Chondroitin sulfate proteoglycan synthesis and reutilization of beta-D-xyloside-initiated chondroitin/dermatan sulfate glycosaminoglycans in fetal kidney branching morphogenesis

Branching morphogenesis and chondroitin sulfate proteoglycan synthesis by explanted fetal mouse kidneys were previously shown to be inhibited by p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside) while glomerular development and heparan sulfate proteoglycan synthesis were unaffected. The metabolic fate of fetal kidney explant proteoglycans was investigated to determine whether or not recovery of proteoglycan synthesis and morphogenesis occur after exposure to beta-D-xyloside. Chondroitin sulfate proteoglycan synthesis resumed within 4 hr of removal of beta-D-xyloside and was enhanced once beta-D-xyloside-initiated chondroitin/dermatan-35SO4 glycosaminoglycans (GAGs) were released from the tissue. Radioactivity incorporated into beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs during labeling in the presence of beta-D-xyloside was reutilized in the synthesis of chondroitin-35SO4 proteoglycan during a 24-hr chase in nonradioactive medium without beta-D-xyloside. Further, highly purified beta-D-xyloside-initiated chondroitin/dermatan-35SO4 GAGs were taken up by kidneys more avidly than was free [35S]sulfate. These 35S-GAGs were degraded and reutilized in the synthesis of chondroitin-35SO4 proteoglycan. Ureteric bud branching resumed 48 hr after beta-D-xyloside was removed from the incubation medium. These findings support the idea that both chondroitin sulfate proteoglycan synthesis and proteoglycan processing may be involved in branching morphogenesis.     

Heparin and heparan sulfate delimit nephron formation in fetal metanephric kidneys

Formation of nephrons from primitive mesenchyme in fetal kidneys is induced by ureteric buds. Nephron induction is closely coordinated with branching morphogenesis of the ureteric bud. Having previously shown that branching of the primitive ureter is associated with de novo synthesis of chondroitin sulfate proteoglycan and release of free heparan sulfate glycosaminoglycan chains, we asked whether glycosaminoglycans influence nephron development. Fetal mouse kidneys were incubated in organ cultures containing heparan sulfate, heparin, chondroitin sulfate, or hyaluronate. After 48 hr the number of nephrons at each developmental stage was enumerated by light microscopic analysis of serial tissue sections. Kidneys incubated in heparin or in heparan sulfate contained up to 10-fold fewer nephrons than did kidneys incubated in control conditions or in chondroitin sulfate or hyaluronic acid. Maturation of nephrons, however, was unaffected. Inhibition of nephron development was associated with binding of labeled heparin to primitive mesenchyme and altered tissue distribution of fibronectin. Branching morphogenesis was impaired in kidneys exposed to heparin but not to heparan sulfate or to de-N-sulfated, N-acetylated heparin. The capacity of glycosaminoglycans to inhibit nephron formation depended on sugar composition and O-sulfation but not GAG chain size or charge density. Thus, heparan sulfate may have the capacity to specifically control formation of nephrons in fetal metanephric kidneys in vitro.     

Effect of beta-D-xyloside on the glomerular proteoglycans. I. Biochemical studies

The effect of p-nitrophenyl-beta-D-xylopyranoside on glomerular extracellular matrices (glomerular basement membrane and mesangial matrix) proteoglycans was studied. The proteoglycans of rat kidneys were labeled with [35S]sulfate in the presence or absence of beta- xyloside (2.5 mM) by using an isolated organ perfusion system. The proteoglycans from the glomeruli and perfusion medium were isolated and characterized by Sepharose CL-6B chromatography and by their behavior in CsCl density gradients. With xyloside treatment there was a twofold decrease in 35S-labeled macromolecules in the tissues but a twofold increase in those recovered in the medium as compared with the control. The labeled proteoglycans extracted from control kidneys eluted as a single peak with Kav = 0.25 (Mr = approximately 130,000), and approximately 95% of the radioactivity was associated with heparan sulfate proteoglycan (HS-PG), the remainder with chondroitin (or dermatan) sulfate proteoglycan (CS-PG). In the xyloside-treated kidneys, the proteoglycans extracted from the tissue eluted as two peaks, Kav = 0.25 (Mr = approximately 130,000) and 0.41 (Mr = approximately 46,000), which contained approximately 40 and approximately 60% of the total radioactivity, respectively. The first peak contained mostly the HS-PG (approximately 90%) while the second peak had a mixture of HS-PG (approximately 70%) and CS-PG (approximately 30%). In controls, approximately 90% of the radioactivity, mostly HS-PG, was confined to high density fractions of a CsCl density gradient. In contrast, in xyloside experiments, both HS- PG and CS-PG were distributed in variable proportions throughout the gradient. The incorporated 35S activity in the medium of xyloside- treated kidneys was twice that of the controls and had three to four times the amount of free chondroitin (or dermatan) sulfate glycosaminoglycan chains. The data suggest that beta-xyloside inhibits the addition of de novo synthesized glycosaminoglycan chains onto the core protein of proteoglycans and at the same time stimulates the synthesis of chondroitin or dermatan sulfate chains which are mainly discharged into the perfusion medium.     

