Incorporation Of 3H-glucosamine Research Articles

Keratinocyte-derived factors that stimulate fibroblast hyaluronan synthesis - the role of a low molecular weight factor

Hyaluronan-rich matrices are associated with cellular proliferation and migration, with high levels being expressed transiently during adult wound healing, and persistent high levels expressed during scar-free foetal wound healing. Cultured allogeneic keratinocytes and lysed keratinocyte extracts promote chronic wound healing, as does addition of exogenous hyaluronan. It is possible that the keratinocyte-stimulated effects on wound healing may be, at least in part mediated by the release of hyaluronan synthesis-stimulating factors. We have demonstrated that serum-free keratinocyte-conditioned medium (KCM), when added to confluent fibroblast cultures for 24 h stimulated up to an 8-fold increase in hyaluronan synthesis, as measured by 3H-glucosamine incorporation into cetylpyridinium chloride-precipitable glycosaminoglycans. This KCM can be fractionated by ultrafiltration into fractions > 30 kD and < 1 kD which are poorly active individually, but synergise strongly to stimulate fibroblast hyaluronan synthesis. We have examined the activity of both platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) to stimulate fibroblast hyaluronan synthesis in the presence and absence of the low molecular weight KCM fraction, and the effect of blocking antibodies to PDGF and bFGF on complete KCM. KCM stimulated a 7-fold increase in 3H-glucosamine incorporation into hyaluronan and a 8.3-fold increase into sulphated glycosaminoglycans. 35SO4 incorporation was increased only 1.5-fold, resulting in a significant decrease in the degree of polymer sulphation of the sulphated glycosaminoglycans. Blocking antibodies to PDGF (all isotypes) reduced the activity of the KCM by 27%, while blocking antibFGF reduced the activity by 58%. Addition of PDGF (20 ng/ml) to the < 1 kD KCM fraction restored full activity, while addition of bFGF had no significant effect. However, addition of heparin with the bFGF resulted in activity being restored. These results demonstrate that cultured human keratinocytes produce potent fibroblast glycosaminoglycan synthesis-stimulating factors. Both PDGF and bFGF appear to be involved, but require the presence of an as yet unidentified KCM low molecular weight factor. Identification of this factor may provide an important therapeutic aid in wound healing

Toremifene decreases type I, type II and increases type III receptors in desmoid and fibroma and inhibits TGFbeta1 binding in desmoid fibroblasts

Tissue infiltration is different in desmoid and fibroma tumours. Both produce high levels of transforming growth factor beta1 (TGFbeta1), which is related to extracellular matrix (ECM) accumulation which in turn regulates cell function and cell migration. Interactions between collagen, proteoglycans and cell surface fibronectin are involved in the assembly and functions of the ECM. As toremifene inhibits collagen and TGFbeta1 synthesis, we tested it in normal, desmoid and fibroma fibroblasts. We will report the changes in glycosaminoglycan (GAG) and collagen synthesis, TGFbeta1 activity, fibronectin mRNA expression and TGFbeta1 receptors after toremifene treatment in normal, fibroma and desmoid fibroblasts. We evaluated GAG and collagen synthesis with 3H-glucosamine and 3H-proline incorporation, TGFbeta1 activity with the ELISA method, TGFbeta1 receptor affinity with 125I-TGFbeta1 binding and total RNA with Northern blot analysis. GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels were higher in fibroma and desmoid than normal fibroblasts. The increase was greater in desmoid than fibroma tumour cells. Toremifene treatment reduced GAG and collagen synthesis, TGFbeta1 activity and fibronectin levels in all cell cultures. The percentage reduction in GAG was similar in all cultures; the reduction in collagen synthesis and TGFbeta1 activity was the highest in desmoid fibroblasts. TGFbeta1 receptors were higher in fibroma and desmoid cells than controls. Toremifene reduced TGFbeta1 receptors only in desmoid fibroblasts, with no effect on the changes in type I, II, and III receptors. Our data show that toremifene modifies the ECM components that regulate cytokine activity and cell migration. The reduction in receptor number only in desmoid cells suggests that toremifene may reduce TGFbeta1's affinity for its receptors. Synthesis of a substance regulating protein kinase activity, which is directly involved in the link between TGFbeta1 and its receptors, cannot be excluded.

