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-labelled glycosaminoglycans showed that heparan sulfate was the major component (approximately 90%) and that small amounts (approximately 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 Kav of 0.28. Gel chromatography of the glycosaminoglycan chains in the latter, isolated after proteolytic digestion, indicated a molecular weight of 17,000.     

Mucopolysaccharide localization in gingival epithelium

Short incubations, at 37°C, of small freshly excised pieces of healthy human gingivae iancorporated [³⁵S]-sulphate and [³H]-acetate into macromolecular material which could be precipitated intercellularly in the epithelium. Following radioactively pulse-chased incubations, such localization was observed by autoradiography on cryostat histological sections fixed in cetylpyridinium chloride. Critical electrolyte salt concentration elution indicated that most of the intercellular material was soluble in 0.63M-MgCl₂, and any material which remained was intracellular. In vitro and in vivo incorporation studies were compared. These data corroborate biochemical studies (Wiebkin, Bartold & Thonard 1979) together with other histochemical observations that proteoglycans (mucopolysaccharides) are a major intercellular component of human gingival epithelium. Molecular conformation and the relatively rapid synthesis and secretion rate for this class of epithelial macromolecule may explain the lack of susceptibility of this material in the intercellular site, both to degradation by some specific enzymes previously reported and to elution with critical salt concentrations from cationic detergent precipitates.
The method described, together with in vivo incorporation studies, provides a useful technique for studying direct effects of some microenvironmental influences on gingival epithelium.     

The effect of interleukin-1 β on hyaluronic acid synthesized by adult human gingival fibroblasts in vitro

The effect of recombinant interleukin-1β (IL-lβ) on hyaluronic acid synthesis by human gingival fibroblasts was studied. IL-1bT caused a dose-dependent increase in the incorporation of (³lucosamine into hyaluronic acid. The ³⁵S/35H ratios of labeled macromolecules did not change regardless of the presence or absence of TL-lβ and indicates stimulation of hyaluronic acid synthesis. Inhibition of cell proliferation by hydroxyurea caused an increase in hyaluronic acid synthesis. The effect of IL-1β on hyaluronic acid synthesis in the presence of hydroxyurea was increased over untreated and IL-lβ-treated controls, but equivalent to the hydroxyurea-treated controls. Thus the effect of IL-1β on hyaluronic acid synthesis may be independent of cell proliferation. Furthermore, inhibition of prostaglandin E2 synthesis by indomethacin abolished the effect of IL-1β on hyaluronic acid synthesis. Inhibition of new protein synthesis by cycloheximide negated the effect of IL-β on hyaluronic acid synthesis. This may be related to inhibition of new hyaluronate synthetase synthesis, since IL-1β stimulated the level of hyaluronate synthetase activity. Sepharose CL-2B chromatography revealed that most of the newly synthesized hyaluronic acid was of large molecular size. The cells exposed to IL-1β retained more large molecular size hyaluronic acid in their cell layer environment than did the control cells. These responses by fibroblasts to IL-1β may be indicative of early tissue repair.     

In vitro study of the giant tubule collagen formation in bovine dentin by [3H]- proline incorporation

Abstract – Unerupted permanent bovine incisors studied by routine autoradiography using radioactive proline incorporation revealed collagen formation within the giant tubules situated in the incisal dentin. A high [³H]-proline labeling was seen within the pulpal vascularized portion of the giant tubules, as well as in association with groups of cells immediately incisal to the blood vessel loops. The incisal portion of the giant tubules showed no or insignificant [³H] -proline labeling.     

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.     

A preliminary study of the effects of laser radiation on collagen metabolism in cell culture

A low power Ga-As pulse laser was used to stimulate cultured human embryonic fibroblast cells. Energy fluencies varied from 0–1 J/cm² over a period of 1–4 days. Fibroblast procollagen production was monitored by the synthesis of [³H] hydroxy-proline, and DNA replication was assessed by [³H] thymidine incorporation. Following laser treatment, controlled pepsin digestion measured the increase in cell biostimulation. Maximum increase in collagen production and cell biostimulation occurred after 4 episodes of laser treatment at 24-hour intervals. Laser doses between 0.099 and 0.522 J/cm² had the most significant stimulatory effects on fibroblast function. Clinical efficacy of the low power Ga-As pulse laser may be related to enhanced connective tissue repair.     

Glycosaminoglycan turnover and synthesis in the rat incisor pulp

abstract – The rate of in vivo turnover and in vitro synthesis of the glycosaminoglycans (GAGs; acid mucopolysaccharides) of the dental pulp loose connective tissue from rat maxillary incisors has been investigated by isotope methods. The biologic half-life of ³⁵SO₄ in whole tissue digests was found to be 4.5 d. Of the sulfated GAGs the chondroitin-4-sulfate fraction was shown to have a more rapid turnover in vivo than the chondroitin-6-sulfate fraction, 4.1 and 5.2 d, respectively. The fraction containing keratan sulfate and glycoproteins had a biologic half-life of 6.8 d. Slices of rat incisor pulps were incubated in vitro with ³⁵SO₄ and the rate of incorporation into the different GAG fractions was determined. After a lag-phase of 15 min, this rate was linear with time. The chondroitin-6-sulfate fraction showed a more rapid uptake than the chondroitin-4-sulfate fraction, and the uptake by keratan sulfate + glycoprotein fraction was much lower. A similar in vitro experiment using [¹⁴C] acetate was performed. Contrary to the sulfate incorporation there was an extended lag-phase, and the total incorporation was much lower.     