1,25-Dihydroxyvitamin D3 inhibits synthesis and enhances degradation of proteoglycans in osteoblastic cells

The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), an active form of vitamin D3, on the metabolism of proteoglycans by an osteoblastic cell line MC3T3-E1 were studied. Cells metabolically labeled with [35S]sulfate and/or [3H]glucosamine synthesized large and small dermatan sulfate proteoglycans and heparan sulfate proteoglycan. The incorporation of [35S]sulfate into proteoglycans for 1 h was reduced by 1,25-(OH)2D3 in a dose-dependent manner with a maximum reduction of 40% obtained at 10(-8)M 1,25-(OH)2D3. This effect was observed for all the proteoglycans with the decrease for the large dermatan sulfate proteoglycan most prominent. Treatment with 1,25-(OH)2D3 did not influence the degree of sulfation nor the molecular size of the glycosaminoglycan chains. Thus, the change in the incorporation of [35S] sulfate reflects net change in the synthesis of proteoglycans. When cells were treated with beta-D-xyloside, 1,25-(OH)2D3 also inhibited net synthesis of dermatan sulfate glycosaminoglycan chains on this exogenous substrate suggesting that it decreases the capacity of the cells for glycosaminoglycan synthesis. The incorporation of [3H]glucosamine into hyaluronic acid was also inhibited up to 70% by 10(-8) M 1,25-(OH)2D3. Treatment with 24,25-dihydroxyvitamin D3 did not cause significant changes in the proteoglycan synthesis. Degradation of proteoglycans associated with the cell layer was enhanced by treatment with 1,25-(OH)2D3 at 10(-8) M. Proteoglycans exogenously added to the culture were also degraded with a cell-mediated process which was stimulated by treatment with 10(-8) M 1,25-(OH)2D3. These results demonstrate that 1,25-(OH)2D3 reduces the synthesis and stimulates the degradation of proteoglycans in osteoblastic cells in culture.     

Sulfated glycosaminoglycan deposition and processing at the basal epithelial surface in branching and beta-D-xyloside-inhibited embryonic salivary glands

We investigated whether the inhibition of proteoglycan synthesis and salivary branching morphogenesis by beta-D-xyloside was related to the deposition and processing of newly synthesized glycosaminoglycans at the basal epithelial surface that correlates with normal branching activity. Forty eight-hour cultures of control and 0.5 mM beta-xyloside-treated submandibular rudiments were labeled for 2 hr with [35S]sulfate and fixed and processed for autoradiography, immediately or after 2, 4, 6, or 8 hr of postlabeling chase in nonradioactive medium. The data demonstrated that deposition of chondroitin sulfate-rich material at the basal epithelial surface was strikingly reduced in beta-xyloside-treated rudiments, while patterns of label loss during postlabeling chase were not altered.     

Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures

The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of [35S]sulfate and [3H]glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on [35S]sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on [35S]sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased [3H]thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.     

