Synthesis of proteoglycans in rat mucosal keratinocytes

The synthesis of hyaluronic acid and proteoglycans by rat mucosal keratinocytes of an established cell line (CCL-10) has been investigated. Proliferating cultures at or near confluency were grown in the presence of [³⁵S] sulfate or D-[1-³H] glucosamine for 24 h, and the glycosaminoglycan composition of cells and medium was determined. Characterization of the ³⁵S-laballed
glycosaminoglycans showed that heparan sulfate was the major component (∼90%) and that small amounts (∼10%) of galactosaminoglycans had also been synthesized. Analysis of cultures labelled with D-[1-³H] glucosamine demonstrated that hyaluronic acid was also present, most prominently in the medium where approximately one third of the radioactivity in the glycosaminoglycan pool was found in the hyaluronic acid fraction. [³⁵S]-labelled proteoglycans extracted from the cell layer in the presence of protease inhibitors showed substantial heterogeneity upon chromatography on Sepharose CL-6B. In contrast, the proteoglycans in the medium gave a major peak which was eluted at a K_av of 0.28. Gel chromatography of the glycosaminoglycan chains in the latter, isolated after proteolytic digestion, indicated a molecular weight of 17,000.     

Effect of p-nitrophenyl-xyloside on the biosynthesis of proteoglycan in rat ovarian granulosa cells--analyses of glycosaminoglycan synthesis in the Golgi apparatus

Biosynthesis of proteoglycans and glycosaminoglycans in the presence of p-nitrophenyl-xyloside was studied using a primary rat ovarian granulosa cell culture system. Addition of p-nitrophenyl-xyloside into cell culture medium caused about a 700% increase of [35S]sulfate incorporation (ED50 at 0.03 mM) into macromolecules, which included free chondroitin sulfate chains initiated on xyloside and native proteoglycans. Free chondroitin sulfate chains initiated on xyloside were almost exclusively secreted into the medium. The molecular size of chondroitin sulfate chains decreased from 40,000 to 21,000 as the total [35S]sulfate incorporation was enhanced, suggesting that enhanced synthesis of chondroitin sulfate perturbed the normal mechanism of glycosaminoglycan chain termination. Biosynthesis of heparan sulfate proteoglycans was reduced by approximately 50%, likely due to competition at the level of UDP-sugar precursors. [35S]Sulfate incorporation was shut down by the addition of cycloheximide with an initial half time of approximately 2 hr in the presence of xyloside, while that in the absence of xyloside was about 20 min. The difference likely reflects the turnover rate of glycosaminoglycan synthesizing capacity as a whole. The turnover rate of glycosaminoglycan synthesizing capacity observed in ovarian granulosa cells was much shorter than that observed in chondrocytes, reflecting the relative dominance of proteoglycan biosynthetic activity in the total metabolic activity of the cells.     

Biosynthesis of versican by rat dental pulp cells in culture

The biosynthesis of proteoglycans by these cultured pulp cells was investigated by metabolic labelling, using [(35)S]sulphate, [(3)H]glucosamine and [(3)H]leucine as precursors. Versican-like large proteoglycan, decorin- and biglycan-like small proteoglycans and a small amount of sulphated protein were released into the culture medium. Heparan sulphate species were also identified in cell-layer extracts. Versican-like proteoglycan had an average molecular mass of approximately 800kDa. The molecular mass of chondroihnase ABC-digested core protein exhibited heterogeneity, ranging from 250 to 400kDa, and the glycosaminoglycan chains had an average molecular mass of approximately 42kDa. These results indicate the presence of 10-13 glycosaminoglycan chains per core protein, consistent with the characteristics of versican. This glycosaminoglycan chain contained approximately 63% 4-sulphated disaccharides.     

Glycosaminoglycan patterns in gingival proteoglycans of rat with age.

Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-, 6-, 8-, 12- and 18-week-old rats were incubated for 4 h in medium containing [3H]-glucosamine and [35S]-Na2SO4, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61-76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development.     