Expression of hyaluronan synthases and hyaluronidases in the MG63 osteoblast cell line

The expression of hyaluronan synthases (1, 2 and 3) and hyaluronidases (1, 2, 3, 4 and PH20) was examined in the MG63 osteoblast cell line induced to mineralize in vitro and compared to the rate of glycosaminoglycan production. Real-time PCR analysis demonstrated a 13-fold decrease in hyaluronan synthase 3 expression in mineralising MG63 cells; no significant change in hyaluronan synthase 2 expression in mineralising cells and hyaluronan synthase 1 was not expressed. In mineralising MG63 cells a 62-fold increase in hyaluronidase 2, a 13-fold increase in hyaluronidase 3, and a 3-fold increase in hyaluronidase 4 expression were observed when compared to non-mineralising cells; hyaluronidase 1 and PH20 expression was not detected. After 5 weeks in mineralising culture conditions a 2-fold increase in total 3H-glucosamine incorporation was observed in cells when compared to 24 h or 5 week control cultures. This was made up of a 5-fold decrease in hyaluronan production, a 2-fold increase in chondroitin sulphate/dermatan sulphate and a 10-fold increase in 3H-glucosamine incorporation into the non-glycosaminoglycan fraction. A 3-fold increase in 35SO4 incorporation into chondroitin sulphate/dermatan sulphate was also observed. Thus there is co-ordinate expression of genes that control hyaluronan metabolism such that there is a general decrease in the expression of hyaluronan synthases, an increase in the expression of hyaluronidases and a corresponding decrease in hyaluronan production by mineralising MG63 cells.

Modulation of cell-phenotype during in vitro aging. Glycosaminoglycan biosynthesis by skin fibroblasts and corneal keratocytes

The aim of this study was to compare keratocyte and fibroblast phenotypes during in vitro aging by comparing their biosynthesis of glycosaminoglycans using explant and cell cultures. Human skin and corneal explant cultures were realised with Dulbecco Modified Eagle's medium containing 3H glucosamine. Sequential cell cultures were studied at different passages for GAGs biosynthesis by 3H glucosamine incorporation followed by selective degradation with specific hydrolases. Radioactivity was determined and each GAG fraction evaluated. KS and DS are the major components synthesised by corneal explant culture. During in vitro aging, keratocytes synthesised 41% less KS between passages 4–9 with a decrease by 26% of the proportion of DS observed in the same conditions. In skin explant cultures, as expected the major components are CS and hyaluronan (HA). In the first cell passage studied compared with skin organ cultures we could notice a strong decrease of the proportions of DS and KS compensated by an increase of the proportion of HA. During the successive passages of fibroblasts, the proportions of DS and HS decreased (−30 and −62%, respectively) and those of KS increased (+90%).These results indicate that there remain measurable differences between keratocyte and fibroblast phenotypes as far as GAG-synthesis is concerned all though the successive passages, starting from explant cultures and up to the limits of in vitro cell passages.

Effects of wheat germ agglutinin and concanavalin A on the accumulation of glycosaminoglycans in pericellular matrix of human dermal fibroblasts. A comparison with insulin.

The effect of insulin, wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (ConA) on [3H]glucosamine incorporation into pericellular glycosaminoglycans (GAGs) was investigated in two lines of cultured human dermal fibroblasts. Insulin and WGA stimulated [3H]glucosamine incorporation into hyaluronic acid (HA) and heparan sulphate (HS) without any alteration of chondroitin sulphate (CS) and dermatan sulphate (DS) contents. ConA increased [3H]glucosamine incorporation into HS, CS and DS, but had no effect on [3Hglucosamine incorporation into HA. PNA affected neither the content, nor the composition of GAGs. In contrast to PNA, ConA and WGA stimulated glycolysis and demonstrated an evident antiproliferative effect on dermal fibroblasts. Thus, both the insulin-like action of WGA and ConA on cultured dermal fibroblasts and the differences between the effects of lectins on modulation of GAGs synthesis appear to be determined by their chemical structure.

Melanoma cell-derived factors stimulate glycosaminoglycan synthesis by fibroblasts cultured as monolayers and within contracted collagen lattices.

