts of adventitial origin have been linked to neointimal formation and remodeling after coronary injury. Accordingly, the goal of this study was to examine whether myofibroblasts contribute to focal accumulation of glycosaminoglycans (GAGs) and lipids during coronary repair. GAG synthesis was assessed by ex vivo labeling of balloon-injured porcine coronary arteries with ¹⁴C-glucosamine. The synthesis of total GAGs transiently increased at 8 days in the normolipemic model (a 2.2-fold increase over baseline, p < 0.05). The majority of newly synthesized GAGs were sensitive to chondroitin ABC lyase (chondroitin/dermatan sulfate GAGs). Versican was localized to myofibroblast-rich regions in the adventitia and neointima [positive for α-smooth muscle (SM) actin, negative for h-caldesmon and SM myosin heavy chain]. In contrast, the adjacent SM-rich media showed no increase in versican expression. The association between injury-induced GAG accumulation and lipid retention was examined at 2 weeks after coronary injury in the hyperlipemic model. Lipid (Oil Red O) accumulated in the neointima and adventitia, but not in the adjacent media. Coronary repair under hyperlipemic conditions was associated with macrophage infiltration (19 ± 5 vs. 3 ± 2% of neointimal cells in normolipemic animals, p < 0.001) and increased neointimal formation (1.8 ± 0.5 vs. 1.0 ± 0.3 mm² in normolipemic animals, p < 0.01). In conclusion, this study demonstrated a transient increase in GAG synthesis following coronary injury. Chondroitin sulfate proteoglycans (e.g., versican) were rapidly synthesized by activated adventitial and neointimal cells which could contribute to early lipid retention in injured vessels.

Verminderte Stimulation der Glycosaminoglycansynthese von kultivierten humanen Hautfibroblasten im Rahmen der In-vitro-Alterung durch Interleukin-1

Addition of human recombinant interleukin-1 (IL-1 alpha or IL-1 beta) to culture medium (supplemented MEM without or with 1%, 10%, or 20% fetal calf serum (FCS)) of human skin fibroblasts exerted a stimulating effect in a dose-dependent manner on glycosaminoglycan (GAG) synthesis, including hyaluronic acid synthesis, of young (Phase II) skin fibroblasts in concentrations of 4, 20, or 100 pg/ml. Stimulation was due to increase in total GAGs, namely extracellular (secreted into culture medium) and cell-bound (pericellular) GAGs. Stimulation with 100 or 200 pg/ml IL-1 beta led to a relative increase (total GAGs: 100%) in hyaluronic acid from 49% to 64 (-FCS) and from 79% to 92% (+10% FCS), respectively. The increase in total GAG synthesis was mainly due to increased synthesis of hyaluronic acid. Maximum stimulation, with and without FCS, was reached by 100 pg/ml IL-1 beta. Compared to young (Phase II) cells senescent (Phase III) cells showed significantly diminished stimulation of hyaluronic acid synthesis by IL-1 beta. Both, IL-1 alpha and IL-1 beta, showed no influence on DNA synthesis. These results suggest that both, IL-1 beta and IL-1 alpha, stimulate GAG and especially hyaluronic acid synthesis, but not DNA synthesis, in vitro. The diminished response of GAG and hyaluronic acid synthesis during ageing of these in vitro cultured fibroblasts gives support to the hypothesis that similar processes might occur during ageing in organisms.

Stimulation of glycosaminoglycan synthesis in cultured fibroblasts by hyperbaric oxygen

In this study we examined the effect of hyperbaric oxygen treatment on the synthesis of glycosaminoglycans by fibroblasts isolated from wounds and normal skin. Fibroblast cultures were exposed to seven treatments of intermittent hyperbaric oxygen, and then metabolically labelled with D-[6-(3)H] glucosamine. Hyaluronic acid and proteoglycan synthesis were determined by measuring the radioactivity precipitated with cetylpyridinium chloride before and after digestion with hyaluronidase. Hyperbaric oxygen treatment resulted in an increased synthesis of hyaluronic acid and proteoglycans by fibroblasts from wounds and normal skin. Overall, the average increase in total glycosaminoglycan synthesis after hyperbaric oxygen treatment was 28%, whereas fibroblast proliferation was decreased by 7%. These results suggest that one of the effects of this treatment on a wound may be to increase the ratio of extracellular matrix to cells. Such a change could have important consequences for cellular activities essential for effective wound healing, such as migration of cells into the wound and control of cell function.