Effects of interleukin-4 on proteoglycan accumulation in human gingival fibroblasts

In inflammatory gingival diseases, cytokines have been demonstrated to play critical roles by coordinating the stimulation of immunological and connective tissue cells. The activities of these cells, degrading and remodeling extracellular matrices, constitute the major pathological and repair processes. Thus, elucidating cellular and molecular events occurring in inflamed connective tissues is crucial for the understanding and treatment of inflammation. In order to test a hypothesis that proinflammatory cytokines affect metabolism of major extracellular matrix molecules, we studied metabolism of proteoglycans (PGs) by human gingival fibroblasts (HGF) under the influence of interleukin-4 (IL-4) as a model of gingivitis. HGF in cell culture were metabolically radiolabeled using [3H]glucosamine and [35S]sulfate in the presence or absence of IL-4, and the labeled PGs were analyzed by chromatographic techniques. The incorporation of 35S into PGs increased with IL-4 both in media and cell layer. At 100 ng/ml of IL-4, the increment of 35S incorporation over control culture was 16-39% (p<0.001) in media and 12-35% (p=0.01) in cell layer. The 35S-labeled macromolecules were PGs containing heparan sulfate (HS) and chondroitin sulfate (CS) chains. From the molecular weight and glycosaminoglycan composition analyses, versican and perlecan-type and biglycan and decorin-type were very likely to be the major PG constituents both in media and cell layer. IL-4 stimulated synthesis of versican and perlecan-type more potently than biglycan and decorin-type. With IL-4 treatment, the ratio of CSPG/HSPG decreased in media and increased in cell layer. This ratio suggested that syndecan family HSPGs were also present in HGF. In conclusion, IL-4 stimulated accumulation of CS/HSPGs in human gingival fibroblasts.     

The effect of the outer membrane fraction of Bacteroides gingivalis W50 on glycosaminoglycan metabolism by human gingival fibroblasts in culture

The synthesis of extracellular [35S]-SO4- and [3H]-glucosamine-labelled glycosaminoglycan (GAG) was studied in confluent human gingival fibroblast cultures in vitro. The differential synthesis of the total chondroitin sulphate/dermatan sulphate (CS/DS) and heparan-sulphate (HS) fraction was measured following chondroitinase-ABC digestion, nitrous-acid treatment and column chromatography on Sephadex G50. Control cultures synthesized a CS/DS fraction that represented 78 per cent of the total [35S]-SO4-GAG; the residual 22 per cent was heparan sulphate. Similar cultures were labelled with [3H]-glucosamine and the proportions of a high molecular-weight hyaluronic acid (HA) and proteoglycan fractions measured by gel-filtration HPLC after papain and hyaluronidase digestions. The HA fraction represented 66 per cent of the total isotope incorporated in control cultures. GAG chains released on treatment with papain (24 per cent of the total label incorporated) were of apparent molecular weight 17-20 kDa. All cultures exposed to Bacteroides gingivalis W50 outer membrane at concentrations between 2 and 50 micrograms ml-1 displayed a decrease in the CS/DS fraction and a reciprocal increase in the HS. However, the proportion of HA synthesized was slightly enhanced with a reciprocal decrease in the proteoglycan (papain-digestible) fraction. There was no alteration in the molecular weight of the papain-digestion products or the size distribution of the hyaluronic-acid fraction.     

Partial characterization of proteoglycans synthesized by human gingival epithelial cells in Culture

Proteoglycans (PGs) were extracted from the [³⁵S]-sulfate labelled medium and cell layer of proliferating human ginigival epithelial cells and anlyzed by ion exchanged and molecular sieve chromatorgraphy, and by SDS-PAGE. The majority of the incorporated radioactivity secreted into the medium eluted from a DEAE Sephacel ion exchange column as a single peak at 0.44 M NaCl with a small shoulder at 0.52 M NaCl. This material, when chromatographed on Sephartose CL-6B contained two spieces – a quantitatively major peak at K_av= 0.30 (M_t? 235 000 on SDS-PAGE) and a qantitatively major peak at K_av= 0.39. The major peak was sensitive to alkaline borohydride, shifting to K_av= 0.45, and nitrous acid degradtion, indicating the presence of heparan sulfate PG with glyscosaminoglycan chins with M_t? 26 000. The minor peak is chondroitin/dermatan sulfateP with glycpsminoglycan chains of M_t= 22 200 as indicted by sensitivity to alkaline borohydride (shifting to K_av= 0.48) and chondroitin ABC lyase digestion. The [³⁵S]-sulfate labelled material from the cell layer eluted in a broad peak between 0–0.50 M NaCL from DEAE Sephacel. Chromatography of this material on Sepharose CL-6B revealed the presence of three peaks at K_av=0.20, 0.31, and 0.75. The largest peak (K_av=0.20 and M_r? 245 000 on SDS-PAGE) shifted elution position to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of M_t? 20000. Two peaks containing [³⁵S]-sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL-6B with K_avs=0.42 (M_r? 30 500) and 0.75 (M_r? 5300). The larger peak was predominately chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase susceptibility to nitrous acid. These results indicate that cultured human gingival epithelial cells synthesize and secrete principally heparan sulfate PGs with small amounts of chondroitin/dermatan sulfate PGs. This work will serve as a basis for future studies designed to examine those factors involved in regulation of PG synthesis by these cells.     