Role of proteoglycans in renal development

The role of proteoglycans (PGs) in morphogenesis was investigated. Fetal kidneys were obtained from 13-day-old mouse embryos and maintained for 7 days in culture. The biosynthesis of PGs was perturbed by addition of p-nitrophenyl-beta-D-xylopyranoside in the culture medium. The kidneys were processed for morphological and biochemical studies. The morphological studies included staining of tissues with anti-basement membrane antibodies and ruthenium red. [35S]sulfate was used as the precursor product for biosynthetic and autoradiographic studies. The kidneys treated with xyloside had loose mesenchyme, inhibition of ureteric bud branching, diminution in the population of developing nephron elements, decreased immunofluorescence with anti-proteoglycan antibodies and staining with ruthenium red, and a reduced [35S]sulfate incorporation into poorly organized extracellular matrices. The biochemical studies included characterization of PGs/glycosaminoglycans (GAGs) by Sepharose CL-4B, -6B, and DEAE-Sephacel chromatographies and cellulose acetate electrophoresis. Under the influence of xyloside, the total radioactivities decreased 2 to 4-fold in tissues and increased 18 to 42-fold in media fractions. A reduction in the size of macromolecular form of PGs, i.e., from MW approximately 2.5 X 10(6) to approximately 2.5 X 10(4), was noted. The PGs/GAGs synthesized were mainly made up of heparan sulfate and small amounts of chondroitin sulfate. They eluted at a lower salt concentration as compared to the controls. A similar diminution in the size of media PGs, i.e., from MW approximately 1.8 X 10(5) to approximately 2.8 X 10(4), was observed. Additional studies with [3H]xyloside indicated that the chains initiated on xyloside residues were similar in size and composition to GAG-chains. These findings indicate that a perturbance in the biosynthesis of PGs/GAGs leads to abnormalities in renal organogenesis.     

Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride

Cultured human melanoma M21 cells were treated with diethylcarbamazine (DEC), an inhibitor of proteoglycan biosynthesis in rat chondrosarcoma cells, to examine the assembly and transport of a chondroitin sulfate proteoglycan to the plasma membrane. Pretreatment of melanoma cells at 37 degrees C for 15 min with increasing doses of DEC followed by a 60-min pulse with [35S]sulfate in the presence of DEC resulted in a dose-related inhibition of incorporation of [35S]sulfate into macromolecules. In cells incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, synthesis and secretion of beta-D-xyloside-bound 35S-glycosaminoglycans were inhibited by more than 80% as compared to cells treated with beta-D-xyloside alone; this inhibition was reversible. As assessed by [3H]serine incorporation into protein, overall protein synthesis was not substantially inhibited by DEC treatment. Detergent lysates from [35S]methionine-labeled melanoma cells were incubated with a monoclonal antibody (9.2.27) that specifically recognizes the peptide core of the melanoma proteoglycan. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate, a 240,000 Mr endoglycosidase H (Endo-H)-sensitive intermediate was the only form of the proteoglycan present inside the cells when the cultures were treated for 60-120 min with 10-15 mM DEC. When the melanoma cells were incubated for 10 min with 15 mM DEC and 100 mu Ci/ml of [35S]methionine, washed, and then chased for 15 min to 4 h in radioactive-free medium, the 240,000 Mr Endo-H-sensitive intermediate was slowly converted to a 250,000 Endo-H-resistant intermediate but not to a mature proteoglycan molecule that possessed chondroitin sulfate glycosaminoglycans. SDS-PAGE analysis of cell surface immunoprecipitates revealed that only a small amount of the 250,000 Mr intermediate was transported to the plasma membrane within 5 h of incubation in the presence of DEC. Proteoglycan synthesis was also inhibited when the melanoma cells were incubated for 60-120 min with ammonium chloride, but unlike DEC-treated cells the majority of the synthesized peptide core was converted to a 245,000 Mr Endo-H-resistant intermediate that was detected on the cell surface. Light and electron microscopic analysis of DEC-treated melanoma cells revealed large vacuoles and a distended Golgi and endoplasmic reticulum. Ammonium chloride-treated cells contained fewer vacuoles than DEC-treated cells but more vacuoles than normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of vanadate on cartilage-matrix proteoglycan synthesis in rabbit costal chondrocyte cultures

The effect of vanadate on proteoglycan synthesis by cultured rabbit costal chondrocytes was examined. Rabbit chondrocytes were seeded at low densities and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of 4 microM vanadate to the culture medium induced a morphologic differentiation of the fibroblastic cells to spherical chondrocytes, and increased by two- to threefold incorporation of [35S]sulfate and [3H]glucosamine into large, chondroitin sulfate proteoglycans. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, in that chemical analyses showed increases in the accumulation of macromolecules containing hexuronic acid and hexosamine in vanadate-maintained cultures. However, vanadate had only a marginal effect on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant material. These results provide evidence that vanadate selectively stimulates the synthesis of proteoglycans characteristically found in cartilage by rabbit costal chondrocyte cultures.     