Effects of All-Trans Retinoic Acid on Glycosaminoglycan Synthesis in Photodamaged Hairless Mouse Skin

All-trans retinoic acid (tRA) was previously shown to be active in wrinkle reduction in the hairless mouse photoaging model. To address the questions of whether tRA also alters glycosaminoglycan (GAG) synthesis and whether observed wrinkle effacement can be attributed to changes in total GAG production, the effects of tRA on de novo GAG synthesis were examined in this model. Sulfated glycosaminoglycans (S-GAG) and the non-sulfated hyaluronic acid (HA) labeled with [3H]-glucosamine or [35S]-sulfate were found to diffuse differentially into the medium during the labeling period in the control animals (9% and 35% of total incorporated label for HA and S-GAG, respectively). Furthermore, the diffusion of HA into media was significantly changed after tRA treatment (from 9% to 24%), but no alteration was observed in the diffusion of S-GAG. Separation of epidermis and dermis indicated that the additional HA in medium after retinoid treatment primarily originated from the dermis. When incorporated label from the medium and skin fractions was combined, both labeling protocols revealed that 10 weeks of tRA treatment did not increase the total (medium plus skin) de novo synthesis of either HA or S-GAG. Wrinkle effacement as induced by retinoids in the photodamaged mouse skin therefore can not be related to an increased total GAG synthesis.

Temporal and spatial differences in glycosaminoglycan synthesis by fetal lung fibroblasts

In studies of the ontogeny of flbroblast-epithelial interactions during late fetal lung rat lung development, we have identified two subpopulations of fibroblasts which differed in their ability to promote epithelial cell proliferation or differentiation. As glycosaminoglycans (GAGs) have been implicated in the regulation of these processes we have tested whether the two fibroblast populations synthesize different GAGs and whether the GAG pattern changes with development. Fibroblasts incorporate more [ 3H]glucosamine and Na 2, 35SO 4 into GAGs than epithelial cells. Both cell types deposited a significant amount of newly synthesized GAGs in the cell-matrix layer. GAGs were lost faster from the cell-matrix layer of fibroblasts ( t 1 2 = 12 h ) than from that of epithelial cells ( t 1 2 = 48 h ). Total GAG synthesis by fibroblasts did not change with advancing gestation, but synthesis of sulfated GAGs by epithelial cells declined with advancing gestation. Independent of gestational age epithelial cells synthesized predominantly heparan sulfate. Depending on their proximity to the epithelium, fibroblasts differed in their production of GAGs. Fibroblasts in close proximity to the epithelium mainly produced and secreted hyaluronan. More distant fibroblasts, from the pseudoglandular stage of lung development synthesized primarily heparan sulfate and chondroitin sulfate. This same population of fibroblasts from the canalicular stage of lung development, produced more hyaluronan. As the shift to hyaluronan occurs with the thinning of the alveolar septal wall, this finding suggests that developmentally regulated GAG production by fibroblasts may facilitate epithelial-fibroblast interaction, thus influencing fetal lung growth and differentiation.

Stimulation of sulfated glycosaminoglycan synthesis by the tripeptide-copper complex Glycyl-L-histidyl-L-lysine-Cu 2+

Glycyl-L-histidyl-L-lysine-copper (II) complex (GHKCu) is a naturally occuring tripeptide with potential healing properties. We studied the effect of GHKCu on the synthesis of glycosaminoglycans (GAGs) by normal human fibroblasts in culture. Cells were incubated with 3H glucosamine and 35S sulfate and the radioactivity of isolated GAGs was determined. GHKCu induced a dose-dependent increase of the synthesis of total GAGs secreted into the culture medium and those associated with the cell layer. The effect of GHKCu was biphasic with a maximal stimulation at 10 −9 to 10 −8 M. At higher concentrations, the rate of synthesis returned progressively to that of controls culture. Electrophoretic analysis of the different GAG populations showed that GHKCu preferentially stimulated the synthesis of extracellular dermatan sulfate and cell layer associated heparan sulfate. No influence of GHKCu on the synthesis of hyaluronic acid was observed. GHKCu stimulation of GAG synthesis may be one of the phenomenons implicated in the wound healing properties of the peptide.