Fibroblastic subpopulations in uninjured and wounded rabbit oral mucosa

Fibroblast cultures derived from uninjured and reparative rabbit buccal mucosa were compared in terms of extracellular glycosaminoglycan (GAG) content and cellular response to interleukin-1 (IL-1). Under identical growth conditions, proliferation of both cell lines was the same. Both lines incorporated [3H]-glucosamine into GAG in cellular, pericellular, and medium fractions, with the majority of incorporated label residing in the medium. Dermatan sulfate (DS) was the predominant GAG in the medium fraction of both normal and wound fibroblast cultures; however, the two cell lines differed in the identity of the medium fraction's secondary GAG: chondroitin sulfate (CS) for normal fibroblasts and hyaluronic acid (HA) for wound-derived cells. The GAG content of the pericellular matrix for all cultures was the same regardless of the tissue of origin: heparan sulfate (HS) accompanied by a very small amount of CS. Exposure to IL-1 produced limited but highly specific effects: It was not mitogenic for either cell line but did cause a quantitative change (increase) in overall incorporation into GAG for medium and pericellular fractions for both cell lines. Further, IL-1 induced a qualitative change in GAG composition for normal mucosal fibroblastic medium fractions by causing the synthesis/release of heparan sulfate (HS) and a variant form of DS. These data support the hypothesis that different fibroblastic substrains can populate a given oral site as a function of variables such as injury and/or healing status.     

钟状期小鼠磨牙牙胚组织中蛋白聚糖类型的检测分析

目的:检测分析钟状期小鼠磨牙牙胚组织中蛋白聚糖的类型。方法:建立钟状期小鼠磨牙牙胚体外培养模型,用核素标记,凝胶层析,碱处理及酶消化的方法分析体外培的养牙胚组织中蛋白聚糖的类型。结果:体外培养的小鼠钟状期磨牙牙胚、成釉器与牙乳头中[35S]标记的大分子经Superose 6层析柱层析后,均得到3个洗脱峰。其中第1个洗脱峰经硫酸软骨素酶ABC消化后完全消失,第2、第3个洗脱峰在硫酸软骨素酶ABC消化后峰值降低,继续用乙酰肝素酶消化后则基本消失。只用硫酸角质素酶处理,上述3个洗脱峰均未发生改变。碱处理后3个洗脱峰均消失,并在有效分配系数Kd=0.47处出现1个新的洗脱峰。结论:小鼠钟状期磨牙牙胚、成釉器与牙乳头组织含有大分子硫酸软骨素蛋白聚糖、小分子硫酸软骨素蛋白聚糖以及硫酸乙酰肝素类蛋白聚糖,但不存在硫酸角质素蛋白聚糖。     

A biochemical and immunohistochemical study of the proteoglycans of alveolar bone

The purpose of this investigation was to study the proteoglycans in alveolar bone of three animal species. Alveolar bone was obtained from humans, pigs, and rabbits. Portions were fixed, sectioned, and stained with monoclonal antibodies against keratan sulfate and chondroitin sulfate. In other samples, biochemical analyses were performed. After removal of the organic matrix by 4 mol/L guanidinium HCl extraction in the presence of proteinase inhibitors, proteoglycans in the mineralized matrix were extracted with 4 mol/L guanidinium HCl/0.5 mol/L EDTA/proteinase inhibitors, and characterized on the basis of their glycosaminoglycan content (cellulose acetate membrane electrophoresis), charge (DEAE-Sephacel and hydroxylapatite chromatography), size (Sepharose CL-6B chromatography and agarose/polyacrylamide gel electrophoresis), and amino acid content. The results indicated that keratan sulfate could be detected immunohistochemically and biochemically in rabbit bone only. The predominant glycosaminoglycan in pig and human alveolar bone was chondroitin sulfate, although some hyaluronate, dermatan sulfate, and heparan sulfate were also detected. The proteoglycans were found to be slightly smaller than gingival proteoglycans, but similar to those in cementum, dentin, and other bones. In addition to intact proteoglycans, some free glycosaminoglycan chains were also extracted from the mineralized matrix. Amino acid analyses showed some subtle differences between alveolar bone proteoglycan and those of the soft tissues of the periodontium.     