Various tumours exhibit glycosaminoglycan rich, and in particular hyaluronan rich matrices surrounding them that facilitate tumour growth and invasion. In many tumours, this matrix is predominantly synthesized by fibroblasts following stimulation by tumour cell-derived factors. To determine what effect tumour cell-conditioned medium has upon fibroblast glycosaminoglycan synthesis when cells were cultured as monolayers and within contracted collagen lattices. Serum-free conditioned medium from melanoma cell lines (C8161, MV3, A375 and Hs294T) was examined for its ability to stimulate the incorporation of 3H-glucosamine and 35SO4 into glycosaminoglycans synthesized by fibroblasts. Conditioned medium from all four melanoma cell lines exhibited potent glycosaminoglycan-stimulating activity. In monolayer culture, C8161-conditioned medium stimulated a 4.2-fold increase in fibroblast hyaluronan, and a 9.9-fold increase in sulphated glycosaminoglycan synthesis, while 35SO4 incorporation was increased only 2.1-fold. In collagen lattice cultures, C8161-conditioned medium stimulated a 4.9-fold increase in hyaluronan synthesis, a 5.4-fold increase in sulphated glycosaminoglycans, and a 1.3-fold increase in 35SO4 incorporation. Melanoma cells produce factors that are potent stimulators of fibroblast glycosaminoglycan synthesis, in both monolayer culture and within contracted collagen lattices. Synthesis of both hyaluronan and sulphated glycosaminoglycans with a reduced degree of polymer sulphation is stimulated. Such changes are likely to promote tumour cell proliferation and migration.

Effect of an α-blocker (Nicergoline ®) and of a β-blocker (Acebutolol ®) on the in vitro biosynthesis of vascular extracellular matrix

The effect of an α-blocking agent and of a β-blocking agent on the biosynthesis of extracellular matrix macromolecules of the arterial wall was investigated. Rabbit aorta explants were cultured up to 48 hours with radioactive proline, lysine or glucosamine. In presence of these drugs, at concentration shown to be effective for the inhibition of platelet-endothelial cell interactions (10 –7M), the incorporation of 14C proline in total macromolecular proline was higher than in macromolecular hydroxyproline suggesting a relatively higher rate of biosynthesis of non-collagenous proteins as compared to collagens. The α-blocking increased the incorporation of 14C proline in collagenous and non-collagenous proteins after 18 hours of incubation. β-blocking also increased the incorporation of proline in macromolecular proline and hydroxyproline as compared to control cultures. Both increased the incorporation of 3H glucosamine in newly synthesised glycosaminoglycans. β-blocking increased mainly the neosynthesis of heparan sulphate, α-blocking that of hyaluronan. The incorporation of 14C-lysine in crosslinked, insoluble elastin was not modified. These experiments confirm that α and β-blocking agents can influence not only the tonus of aortic smooth muscle cells but also the relative rates of biosynthesis of extracellular matrix macromolecules. This effect should be taken in consideration for the evaluation of the long range effect of α and β-blocking drugs on the vascular wall.

A Simple Method to Establish Short-Term Cultures of Normal Human Colonic Epithelial Cells from Endoscopic Biopsy Specimens: Comparison of Isolation Methods, Assessment of Viability and Metabolic Activity

Background: Abnormalities in colonic epithelial cell function have been implicated in the pathogenesis of various intestinal disorders, especially inflammatory bowel disease (IBD). The mechanisms, however, remain obscure owing to the lack of representative human colonic epithelial cell models. The aim of this study was to develop and validate a method for establishment of short-term culture of normal human colonic epithelial cells from endoscopic biopsies. Methods: Epithelial cells were isolated from colonoscopic biopsies by means of ethylenediaminetetraacetic acid/ethylene glycol tetraacetic acid (EDTA/EGTA) (10 or 60 min) or by enzyme treatment and cultured in collagen-coated wells. Viability was measured with a methyltetrazoleum conversion assay, confocal laser, and electron microscopy. Metabolic function was measured by means of butyrate oxidation, 14C-leucine and 3H-glucosamine incorporation; DNA synthesis by means of 3H-thymidine incorporation, and apoptosis with an enzyme-linked immunosorbent assay (ELISA) for histone-associated DNA fragments. Cell types were identified by immunocytochemistry. Results: Ten minutes of EDTA/EGTA treatment released intact crypts and was superior to both the 60-min treatment and enzymatic treatment in terms of viability and nonepithelial cell contamination, respectively. Despite activation of detachment-induced apoptosis, a median 51% of the isolated cells was viable after 24 h of culture and metabolically active as judged by 3H-thymidine, 14C-leucine, and 3H-glucosamine incorporation. Butyrate oxidation followed more complex kinetics (substrate activation) than observed previously in other models. The apparent Km values (medians) were 0.7 mM and 4.5 mM in low and high concentration ranges, respectively. Conclusion: We report a simple method to establish culture of human colonic epithelial cells from endoscopically obtained biopsy specimens, producing sufficient viable cells to perform metabolic studies pertinent to the pathogenesis of IBD and related human disorders.