Transforming growth factor-β modulation of glycosaminoglycan production by mesenchymal cells of the developing murine secondary palate

Development of the mammalian secondary palate requires proper production of the extracellular matrix, particularly glycosaminoglycans (GAGs) and collagen. Endogenous factors that regulate the metabolism of these molecules are largely undefined. A candidate for a locally derived molecule would be transforming growth factor β 1(TGF β 1) by virtue of its potency as a modulator of extracellular matrix metabolism by several cell lines. We have thus attempted to assign a regulatory role for TGF β 1 in modulation of GAG production and degradation by mesenchymal cells of the murine embryonic palate (MEPM). Treatment with TGF β 1 or TGF β 2, but not IGF-II, resulted in a stimulation of total GAG synthesis. Furthermore, cells treated with bothTGF β 1 and TGFα showed a synergistic increase in GAG synthesis if pretreated with TGF β 1 but not TGFα. Simultaneous stimulation with TGF β 1 and TGF β 2 did not elicit a synergistic response. These studies demonstrate the ability of TGFβ, synthesized by embryonic palatal cells, to specifically stimulate GAG synthesis by MEPM cells. Other growth factors present in the developing craniofacial region may also modulate TGFβ-induced GAG synthesis, a biosynthetic process critical to normal development of the embryonic palate.

Modulation of extracellular matrix metabolism in rabbit articular chondrocytes and human rheumatoid synovial cells by the non-steroidal anti-inflammatory drug etodolac. II: Glycosaminoglycan synthesis

The effect of Etodolac on glycosaminoglycan (GAG) synthesis by human synovial cells and rabbit articular chondrocytes in culture was studied at doses ranging from 0.01 to 10 micrograms/ml. In chondrocyte cultures, short-term exposure to Etodolac decreased the total amount of GAGs (mainly chondroitin sulfate) affecting essentially the cell layer-associated fraction. When cells were incubated with Interleukin-1 (IL-1), Etodolac still exerted its inhibiting effect on GAG synthesis. Long-term exposure of chondrocytes to Etodolac caused a diminution of the total GAG synthesis for the highest concentration studied (1 micrograms/ml). IL-1 alone or in combination with Etodolac decreased the synthesis of GAGs suggesting that pretreatment with Etodolac cannot prevent the action of IL-1. In synoviocyte cultures, the drug caused an inhibition of the GAG biosynthesis (mainly hyaluronic acid). In presence of IL-1, Etodolac at the highest concentration partially diminished the stimulatory effect of IL-1. A 8-day exposure of the cells to Etodolac led to a decrease of GAG synthesis. In the same experiment, IL-1 alone caused a slight increase of GAG production that was not abolished by Etodolac treatment.

Effect of Transforming Growth Factor Beta on Synthesis of Glycosaminoglycans by Human Lung Fibroblasts

The processes of lung growth, injury, and repair are characterized by alterations in fibroblast synthesis and interstitial distribution of extracellular matrix components. Transforming growth factor beta (TGF-beta), which is postulated to play a role in modulating lung repair, alters the distribution of several matrix components such as collagen and fibronectin. We studied the effect of TGF-beta on the synthesis and distribution of the various glycosaminoglycans (GAGs) and whether these effects may explain its role in lung repair. Human diploid lung fibroblasts (IMR-90) were exposed to various concentrations of TGF-beta (0-5 nM) for variable periods of time (0-18 h). Newly synthesized GAGs were labeled with either [3H]glucosamine or [35S]sulfate. Individual GAGs were separated by size exclusion chromatography after serial enzymatic and chemical digestions and quantitated using scintillation counting. There was a dose-dependent increase in total GAG synthesis with maximal levels detected after 6 h of exposure. This increase was noted in all individual GAG types measured and was observed in both the cell associated GAGs (cell-matrix fraction) as well as the GAGs released into the medium (medium fraction). In the cell-matrix fraction, TGF-beta increased the proportion of heparan sulfate that was membrane bound as well as the proportion of dermatan sulfate in the intracellular compartment. In the medium fraction, TGF-beta increased the proportion of hyaluronic acid, chondroitin sulfate and dermatan sulfate released. We conclude that the role of TGF-beta in lung growth and repair may be related to increased synthesis of GAGs by human lung fibroblasts as well as alterations in the distribution of individual GAGs.

Exogenous glycosaminoglycans modulate extracellular and intracellular accumulation of newly synthesized glycosaminoglycans by embryonic chick skin fibroblasts

The effects of exogenous glycosaminoglycans (GAG) on newly synthesized GAG accumulation in intra- and extra-cellular compartments of 17 incubation days chick embryonic skin fibroblasts have been examined. Exogenous GAG are able to act on both total GAG synthesis and secretion by embryonic fibroblasts and on the relative concentration of individual GAG. A decline in hyaluronic acid and an increase in chondroitin sulfate plus dermatan sulfate in the cellular compartment for all GAG administered have been detected. There was also similar behaviour in the extracellular compartment after sulfated GAG administration.

Changes in the biosynthesis of glycosaminoglycans in polymorphonuclear leukocytes in relation to their induction into the peritoneal cavity.