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.     

伴刀豆蛋白A及其衍生物对培养软骨细胞蛋白多糖代谢的影响

观察了ＣｏｎＡ对培养软骨细胞ＰＧ合成代谢的影响。证实ＣｏｎＡ能够使培养的软骨细胞高分子硫酸化ＰＧ的合成增加３～４倍,其分子量、硫酸化部位和硫酸化程度与对照组相比无明显差异,是具有正常结构的软骨型ＰＧ。ＣｏｎＡ对低分子型ＰＧ的合成未见明显的影响。ＣｏｎＡ促进ＰＧ合成的作用可由ＭｅＭａｎ完全解除,比具有同样效应的激素、生长因子都强,并有明显的凝集素特异性。推测ＣｏｎＡ的作用可能与软骨细胞膜或细胞内的分化诱导因子的受体或软骨中存在的ＣｏｎＡ软骨细胞分化因子有关。     

Effects of inhibition of proteoglycan synthesis on the differentiation of cultured rat Schwann cells

Schwann cells synthesize two heparan sulfate proteoglycans, one that is a component of the Schwann cell basement membrane and a smaller one that is an integral component of the Schwann cell plasma membrane. To determine the functions of these molecules, Schwann cell-nerve cell cultures were grown in medium containing a specific inhibitor of proteoglycan biosynthesis, 4-methylumbelliferyl-beta-D-xyloside. Treatment with 1 mM beta-D-xyloside caused a 90% reduction in the accumulation of 35SO4-labeled proteoglycans in the cell layer of the cultures. Gel filtration analysis revealed that both the basement membrane and plasma membrane proteoglycans were affected. Inhibition of proteoglycan biosynthesis was accompanied by an inhibition of laminin deposition into extracellular matrix as determined by immunostaining of cultures and by immunoblotting of cell-associated proteins. This occurred even though there was no decrease in the amount of laminin detected in the medium of beta-D-xyloside-treated cultures. Deposition of collagen type IV was similarly affected. In addition, there was no myelin produced in beta-D-xyloside treated cultures. However, when beta-xyloside-treated cultures were supplied with exogenous basement membrane, Schwann cells produced numerous myelin segments. These results indicate that Schwann cell proteoglycans play an essential role in basement membrane assembly, and that the integral plasma membrane proteoglycan is not required for the basement membrane to exert its effects on Schwann cell differentiation.     

Novel inhibition of proteoglycan synthesis and exocytosis by diethylcarbamazine in the Swarm rat chondrocyte

Pretreatment of cultured chondrosarcoma chondrocytes at 37 degrees C for 15 min with 15 mM diethylcarbamazine (DEC) followed by a 60-min pulse with [35S] sulfate in the presence of DEC resulted in an approximate 40% inhibition of synthesis and a 75% inhibition of secretion of 35S-proteoglycan. The inhibition was dose-related and was not due to a decrease in protein synthesis. Chondrocytes exposed for 75 min to 15 mM DEC, washed, incubated for 17 h in DEC-free medium, and then pulsed with [35S]sulfate showed no inhibition in the rate of synthesis of proteoglycan or in the per cent of radiolabeled proteoglycans exocytosed into the culture medium, indicating full reversibility of the inhibitory effect. When chondrocytes were incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, secretion of beta-D-xyloside-bound 35S-glycosaminoglycan was inhibited by more than 70% despite an approximate 3-fold increase in intracellular 35S-macromolecules, as compared to cells exposed to beta-D-xyloside alone. Upon removal of DEC, the block in the secretion of beta-D-xyloside-bound 35S-glycosaminoglycans was reversed, although there was a 15-30-min lag in the initiation of exocytosis. Light and electron microscopic examination of chondrocytes after 75 min of incubation with 15 mM DEC revealed large vacuoles, a distended Golgi apparatus, and a distended endoplasmic reticulum which contained electron dense material. Upon removal of DEC, the vacuoles disappeared and distended organelles returned to their normal appearance between 15 and 30 min, coincident with the start of exocytosis of 35S-proteoglycan and beta-D-xyloside-bound 35S-glycosaminoglycan. These biochemical and morphological studies indicate that DEC treatment of chondrosarcoma chondrocytes alters the transport of molecules from the endoplasmic reticulum to the Golgi and the transport of molecules from the Golgi to the cell surface.     