Stimulation of proteoglycan and DNA syntheses in chondrocytes by centrifugation

So that the effects of biomechanical forces on the proliferation of chondrocytes and their proteoglycan synthesis could be studied, growth-plate and articular chondrocytes were maintained separately as packed masses in centrifuge tubes in the presence of 10% serum. In these conditions, the cells became re-organized into cartilaginous tissue in seven days. After ten days, they were centrifuged at gravities (g) of 1.3-27 for 24 h in a CO2 incubator. Control cells were maintained in the CO2 incubator without centrifugation. Centrifugation of growth-plate chondrocytes at 3 g resulted in a two-fold increase in incorporation of [35S]sulfate into proteoglycans, but had little effect on their [3H]thymidine incorporation into DNA. On the other hand, centrifugation of articular chondrocytes at 3 g for 24 h caused 1.5-fold increases in both [35S]sulfate incorporation into proteoglycans and [3H]thymidine incorporation into DNA. These results suggest that biomechanical forces have different effects on the growth and differentiation of articular and growth-plate chondrocytes.     

The effect of chronic inflammation on gingival connective tissue proteoglycans and hyaluronic acid

Proteoglycans have been isolated and analysed from extracts of normal and chronically inflamed human gingiva in order to determine the effects of chronic inflammation on these important soft connective tissue extracellular macromolecules. The uronic acid content of glycosaminoglycans isolated by papain digestion of normal and inflamed gingiva did not differ significantly. Likewise, electrophoretic analysis revealed that the content of hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfute was similar. The sulfated glycosaminoglycans from both sources eluted from a Sepharose C1-6B column with a Kav of 0.45 (approximate M_r 25,000). However, hyaluronic acid from normal gingiva was predominantly of a large size eluting in the void volume of a Sepharose. CL-6B column, while that isolated from inflamed tissue was mostly a small molecular weight species which elutccl in the included volume of a Sepharose CL-6B column. Using dissociative conditions, intact proteoglycans could be more readily extracted from inflamed tissues (90% of the total tissue uronic acid) than from normal tissues where only 80% of the total tissue uronic acid was extractable. Even though DEAE-Sephacel ion-exchange chromatography revealed no differences in charge between normal and inflamed gingival proteoglycans, Sepharose CL-4B chromatography revealed more molecular size polydispersity in samples from inflamed tissue than from normal tissue. Taken together, these results indicate that while hyaluronic acid is depolymerized in inflamed tissue, no evidence of sulfated glycosaminoglycan degradation was found. Therefore, the most likely cause for disruption to the molecular integrity of the proteoglycans is via proteolytic alteration to the proteoglycan core protein.     

Inhibition of synthesis of rat parotid secretory proteoglycan in a gland slice system.

The chondroitin sulphate contained within the secretory granules of the rat parotid gland and its saliva was shown to be in the form of a proteoglycan by using inhibitors of proteoglycan synthesis in a gland slice system. Gland slices were incubated in either p-nitrophenyl-beta-D-xyloside or chlorate in the presence of both [3H]-leucine and [35S]-sulphate. The slices were next homogenized and either the 250 g supernatant fraction (for initial experiments) or secretory granule-containing fractions were isolated. Protein and proteoglycans of these fractions were precipitated in 10% trichloracetic acid (TCA), and glycosaminoglycans in cetylpyridinium chloride. [3H]-leucine and [35S]-sulphate were quantitated in each type of precipitate by scintillation counting. The results showed that 1 mM xyloside had no effect on protein or glycosaminoglycan synthesis but blocked incorporation of radiosulphate into TCA-precipitable material. Sixteen mM chlorate almost totally inhibited incorporation of radiosulphate into glycosaminoglycan and TCA-precipitable material. These findings demonstrate that the rat parotid secretory chondroitin sulphate is indeed a proteoglycan because its synthesis is blocked by the protein-core analogue acceptor, p-nitrophenyl-beta-D-xyloside. This system offers opportunities for exploring the functional role of chondroitin sulphate proteoglycan in this salivary gland.     