The synthesis and function of dermatan sulfate in peritoneal polymorphonuclear (PMN) leukocytes were studied. The peritoneal PMN leukocytes were obtained at 4,8, and 16 h after guinea pigs were injected intraperitoneally with caseinate solution, and were incubated with [35S] sulfate or [3H] glucosamine on plastic. The total glycosaminoglycan synthesis and the proportion of dermatan sulfate to total glycosaminoglycans linearly increased with time. In order to clarify the function of the increased dermatan sulfate, peritoneal PMN leukocytes were cultured with [35S] sulfate on plastic or collagen gel. The total glycosaminoglycan synthesis on the collagen gel culture increased 1.5 times compared with that on the plastic culture, and especially, dermatan sulfate synthesis increased twofold. In addition, 65% of dermatan sulfate on the collagen gel culture was found in the cell and the collagenase-soluble fractions. These results indicate that proteodermatan sulfate synthesized by PMN leukocytes interacts with collagen in vitro, and suggest that PMN leukocytes, which migrated to the inflammatory locus, lastly adhere to the injured tissue through the interaction of proteodermatan sulfate synthesized by themselves with collagen fibers exposed in the inflammatory locus.

Proteochondroitin sulfate is the main proteoglycan synthesized in fetal hepatocytes.

The synthesis of total and specific types of glycosaminoglycans (GAG) with emphasis on proteochondroitin sulfate (PCS) was studied in late embryonic and early postnatal liver parenchymal cells. In contrast to adult hepatocytes, which synthesize almost exclusively proteoheparan sulfate (PHS), PCS proved to be the major type of GAG synthesized in fetal hepatocytes (more than 60% of total GAG) whereas PHS contributes less than 40% of total GAG synthesis. Starting immediately after birth PCS synthesis in hepatocytes declines progressively, at the 6th postnatal day PCS formation is one-fifteenth of that measured in embryonic liver cells. Adult levels are reached around the 10th postnatal day. A significant portion of plasma membrane-associated proteoglycans in fetal hepatocytes is represented by PCS, its fraction declines in early postnatal life. Between the synthesis rate of PCS and [3H]thymidine incorporation into DNA exists a strong positive statistic correlation (r = 0.949). In conclusion, fetal hepatocytes have a completely different profile of GAG synthesis characterized by preponderant production of PCS. This ability is lost early after birth but might be regained in hepatocellular carcinoma cells and parenchymal cells in chronically injured liver tissue developing fibrosis.

Developmental changes of proteoglycan synthesis in rat liver and isolated hepatocytes

The synthesis of sulfated proteoglycans in late fetal (19th to 22nd day of intrauterine life), early postnatal, and adult liver tissue as well as in hepatocytes and their distribution in plasma membranes were studied. Overall proteoglycan production is enhanced two-fold in fetal as compared with adult liver tissue. In contrast to slices from adult liver, in which the synthesis of heparan [ 35S]-sulfate comprises more than 80% and chondroitin sulfate less than 5% of total glycosaminoglycans, chondroitin [ 35S]sulfate is the major type of glycosaminoglycans synthesized in fetal liver representing about 50% of total sulfated glycosaminoglycans. Thus, the synthesis of chondroitin sulfate is elevated nearly 30-fold in fetal liver as compared with the adult counterpart. Immediately after birth chondroitin sulfate formation decreases rapidly reaching adult levels between the 10th and 15th day of postnatal life. The production of heparan sulfate is almost unchanged during perinatal liver development due to a relatively low fractional synthesis of heparan [ 35S]sulfate in fetal liver. Hepatocytes were identified as the cell type responsible for elevated chondroitin sulfate production in fetal liver. Erythroblasts, which synthesize chondroitin sulfate, contribute less than 10% to total glycosaminoglycan synthesis in embryonic liver. Plasma membranes of adult liver contain exclusively heparan sulfate whereas in neonatal liver cell membranes 25% of labeled glycosaminoglycans is represented by chondroitin sulfate, a fraction which decreases rapidly after birth. In parallel to the postnatal shut down of chondroitin sulfate synthesis the activity of the UDPxylose:coreprotein xylosyltransferase (EC. 2.4.2.26) decreases from 4.8±0.5 dpm/h per μg protein to 0.3±0.1 dpm/h per μg protein suggesting a regulatory function of the enzyme for proteochondroitin sulfate synthesis in developing liver. The formation of both heparan sulfate and chondroitin sulfate is dependent on functioning protein synthesis, which indicates, together with double labeling experiments using [ 3H]serine and [ 14C]glucosamine as isotopic precursors, their synthesis as proteoglycans. The positive correlation ( r − 0.949) between the incorporation of [ 3H]thymidine into DNA and chondroitin [ 35S]sulfate production supports the assumption of a cell growth promoting activity of chondroitin sulfate and points to a significant role of the glycosaminoglycan in the process of cellular proliferation and tissue differentiation.