Partial characterization of proteoglycans synthesized by human glomerular epithelial cells in culture

Confluent adult and fetal human glomerular epithelial cells were incubated for 24 h in the presence of [3H]-amino acids and [35S]sulfate. Two heparan-35SO4 proteoglycans were released into the culture medium. These 35S-labeled proteoglycans eluted as a single peak from anion exchange chromatographic columns, but were separable by gel filtration on Sepharose CL-6B columns. The larger heparan-35SO4 proteoglycan eluted with the column void volume and at a Kav of 0.26 from Sepharose CL-4B columns. The most abundant medium heparan-35SO4 proteoglycan was a high buoyant density proteoglycan similar in hydrodynamic size (Sepharose CL-6B Kav 0.23) to those previously described in glomerular basement membranes and isolated glomeruli. Heparan-35SO4 chains from both proteoglycans were 36 kDa. A smaller proportion of Sepharose CL-6B excluded dermatan-35SO4 proteoglycan was also synthesized by these cells. The predominant protein cores of both medium heparan-35SO4 proteoglycans were approximately 230 and 180 kDa. A hybrid chondroitin/dermatan-heparan-35SO4 proteoglycan with an 80-kDa protein core copurified with the smaller medium heparan-35SO4 proteoglycan. This 35S-labeled proteoglycan appeared as a diffuse, chondroitinase ABC sensitive 155-kDa fluorographic band in sodium dodecyl sulfate-polyacrylamide gels after the Sepharose CL-6B Kav 0.23 35S-labeled proteoglycan fraction was digested with heparitinase. The heparitinase generated heparan sulfate proteoglycan protein cores and the 155-kDa hybrid proteoglycan fragment had molecular weights similar to those previously identified in rat glomerular basement membrane and glomeruli using antibodies against a basement membrane tumor proteoglycan precursor (Klein et al. J. Cell Biol. 106, 963-970, 1988). Thus, human glomerular epithelial cells in culture are capable of synthesizing, processing, and releasing heparan sulfate proteoglycans which are similar to those synthesized in vivo and found in the glomerular basement membrane. These proteoglycans may belong to a family of related basement membrane proteoglycans.     

Long acting cAMP analogues enhance sulfate incorporation into matrix proteoglycans and suppress cell division of fetal rat chondrocytes in monolayer culture.

The relationship between replication and the synthesis of matrix sulfated proteoglycans was investigated with fetal rat chondrocytes grown in monolayer culture. The effect of N6 O2' dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP), adenosine 3', 5' cyclic monophosphate (cAMP), 8 Bromo adenosine 3', 5' cyclic monophosphate (8 Br-cAMP), sodium butyrate and hydroxyurea was examined. Between 0.05 and 0.5 mM DBcAMP, a dose related inhibition of cell division and stimulation of [35SO=/4] incorporation into matrix proteoglycans was demonstrated. At the higher concentrations of DBcAMP, cell division was completely inhibited and the enhancement of [35SO=/4] incorporation into matrix proteoglycans ranged between 40 and 120% (P less than 0.01). Utilizing 14C-glucosamine and photometric determination of proteoglycans with Alcian Blue, it was demonstrated that the increase in sulfate incorporation reflected enhanced accumulation of extracellular matrix. The effects of DBcAMP were mimicked by 8 Br-cAMP, suggesting they were mediated by the adenylyl cyclase system. cAMP (0.05-0.5 mM), sodium butyrate (0.1-0.5 mM) and hydroxyurea (0.5-5 mM) partially or fully inhibited cell division, but either failed or only slightly enhanced sulfate incorporation. The enhanced sulfated proteoglycan deposition promoted by DBcAMP began 8 to 12 hours after serum stimulation, its onset occurred prior to thymidine incorporation and the effect persisted for 28 hours. Determination of cell volume demonstrated an increase in size of DBcAMP treated chondrocytes between 8 to 12 hours, coincident with the onset of increased sulfate incorporation. These results are consistent with a model where matrix sulfated proteoglycan deposition by chondrocytes is mediated by intracellular cAMP levels and occurs in the G1 phase of the cell cycle.     