Regulation of collagen production in fibroblasts cultured from normal and phenytoin-induced hyperplastic human gingiva

We have studied how collagen production is regulated in fibroblasts obtained from normal and phenytoin-induced hyperplastic human gingiva. Collagen production was determined as collagenase digestible radioactivity and degradation was examined by adding labelled procollagen to the cultures and by pulselabelling in the presence of lysosomal inhibitors. Collagen mRNA levels were measured using a [³⁵S]-UTP labelled proα[1] probe. The normal and phenytoininduced fibroblasts did not degrade collagen extracellularly and lysosomal inhibitors did not enhance collagen production in either culture. Collagen production by the cultures correlated with mRNA levels, and in 2 of 3 phenytoin-induced fibroblasts, which produced more collagen than other cells. collagen mRNA levels were higher. We conclude that collagen production in gingival fibroblasts is primarily regulated by the mRNA levels and that overproduction of collagen by cells from phenytoin-induced hyperplastic gingiva results from an increased steady state level of collagen mRNA and not decreased collagen degradation.     

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.     

Characterization of hydrogen sulphide reaction with rat-tail tendon Type I collagen in vitro

Type I acid-soluble rat-tail tendon collagen was reacted up to four days with hydrogen sulphide (H₂S) concentration equivalent to that produced by putrefying saliva in in vitro systems. After reaction, the head-space was assayed for H₂S by gas chromatography (GC), and the reaction mixture was separated into salt-soluble supernatant fraction and sediment which was solubilized in 0.5 M acetic acid. Both fractions were analyzed by SDS-polyacrylamide gel disc electrophoresis (SDS-PAGE) and assayed for free aldehyde, free thiol, total available thiol, and hydroxyproline content. The GC analysis of head-space samples showed that more than 85% of the added H₂S was absorbed within the initial 24 h by the reaction medium and totally removed from the head-space in four days. Although no discernible qualitative or quantitative changes were visually detected in H₂S-treated systems by SDS-PAGE, increased levels of hydroxyproline in the supernatant fraction indicated production of a neutral salt-soluble product. Chemical analysis showed that the reaction resulted in the incorporation of H₂S in free thiol and disulphide forms in both fractions. [³⁵S]-H₂S/paper chromatography methods confirmed that H₂S was incorporated into collagen. An increase in free aldehyde groups of both fractions inferred exposure of aldehyde groups presumably through cleavage of intramolecular cross-linkages.
The reversion of acid-soluble collagen to a more soluble product, which is considered more susceptible to enzymatic degradation, suggests one mechanism how the volatile thiol compounds may contribute to the etiology of periodontal disease.     

Effect of macrophage depletion on growth and neovascularization of hamster buccal pouch carcinomas

The effect of macrophage depletion on growth and neovascularization of 7, 12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinomas (HBPC) was evaluated by quantitating tritiated thymidine (³[H]TdR) incorporation by tumor cells and microvascular endothelium in light microscopic autoradiographs. Tumors that were depleted of macrophages with systemic hydrocortisone acetate (HA) and intratumor injections of antimacrophage serum (AMS) were examined 7 days after treatment. In control animals 30% of infiltrating host cells were esterase-positive tumor-associated macrophages (TAM) and 28.14% of tumor cells and 14.45% of endothelial cells were ³[H]TdR labelled. Hamsters treated with HA or HA and control serum showed no significant reduction in either the number of TAM or proportion of [³H]TdR labelled tumor of endothelial cells. AMS administered alone had no effect on either the content of TAM or ³[H]TdR labelling. In contrast hamsters treated with HA and AMS showed a 54% decrease in TAM and a 50% and 63% reduction in tumor and endothelial cell labelling respectively. These results suggest that growth and neovascularization of these tumors is mediated in part by macrophages.     

Cytotoxicity of root canal sealers: a study using HeLa cells and fibroblasts

Summary. The relative cytotoxicity of five root canal sealers was measured by adding various concentrations of freshly mixed and set sealers to cultures of either HeLa or human fibroblast cells. The toxicity was determined by cell survival, which was measured by subsequent incorporation of [³H]-thymidine into the nucleic acid components of each cell type used. The results showed that all sealers are cytotoxic in both fresh and set states. The ranking of toxicity of sealers on HeLa cells was similar to the one obtained for fibroblasts. Comparison of the results of this study with those of previous investigations questions the value of ranking sealers based only upon their effects in vitro.     

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.     

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.     

Effect of glucocorticoids on the 35SO4 incorporation in the rat incisor pulp

abstract – The effects of cortisone acetate and hydrocortisone acetate on the incorporation of ³⁵S-labelled sulfate in the loose connective tissue of the rat incisor pulp have been studied. A reduction by 25 % and 50 %, respectively was found. This was interpreted as representing a decrease in the synthesis of sulfated glycosaminoglycans (mucopolysaccharides), especially the chondroitin sulfates which are the main glycosaminoglycan fraction in this tissue. The effects of glucocorticoids on the chondroitin sulfate synthesis are discussed in relation to dentinogenesis and tooth eruption.     