Melanoma cell-derived factor stimulation of fibroblast glycosaminoglycan synthesis—The role of platelet-derived growth factor

The hyaluronan-rich matrix surrounding many tumours may facilitate tumour growth, invasion and angiogenesis, with the majority of this hyaluronan apparently being synthesised by normal fibroblasts, stimulated to do so by tumour cell-derived factors. Melanoma cell-conditioned medium (CM) stimulates up to a 6-fold increase in fibroblast glycosaminoglycan (GAG) synthesis, with the active factors being present in tumour CM ultrafiltration fractions >30 kDa and <1 kDa. These fractions are poorly active individually, but when recombined, the activity is substantially greater than the additive effect. The objective of this study was to identify the factors present in the ultrafiltration fraction >30 kDa that produce a greater than additive effect with the fraction <1 kDa in stimulating the incorporation of 3H glucosamine into fibroblast GAGs. A number of factors including basic fibroblast growth factor (bFGF), interleukin (IL)-1β, pleiotrophin, platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), tumour necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) failed to stimulate any significant increase in GAG synthesis, but when added to the <1 kDa tumour CM fraction, both PDGF and to a lesser extent, bFGF, exhibited potent stimulating activities. Neutralising antibodies to PDGF and bFGF added to the melanoma CM decreased the fibroblast GAG-stimulating activity by 29% and 40%, respectively, in C8161 melanoma CM and by 47% and 45%, respectively, in Hs294T melanoma CM. The activities of PDGF-AA and PDGF-BB isoforms were indistinguishable, suggesting the PDGF-α receptor plays a role in the GAG-stimulatory response. Western analysis following treatment with PDGF, bFGF or melanoma CM revealed banding patterns for PDGF and melanoma CM that were similar. Immunoprecipitation of the PDGF-α receptor revealed it to be phosphorylated in fibroblasts treated with PDGF and melanoma CM, but not with control fibroblast CM. These studies suggest that PDGF plays an important role in the GAG-stimulating activity of the melanoma CM, but requires the presence of an as yet unidentified novel low molecular weight factor for full activity.

The biosynthesis and post-translational modification of Pbs21 an ookinete-surface protein of Plasmodium berghei

Radiolabelled methionine incorporation into synchronised Plasmodium berghei gametocytes or ookinete cultures, showed that Pbs21 is not synthesised in bloodstage parasites; synthesis was detected within three hours of induction of gametogenesis; synthesis was triggered at gametogenesis, not by fertilisation. We show native Pbs21 to be a hydrophobic membrane protein that was insensitive to cleavage by phosphatidylinositol phospholipase C (PI-PLC), but sensitive to alkaline hydroxylamine, and partially sensitive to glycosylphosphatidylinositol-dependent phospholipase D (GPI-PLD) and HNO 2. 3H-myristic and palmitic acid, 3H-glucosamine and mannose incorporation indicated Pbs21 was acylated and glycosylated. Linkage of the acyl group was sensitive to HNO 2, which released an acyl-phosphatidylinositol more hydrophobic than that released from P3 of Trypanosoma brucei. All these properties are consistent with the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. In contrast recombinant Pbs21 (rPbs21), expressed in Spodoptera frugiperda cells, was sensitive to both PI-PLC and GPI-PLD, consistent with the protein being modified by a different ( S. frugiperda) GPI anchor. Brefeldin A blocked secretion of rPbs21 within a cytoplasmic reticular compartment. Following deletion of the putative GPI anchor addition site (amino acids 189–213), the protein was transported to the cell surface and secreted directly into the aqueous phase of the culture medium. Deletion of amino acids 205–213 disrupted Pbs21 processing, transport through the ER and distribution onto the cell surface. Deletion of amino acids 1–28 prevented transport of Pbs21 into the ER. This suggests that correct processing of the GPI anchor in the ER-Golgi network is essential for the successful secretion of the recombinant protein, which is additionally dependent upon an N-terminal secretory signal sequence.