Three distinct molecular species of proteoglycan synthesized by the rat limb bud at the prechondrogenic stage

To characterize proteoglycans in the prechondrogenic limb bud, proteoglycans were extracted with 4 M guanidine HCl containing a detergent and protease inhibitors from Day 13 fetal rat limb buds which had been labeled with [35S]sulfate for 3 h in vitro. About 90% of 35S-labeled proteoglycans was solubilized under the conditions used. The proteoglycan preparation was separated by DEAE-Sephacel column chromatography into three peaks; peak I eluted at 0.45 M NaCl concentration, peak II at 0.52 M, and peak III at 1.4 M. Peaks I and III were identified as proteoglycans bearing heparan sulfate side chains. The heparan sulfate proteoglycan in peak III was larger in hydrodynamic size than the proteoglycan in peak I. The heparan sulfate side chains of peak III proteoglycan were smaller in the size and more abundant in N-sulfated glucosamine than those of peak I proteoglycan. Peak II contained a chondroitin sulfate proteoglycan with a core protein of a doublet of Mr 550,000 and 500,000. The chondroitin sulfate proteoglycan was easily solubilized with a physiological salt solution and the heparan sulfate proteoglycan in peak I was partially solubilized with the physiological salt solution. The remainder of the proteoglycan in peak I and the heparan sulfate proteoglycan in peak III could be solubilized effectively only with a solution containing a detergent, such as nonanoyl-N-methylglucamide. This observation indicates the difference in the localization among these three proteoglycans in the developing rat limb bud.     

In vivo turnover of the basement membrane and other heparan sulfate proteoglycans of rat glomerulus

The metabolic turnover of rat glomerular proteoglycans in vivo was investigated. Newly synthesized proteoglycans were labeled during a 7-h period after injecting sodium [35S]sulfate intraperitoneally. At the end of the labeling period a chase dose of sodium sulfate was given. Subsequently at defined times (0-163 h) the kidneys were perfused in situ with 0.01% cetylpyridinium chloride in phosphate-buffered saline to maximize the recovery of 35S-proteoglycans. Glomeruli were isolated from the renal cortex and analyzed for 35S-proteoglycans by autoradiographic, biochemical, and immunochemical methods. Grain counting of autoradiographs revealed a complex turnover pattern of 35S-labeled macromolecules, commencing with a rapid phase followed by a slower phase. Biochemical analysis confirmed the biphasic pattern and showed that the total population of [35S]heparan sulfate proteoglycans had a metabolic half-life (t1/2) of 20 and 60 h in the early and late phases, respectively. Heparan sulfate proteoglycans accounted for 80% of total 35S-proteoglycans, the remainder being chondroitin/dermatan sulfate proteoglycans. Whole glomeruli were extracted with 4% 3-[(cholamidopropyl)dimethy-lammonio]-1-propanesulfonate-4 M guanidine hydrochloride, a procedure which solubilized greater than 95% of the 35S-labeled macromolecules. Of these 11-13% was immunoprecipitated by an antiserum against heparan sulfate proteoglycan which, in immunolocalization experiments, showed specificity for staining the basement membrane of rat glomeruli. Autoradiographic analysis showed that 18% of total radioactivity present at the end of the labeling period was associated with the glomerular basement membrane. The glomerular basement membrane [35S]heparan sulfate proteoglycans, identified by immunoprecipitation, have a very rapid turnover with an initial phase, t1/2 = 5 h, and a later phase t1/2 = 20 h.     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.