Chemistry of the sulfate groups in a sulfated glycoprotein from rabbit submandibular gland

abstract – A ³⁵S-labeled sulfated glycoprotein was isolated from rabbit submandibular glands. Acid stability studies on the ³⁵sulfate groupings present in the intact glycoprotein gave a half-life of 45 min. Partial acid hydrolysis of the ³⁵S-labeled glycoprotein in 0.1 M HCl for 90 min at 100°C liberated a radioactive fraction which was free from peptide and fractionated in the monosaccharide range of a Sephadex G-15 column. Examination of this fraction by paper chromatography revealed the presence of a major component having the characteristics of N-acetylglucosaminc 6-0-sulfate and a minor component having the properties of N-acetylgalactosaminc 6-0-sulfate. The presence of ester sulfate groups in the intact glycoprotein was confirmed by infrared spectroscopy.     

Effect of calcium, given before or after a fluoride insult, on hamster secretory amelogenesis in vitro

We tested the hypothesis that high-calcium medium given prior to or immediately after exposure to fluoride (F) reduces the negative effects of F on secretory amelogenesis. Hamster molar tooth germs were grown in organ culture in media with different calcium levels. Deposition of enamel matrix and matrix mineralization were monitored by incorporation of [³H]proline and uptake of ⁴⁵Ca and acid-soluble ³²PO₄. Ameloblast structure and the occurrence of a fluorotic enamel matrix were examined by light and electron microscopy. A preculture of explants in high-calcium medium partially prevented the formation of fluorotic (non-mineralizing) enamel matrix, increased matrix secretion but could not prevent F-induced hypermineralization of the pre-exposure enamel. High-calcium medium, applied after F insult, accelerated the recovery of fluorotic matrix, improved ameloblast structure, enhanced amelogenin secretion, and increased enamel thickness. The data indicate that it might be the balance between the amount of mineral deposition and that of matrix secretion which is critical for the mineralization of newly secreted enamel. Exposure to F disturbs this balance by enhancing mineralization of the pre-exposure enamel, probably generating an excess of protons. High calcium may protect against F exposure by enhancing amelogenin secretion into the enamel space, thereby increasing the local buffering capacity at the mineralization front.     

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.     

Effects of epidermal growth factor and nerve growth factor on isoproterenol-induced DNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

When epidermal (EFG) (10 ng/kg body wt) or nerve growth factor (NGF) (1 ng/kg body wt) was given intraperitoneally to sialadenectomized young rats (submandibular-sublingual (SM-SL) glands removed) prior to injection of isoproterenol (ISO) (50 mg/kg body wt), the inhibition of ISO-induced thymidine incorporation into DNA of parotid gland and pancreas caused by removal SM-SL glands was reversed, and thymidine incorporation of sialadenectomized ISO-treated organs was as high as that of parotid and pancreas of surgically intact animals given ISO. EOF alone caused an increase in [³H] thymidine incorporation into DNA of parotid (63%) and pancreas (59%); removal of the SM-SL glands caused a decrease of 57–70% in thymidine incorporation into DNA of parotid, pancreas, liver, lung, kidney, and spleen. A growth effect attributable to the EGF and NGF of the submandibular gland was thus apparent for all organs examined, but even if they had large complements of β₁, adrenoceptors, only the exocrine organs showed the ISO-induced β₁, adrenoceptor response to EOF and NGF. EGF and NGF thus interact only with β₁ adrenoceptors of exocrine organs to cause marked increase in [³H] thymidine incorporation of these organs.     

Cyclosporin A upregulates prostaglandin E2 production in human gingival fibroblasts challenged with tumor necrosis factor alpha in vitro

The effect of Cyclosporin A (CsA) on prostaglandin E₂ (PGE₂) production in human gingival fibroblasts challenged with tumor necrosis factor alpha (TNF-α) was studied. TNF-α (1-100 ng/ml) dose-dependently stimulated PGE₂; formation in 24 h cultures. CsA (1-100 ng/ml) did not induce PGE₂; formation itself but potentiated TNF-α induced PGE; formation in gingival fibroblasts in a manner dependent on the concentrations of both CsA and TNF-α. TNF-α (10 ng/ml) stimulated the release of [³H]-arachidonic acid (A.A) from prelabelled fibroblasts that was potentiated by CsA (100 ng/ml). Addition of exogenous unlabelled AA (5-20 μM/ml) to the cells resulted in enhanced PGE₂: formation that was not potentiated by CsA (100 ng/mi). Furthermore. CsA (100 ng/ml) did not further increase the level of cyclooxygenase-2 mRNA induced by TNF-α (10 ng/ml). although PGE₂ formation was enhanced. The results indicate that CsA and TNF-α act in concert on PGE₂ formation in gingival fibroblasts. which may be of importance in the pathogenesis of gingival overgrowth induced by the drug.