Quantification of in vitro growth and synthesis in rabbit middle ear epithelial cells.

The purpose of the present study was a quantitative description of middle ear epithelial cell reactions in a standardized model system under controlled conditions. The model is based on a newly developed culture method for rabbit middle ear epithelial cells. The growth pattern of identical subcultures was determined by measurement of total cell protein, DNA synthesis and DNA amount during a 15-day period. The secretory product was quantified by means of 3H-glucosamine incorporation. Epithelial cells from the auditory meatus were examined by identical methods. The results proved the method to be reproducible regarding all parameters. The DNA amount and total cell protein increased throughout the period, whereas DNA synthesis remained constant. Secretion increased linearly for 9 days and then diminished. All parameters were without significant changes when related to DNA amount, reflecting a uniform cellular activity throughout the period. The DNA amount was the most precise parameter with a mean variation coefficient of 6.0% (SD 3.1%). With the baseline values found to be reproducible for the parameters chosen, our model system is considered to be usable for quantifying various factors influencing the proliferation and activity of middle ear epithelium.

Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture.

We studied the influence of hydrocortisone (HC) on hyaluronan (HA) metabolism in explants of human skin, a model retaining normal three-dimensional architecture of dermal connective tissue and dynamic growth and stratification of epidermal keratinocytes. The synthesis of hyaluronan and proteoglycans (PGs), and DNA, were determined with 3H-glucosamine and 3H-thymidine labelings, respectively. The total content and histological distribution of hyaluronan was studied utilizing a biotinylated aggrecan-link protein complex. A low concentration of HC (10(-9) M) stimulated the incorporation of 3H-glucosamine into hyaluronan in epidermis by 23% and reduced the disappearance rate of hyaluronan by 25% in chase experiments, resulting in a 74% increase in total hyaluronan (per epidermal dry weight) after a 5-day culture in 10(-9) M HC. On the other hand, a high concentration of HC (10(-5) M) reduced both synthesis (-42%) and degradation (-46%) of epidermal hyaluronan during 24 h labeling and chase periods. The cumulative effect of a 5-day treatment was a 24% decrease of total epidermal hyaluronan. The high dose (10(-5) M) also reduced keratinocyte DNA synthesis and epidermal thickness. In dermis, only the high (10(-5) M) concentration of HC was effective, inhibiting the incorporation of 3H-glucosamine into hyaluronan by 28%. No significant influences on total hyaluronan content or the disappearance rate of hyaluronan in dermal tissue was found. All HC concentrations lacked significant effects on newly synthesized PGs in epidermal and dermal tissues, but reduced the labeled PGs diffusing into culture medium. A low physiological concentration of HC thus maintains active synthesis and high concentration of hyaluronan in epidermal tissue, while high pharmacological doses of HC slows hyaluronan turnover and reduces its content in epidermis, an effect correlated with enhanced terminal differentiation, reduced proliferation rate and reduced number of vital keratinocyte layers.

BIOCHEMICAL EFFECTS OF DIFLUBENZURON ON CHITIN SYNTHESIS IN THE POSTMOLT BLUE CRAB CALLINECTES SAPIDUS

ABSTRACT In vivo and in vitro metabolic studies were conducted on the effects of the insect growth regulator diflubenzuron (DFB) on chitin synthesis in the postmolt blue crab Callinectes sapidus. The effects of the pesticide on the incorporation of either 3H-glucosamine or 3H-N-acetylglucosamine into sodium dodecyl sulfate (SDS) insoluble cuticular residue were examined. The radiolabeled product formed was identified as chitin by chemical and enzymatic criteria. 3HN-acetylglucosamine was found to be incorporated to a greater extent than 3H-glucosamine during metabolic studies. During in vitro explant culture experiments, the highest concentration of DFB tested (1 ppm) caused 98% inhibition of chitin synthesis; 64% inhibition was observed at concentrations as low as 0.7 parts per billion. The results indicate that diflubenzuron inhibits the incorporation of 3H-N-acetylglucosamine into cuticular chitin in postmolt blue crabs. The data are consistent with our previous autoradiographic and ultrastructural studies on the effects of DFB in the blue crab; i.e., that the Golgi complex is disrupted by DFB treatment.