Isolation and characterization of the proteoglycans synthesized by adult human pulp fibroblasts in vitro

The proteoglycans synthesized by fibroblasts derived from healthy human adult dental pulps have been isolated and characterized on the basis of their glycosaminoglycan content, molecular size and charge. The proteoglycans were identified by their labelling with [³⁵S] sulphate and susceptibility to digestion by papain. The sulphated glycosaminoglycans associated with the proteoglycans were identified following specific enzymatic and chemical degradations as chondroitin sulphate, dermatan sulphate and heparan sulphate. Dermatan sulphate and chondroitin sulphate were identified as the major glycosaminoglycans secreted into the medium, whereas chrondroitin sulphate and heparan sulphate were the principal glycosaminoglycans associated with the cell layers. The proteoglycans could be fractionated on the basis of their charge and size into a number of heterogeneous pools. The principal proteoglycans isolated were small and contained either chondroitin sulphate or dermatan sulphate and most likely correspond to decorin and biglycan. Other molecules with features similar to versican and syndecan were also identified.     

Role of sulphation in post-translational processing of rat salivary mucins.

Segments of rat submandibular salivary gland were incubated in MEM supplemented with 10-800 microM sulphate in the presence of [3H]-glucosamine, [3H]-proline and [35S]-Na2SO4, with 0-8 mM chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulphate formation. Incorporation of glucosamine and sulphate depended upon the sulphate content of the medium and reached a maximum at 400 microM sulphate. The introduction of chlorate into the medium, while having no effect on the protein synthesis as shown by [3H]-proline incorporation, caused, at its optimal concentration of 4 mM, a 90% decrease in mucin sulphation and a 29% drop in mucin glycosylation. At low sulphate content in the medium and in the presence of chlorate the incorporation of sulphate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulphate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulphation in its carbohydrate chain. This effect of sulphate was, however, inhibited by chlorate. The results suggest that sulphation takes place at an early stage of mucin assembly and that sulphate availability is essential for the formation of the high molecular-weight mucin.     

Molecular size distribution analysis of human gingival glycosaminoglycans in cyclosporin- and nifedipine-induced overgrowths

Glycosaminoglycans are thought to accumulate in formative lesions like drug-induced gingival overgrowth. Recent evidences, however, suggest that the amounts of glycosaminoglycans are comparable in overgrown and healthy gingiva. Besides, alterations in the size distribution of glycosaminoglycan molecules isolated from phenytoin-induced overgrown samples have also been suggested. Therefore, we sought to determine possible differences in molecular size distribution of gingival glycosaminoglycans in other types of drug-induced overgrowths. Purified gingival glycosaminoglycans from healthy and cyclosporin- and nifedipine-induced overgrown gingival tissues were analyzed by agarose gel electrophoresis and their molecular-size distribution was evaluated by both gel filtration chromatography and polyacrylamide gel electrophoresis. Our results on the gingival glycosaminoglycan composition showed presence of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in all types of gingival tissues examined. In addition, hyaluronic acid was predominantly of a large size eluting near to the void volume of a Superose-6 column, while the sulfated glycosaminoglycans were mainly composed of low molecular size glycosaminoglycans. Our results show no differences in the molecular-size distribution of hyaluronic acid and sulfated glycosaminoglycans among healthy and drug-induced overgrown gingival tissues.     

Human gingival glycosaminoglycans in cyclosporin-induced overgrowth

Glycosaminoglycans in normal and cyclosporin‐induced gingival overgrowth were extracted by papain digestion and purified by Mono Q‐FPLC chromatography. The purified glycosaminoglycans were analyzed by agarose gel electrophoresis and by the pattern of degradation products formed by chondroitin lyases on HPLC chromatography. Our results on the glycosaminoglycan composition showed presence of chondroitin 4‐ and 6‐sulfate, dermatan sulfate, heparan sulfate and hyaluronic acid in both normal gingiva and cyclosporin‐induced gingival overgrowth. The total and relative amounts of glycosaminoglycans were similar between normal and overgrown gingiva. This suggests that the glycosaminoglycan composition is not changed in cyclosporin‐induced gingival overgrowth. Our present biochemical results conflict with histochemical and biosynthetic data previously reported by other groups. Those studies suggested that the affected tissues contained higher levels of glycosaminoglycans and that cyclosporin induced comparably high levels of these compounds in in vitro cultures of gingival fibroblasts. Therefore, these discrepant results suggest that a cyclosporin‐induced increase on gingival glycosaminoglycans still remains an open question. The implications of these conflicting results are discussed.     