Proteoglycan and glycosaminoglycan synthesis in embryonic mouse salivary glands: effects of beta-D-xyloside, an inhibitor of branching morphogenesis.

The proteoglycans and glycosaminoglycans synthesized by embryonic mouse salivary glands during normal morphogenesis and in the presence of beta-xyloside, an inhibitor of branching morphogenesis, have been partially characterized. Control and rho-nitrophenyl-beta-D-xyloside-treated salivary rudiments synthesize proteoglycans that are qualitatively similar, based on mobility on Sepharose CL-4B under dissociative conditions and glycosaminoglycan composition. However, beta-xyloside inhibits total proteoglycan-associated glycosaminoglycan synthesis by 50%, and also stimulates synthesis of large amounts of free chondroitin (dermatan) sulfate. This free glycosaminoglycan accounts for the threefold stimulation of total glycosaminoglycan synthesis in beta-xyloside-treated cultures. Several observations suggest that the disruption of proteoglycan synthesis rather than the presence of large amounts of free glycosaminoglycan is responsible for the inhibition of branching morphogenesis. (a) We have been unable to inhibit branching activity by adding large amounts of chondroitin (dermatan) sulfate, extracted from beta-xyloside-treated cultures, to the medium of salivary rudiments undergoing morphogenesis. (b) In the range of 0.1-0.4 mM beta-xyloside, the dose-dependent inhibition of branching morphogenesis is directly correlated with the inhibition of proteoglycan synthesis. The stimulation of free glycosaminoglycan synthesis is independent of dose in this range, since stimulation is maximal even at the lowest concentration used, 0.1 mM. The data strongly suggest that the inhibition of branching morphogenesis is caused by the disruption of proteoglycan synthesis in beta-xyloside-treated salivary glands.

Influence of calcium concentration in the medium on the cell proliferation and glycosaminoglycan synthesis of human diploid skin fibroblasts in culture

Changes of the glycosaminoglycan (GAGs) fractions in the medium, the cell surface of human diploid skin fibroblasts and the relation between GAGs synthesis and cell growth in cultured media containing various Ca++ concentrations were investigated by means of electrophoretic fractionation. The results demonstrated that the synthesis of the hyaluronic acid (HA) fraction was increased with increase of the Ca++ concentration in the medium, and this change appeared to be linked to the condition of cell growth. The synthesis of the heparan sulfate and dermatan sulfate (HS + DS) fraction was decreased with increase of the Ca++ concentration in the medium, and this change depended largely on the Ca++ concentration in the medium rather than the cell growth. The synthesis of the chondroitin sulfate (Ch-s) fraction was also decreased with increase of the Ca++ concentration in the medium. However, this change was neither controlled especially by the Ca++ concentration in the medium nor by the condition of cell growth, but reflected the decrease of total GAGs synthesis by the increase of the Ca++ concentration in the medium.

The synthesis and localization of glycosaminoglycans in striated muscle differentiating in cell culture.

AbstractDay 3 mass muscle cultures incorporated 35SO4= and 3H· glucosamine into specific glycosaminoglycans that were identified by a variety of methods (enzyme susceptibility, nitrous acid degradation, electrophoresis, thin layer chromatography). More than 75% of the newly synthesized glycosaminoglycans appeared in the medium. In the medium, the glycosaminoglycans consisted of primarily hyaluronic acid (∼35%) and chondroitin sulfate (∼40%) with smaller amounts of chondroitin (∼15%) and heparan sulfate (∼10%). Among the glycosaminoglycans that remained in the cell layer, chondroitin sulfate (∼45%) and heparan sulfate (20‐40%) predominated and only a small amount of chondroitin (∼15%) and hyaluronic acid (∼5%) were present. In both the medium and cell layer, greater than 80% of the chondroitin sulfate was chondroitin‐6‐sulfate. Although mass cultures contain some non‐muscle cell types which undoubtedly contributed to total glycosaminoglycan synthesis, susceptibility of the autoradiographic grains associated with cells in muscle clones to testicular hyaluronidase digestion suggests that at least some glycosaminoglycans (chondroitin sulfate, in particular) were synthesized by muscle cells.Transmission electron microscopy revealed the presence of ruthenium red staining globular units along the surfaces of myotubes. Destruction of the integrity of surface associated ruthenium red stained material by both Streptomyces hyaluronidase and testicular hyaluronidase suggests that both hyaluronic acid and chondroitin sulfate are present on muscle cell surfaces.