Exogenous heparin induces an increase in glycosaminoglycans of the chick embryo chorioallantoic membrane: its possible role in the regulation of angiogenic processes.

In this study heparin (HE) was injected into the allantoic sac of chick embryo eggs at 5, 9, 14 days of incubation. 48 h after injection glycosaminoglycan (GAG) concentration was measured in the chorioallantoic membrane (CAM) in order to verify if HE-related CAM angiogenic activity previously demonstrated [Ribatti et al: Acta Anat 1987; 130:257-263] might be correlated with changes in GAG concentration. The results showed that HE inoculation induced an increase of 3H-glucosamine incorporation into total GAGs in comparison to control CAMs. Furthermore, HE altered the balance between the GAG classes, and in particular it produced a significant increase in the accumulation of hyaluronic acid and heparan sulfate between 7 and 11 days of incubation in comparison to control CAMs.

Bradykinin modulates mucin secretion but not synthesis from an intestinal goblet cell line.

The effect of the inflammatory mediator bradykinin on glycoprotein synthesis and mucin secretion in the human colonic adenocarcinoma cell line HT29-18N2 was examined. Bradykinin, at a threshold of 0.01 microM, accelerated the rate of mucin discharge as assessed by a mucin-specific ELISA. Using immunofluorescence microscopy, a thick meshwork of extracellular mucus was observed over bradykinin-treated monolayers but not mock-treated controls. Morphometric analysis of bradykinin-treated monolayers revealed no decreases in intracellular mucin stores or any other easily discernable morphological alteration. The ability of the cyclooxygenase inhibitors indomethacin and naproxen to decrease the response to bradykinin by approximately 68% indicates the effect is mediated, at least partially, through the generation of prostaglandins. Bradykinin did not alter the rate of incorporation of 3H-glucosamine into newly synthesized glycoproteins. Bradykinin-accelerated mucin secretion may be linked to the depletion of intracellular mucin stores in the inflammatory bowel disease ulcerative colitis.

Characterization of excretory‐secretory products of adult Dictyocaulus viviparus and the antibody response to them in infection and vaccination

In vitro released products of the adult stage of the bovine lungworm. Dictyocaulus viviparus, were characterized according to their SDS-PAGE profile, glycosylation pattern, in vitro synthesis and antigenicity in the context of infection and vaccination with irradiated larvae. Biosynthetic labelling experiments with 35S-methionine indicated active synthesis of ES throughout this time. There was, however, little incorporation of 3H-glucosamine into ES products, and lectin affinity chromatography and glycopeptidase F digestion identified only one glycosylated component. Immunoprecipitation of 125I-labelled ES products with sera from calves patently infected with D. viviparus demonstrated that all of these, with the exception of two components, are antigenic to the bovine host. One of those not immunoprecipitated was shown to be host serum albumin carried over into culture. A limited degree of cross-reactivity between nematode species was observed, with a D. viviparus female-specific antigen of 290 kDa being recognized by serum antibody from calves infected with the gastrointestinal nematodes Cooperia oncophora and Ostertagia ostertagi. Calves vaccinated with irradiated larvae of D. viviparus, despite not being exposed to the adult stage of the parasite, also showed some recognition of adult ES products. This might suggest that vaccination with irradiated larvae operates against both pre-pulmonary and pulmonary stages of the infection.

Effects of hyaluronic acid on the release of proteoglycan from the cell matrix in rabbit chondrocyte cultures in the presence and absence of cytokines

To investigate the effects of hyaluronic acid (HA) on the release of proteoglycan by cultured rabbit chondrocytes. Articular cartilage chondrocytes were isolated from the knee joints of New Zealand white rabbits. Proteoglycan synthesis after incubation with HA was determined by measuring 35S-sulfate incorporation. Cells incubated with HA were labeled with 3H-glucosamine and applied to a Sepharose CL-2B column. After incubation of confluent cells with 35S-sulfate and then with HA in various concentrations in the presence or absence of cytokines, proteoglycan release from the cell matrix layer was measured. HA (M(r) 3 x 10(5) to 19 x 10(5)), at 10 micrograms/ml to 1 mg/ml, had little effect on the incorporation of 35S-sulfate or 3H-glucosamine into cartilage matrix proteoglycans, or on the hydrodynamic size of proteoglycan monomers, in rabbit chondrocyte cultures. However, at 10-1,000 micrograms/ml, HA suppressed the release of 35S-proteoglycans from the cell matrix layer into the medium in the presence and absence of interleukin-1, tumor necrosis factor alpha, or basic fibroblast growth factor. These results suggest that HA is a potent inhibitor of the displacement of matrix proteoglycan into culture medium.