Glycosaminoglycan synthesis by adult rat submandibular salivary-gland secretory units

The synthesis of glycosaminoglycans (GAG) by a preparation of purified, functional submandibular-gland secretory units (acini and intercalated ducts) was examined. Such units were isolated from Sprague-Dawley rats by digestion of minced gland with hyaluronidase and collagenase followed by gentle sieving of the digest through a graded series of Teflon screens. They incorporated amino acids into exocrine proteins which could be released by stimulation with isoproterenol as in vivo, indicating their functional integrity. Secretory units, incubated for 2 h in medium containing [35S]-sodium sulphate alone or in combination with [3H]-glucosamine, were then washed, homogenized and digested in pronase. The resulting material was then sequentially digested by specific enzymic and chemical procedures and analysed by chromatography on Sephadex G-50 columns to identify the various GAG synthesized. Secretory units synthesized a GAG mixture which was 20-25 per cent hyaluronic acid, 70-75 per cent heparan sulphate, and only 3-5 per cent chondroitin or dermatan sulphates, similar to that synthesized in vivo. No GAG was present in the secretory material, suggesting that all the GAG synthesized was destined for the basement membrane or cell surface.     

Comparison in vitro of the incorporation of D-[2-3H(N)]-mannose and D-[1-14C]-glucosamine into glycoproteins of dispersed rat submandibular salivary gland cells

The incorporation of two different radiolabelled sugars, D-[14C]-glucosamine and D-[2-3H(N)]-mannose, into cellular and secretory glycoproteins was compared using dispersed rat submandibular cells. Most of the de-novo biosynthesis appeared to be directed toward the synthesis of secretory material as the molecular profile of the 3H-labelled material released following sympathomimetic stimulation and the percentage of total 3H-labelled acid--precipitable material secreted following cholinergic- or adrenergic-receptor stimulation coincided with the data obtained from similar studies using [14C]-glucosamine. The [3H]-mannose label was found in the neutral sugars mannose, galactose, glucose and fucose, with trace amounts of radiolabel in the amino sugars, whereas the [14C]-glucosamine label was present in three different amino sugars; glucosamine, galactosamine and sialic acid.     

Interleukin-1 stimulates proteoglycan and hyaluronic acid production by human gingival fibroblasts in vitro

The effect of recombinant interleukin 1β [IL-1β] on proteoglycan and hyaluronic acid synthesis by human gingival fibroblasts has been investigated. It was found to stimulate gingival fibroblast proliferation in a dose dependent fashion with the midpoint of this response being in the 10⁻¹¹ mol/L range. At a concentration of 10⁻¹¹ mol/L, IL-1β stimulated proteoglycan synthesis by 40 per cent. Although IL-1β can stimulate cell proliferation and prostaglandin synthesis, its effect on proteoglycan synthesis was independent of these parameters. The kinetics of proteoglycan degradation in the presence or absence of IL-1β was monitored by pulse chase experiments and were found not to differ between treated and untreated cultures. The molecular size and carbohydrate composition of the proteoglycans was not affected by IL-1β. Additional studies revealed the synthesis of hyaluronic acid was also stimulated by IL-1β. As for the proteoglycans, inhibition of cell proliferation did not affect the stimulatory effect of IL-1β. However, blockage of prostaglandin synthesis abolished the stimulatory effect of IL-1β on hyaluronic acid synthesis. The effect of IL-1β on hyaluronic acid synthesis was found to be related to elevated levels of the enzyme hyaluronate synthetase. Molecular size analysis of newly synthesized hyaluronic acid revealed that cells treated with IL-1β synthesized more large molecular mass hyaluronic acid. Taken together, these findings are considered to reflect the ability of gingival fibroblasts to respond to inflammatory mediators in a manner indicative of early tissue repair.