Stimulation of cell proliferation by hyaluronidase during in vitro aging of human skin fibroblasts

The effect of the degradation of extracellular hyaluronan on the proliferation of human skin fibroblasts in serial cultures during in vitro aging was investigated. Human skin fibroblasts at different time intervals from 3rd to 36th passages were exposed after plating to bovine testicular hyaluronidase. The enzyme treatment resulted in an increase in cell proliferation (cell number vs. time) as compared to the untreated control fibroblasts. The effect was dose dependent, reversible, and was independent of the type of the glycosidic linkage cleaved in hyaluronan. The increased proliferation was observed at all passages when untreated cells underwent mitosis. The degradation of hyaluronan induced cell proliferation up to the presenescent phase. Depletion of hyaluronan did not induce proliferation of postmitotic fibroblasts. The incorporation of 3H-glucosamine into hyaluronan decreased with increasing cell passages (increase of the number of population doublings). Twenty-fourth passage fibroblasts accumulated about two time less hyaluronan in the medium than ninth passage cultures. Following hyaluronidase treatment, the amount of newly synthesized, labeled hyaluronan increased in the medium. Accordingly, the fibroblasts restored the degraded hyaluronan even in the declining phase of proliferation (phase III according to Hayflick).

Cultured fibroblasts in avian scleroderma, an autoimmune fibrotic disease, display an activated phenotype

University of California, Davis, line 200 and 206 chickens spontaneously develop an autoimmune syndrome that has many features analogous to human scleroderma, including dermal fibrosis, antinuclear antibodies and antibodies to type II collagen. These birds also have thymic subcapsular epithelial defects and an abnormality in T cell calcium influx and proliferation in response to both T cell receptor-dependent and -independent activators. To determine whether fibroblast activation is a contributing factor to development of skin fibrosis in line 200 206 chickens, as it is in human scleroderma, we studied the collagen, non-collagenous protein and glycosaminoglycan (GAG) production of 34 separate fibroblast lines derived from the normal and fibrotic skin of line 200 and 206 chickens and from the skin of control chicken lines 058 and 254. The mean ± SEM 24-h incorporation of 3H-proline or 3H-glucosamine into extracellular collagen, non-collagenous protein or GAG by first passage fibroblast lines derived from the fibrotic skin of diseased birds was 1.526 ± 136, 859 ± 82 and 25.7 ± 1.3 dpm 10 3 cells , respectively, while fibroblast lines derived from the skin of control birds produced only 341 ± 36, 343 ± 42 and 15.2 ± 1.4 dpm 10 3 cells . Similar differences in results were recorded for cell-associated production, and when collagen and non-collagenous protein production were assessed using non-radioactive electrophoretic methods. The activated phenotype of the fibroblast lines derived from the fibrotic skin of diseased birds persisted through 10 cell doublings in tissue culture. However, the ratio of type I:III collagen and the profile of GAG types produced were similar in all fibroblast lines studied. These results suggest that fibroblast activation is responsible for the skin fibrosis observed in this avian model of scleroderma.

Human melanoma cell-derived factor(s) stimulate fibroblast glycosaminoglycan synthesis.

Conditioned media from cultures of human metastatic Hs294T melanoma cells contain a factor/factors that promote(s) fibroblast-mediated contraction of collagen lattices, and stimulate(s) fibroblast glycosaminoglycan (GAG) synthesis. Complete medium from melanoma cell cultures stimulated fibroblast hyaluronate synthesis 9.3-fold, and sulphated GAG synthesis 2.6-fold, as measured by 3H-glucosamine incorporation. 35SO4 incorporation into sulphated GAGS was essentially unaltered, the net result being a decrease in the degree of sulphation. Fibroblasts synthesized hyaluronate with an increased molecular weight when grown in the presence of the melanoma-cell culture medium, while the molecular weights of heparan and chondroitin sulphates remained essentially unaltered. Our results indicate that the tumour-cell-derived factor(s) stimulate(s) changes in fibroblast glycosaminoglycan synthesis, and that these changes may facilitate tumour cell invasion in vivo.