scholarly journals Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels

1983 ◽  
Vol 215 (1) ◽  
pp. 107-116 ◽  
Author(s):  
J T Gallagher ◽  
N Gasiunas ◽  
S L Schor
Keyword(s):  
Human Skin ◽  
Heparan Sulphate ◽  
Surface Membrane ◽  
Skin Fibroblasts ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Cellular Functions ◽  
Iduronic Acid ◽  
Sulphated Glycosaminoglycans

Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium. Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium. Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels. In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface. The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate. The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions.

Thyroid hormone excess stimulates the synthesis of proteoglycan in human skin fibroblasts in culture

1990 ◽  
Vol 123 (5) ◽  
pp. 541-549 ◽  
Author(s):  
Yoshimasa Shishiba ◽  
Yasuhiro Takeuchi ◽  
Noriko Yokoi ◽  
Yasunori Ozawa ◽  
Taeko Shimizu
Keyword(s):  
Thyroid Hormone ◽  
Human Skin ◽  
Specific Activity ◽  
Heparan Sulphate ◽  
Skin Fibroblasts ◽  
Proteoglycan Synthesis ◽  
Heparan Sulphate Proteoglycan ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Sulphate Proteoglycan

Abstract We previously demonstrated that proteoglycan accumulated in the affected skin of circumscribed pretibial myxedema of Graves' disease. As an underlying mechanism responsible for the accumulation, we sought to determine whether excess thyroid hormone was partially responsible for the increase in proteoglycan synthesis. Human skin fibroblasts were cultured in Ham's F-10 medium containing 1% Nutridoma with graded doses of T3 (0.184 × 10−9 to 46 × 10−9 mol/l) and were labelled with [35S]sulphate and [3H]glucosamine. Proteoglycans were purified by Sephadex G-50, Q-Sepharose chromatography with NaCl-gradient and Sepharose CL-6B chromatography. 35S and 3H incorporated into dermatan sulphate proteoglycan and heparan sulphate proteoglycan and 3H incorporated into hyaluronan were measured. 35S and 3H incorporation into dermatan sulphate proteoglycan was minimum at a T3 concentration of 0.184 × 10−9 mol/l, and increased with increasing doses of T3 up to 46 × 10−9 mol/l. 35S and 3H incorporation into heparan sulphate proteoglycan also increased with increasingdoses of T3. 3H incorporation into hyaluronan was not influenced at all by T3. The increased incorporation of 35S into proteoglycan in high-T3 culture reflects the increased synthesis of proteoglycan because 1. the extent of sulphation of disaccharides examined by thin-layer chromatography was not altered by T3; 2. the specific activity of [35S]sulphate was not influenced by T3, and 3. T3 did not decrease the degradation rate of cell-associated proteoglycan.

scholarly journals Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

1980 ◽  
Vol 190 (2) ◽  
pp. 243-254 ◽  
Author(s):  
J T Gallagher ◽  
N Gasiunas ◽  
S L Schor
Keyword(s):  
Hyaluronic Acid ◽  
Salt Concentration ◽  
Heparan Sulphate ◽  
Collagen Gel ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Sulphated Glycosaminoglycans ◽  
Sulphated Glycosaminoglycan ◽  
Spent Media

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.

scholarly journals Interferon γ differentially affects the synthesis of chondroitin/dermatan sulphate and heparan sulphate by human skin fibroblasts

1996 ◽  
Vol 318 (3) ◽  
pp. 863-870 ◽  
Author(s):  
Christel PRAILLET ◽  
Hugues LORTAT-JACOB ◽  
Jean-Alexis GRIMAUD
Keyword(s):  
Human Skin ◽  
Normal Skin ◽  
Heparan Sulphate ◽  
Periodate Oxidation ◽  
Interferon Γ ◽  
Skin Fibroblasts ◽  
Size Exclusion ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Induced Changes

Interferon γ (IFNγ) is often considered to be an antifibrotic cytokine because it inhibits collagen synthesis in fibroblasts. Here we report the effects of recombinant human IFNγ on sulphated glycosaminoglycan chains produced by normal skin fibroblasts from adult donors. IFNγ (250 i.u./ml) induced an increase in incorporation of d-[1-3H]glucosamine into glycosaminoglycans, either secreted into the culture medium or associated with the cell layer. The structures of these molecules were analysed by using various cleavage agents (heparinases I and II, heparitinase/chondroitinases ABC and AC/periodate oxidation) followed by size-exclusion and anion-exchange HPLC. No modification was detected in the structure of the heparan sulphate chains. In contrast, the cytokine induced changes in the microcomposition of chondroitin/dermatan sulphate chains. More precisely, we found a decrease in the iduronic acid content, associated with down-regulation of the 4-O-sulphation on the GalNAc residues. In contrast, the 6-O-sulphation on these GalNAc residues was potentiated by the cytokine. These results indicate that IFNγ is able to modulate not only collagen but also the structure of galactosaminoglycans synthesized by human skin fibroblasts.

scholarly journals Identification of N-sulphated disaccharide units in heparin-like polysaccharides

1979 ◽  
Vol 179 (1) ◽  
pp. 77-87 ◽  
Author(s):  
I Jacobsson ◽  
M Höök ◽  
I Pettersson ◽  
U Lindahl ◽  
O Larm ◽  
...  
Keyword(s):  
Human Skin ◽  
Paper Chromatography ◽  
Uronic Acid ◽  
Heparan Sulphate ◽  
Paper Electrophoresis ◽  
Exchange Chromatography ◽  
Skin Fibroblasts ◽  
Gel Chromatography ◽  
Human Skin Fibroblasts ◽  
Iduronic Acid

1. Preparations of heparin and heparan sulphate were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and were then fractionated into non-sulphated, monosulphated and disulphated species by ion-exchange chromatography or by paper electrophoresis. The non-sulphated disaccharides were separated into two, and the monosulphated disaccharides into three, components by paper chromatography. 2. The uronic acid moieties of the various non- and mono-sulphated disaccharides were identified by means of radioactive labels selectively introduced into uronic acid residues (3H and 14C in D-glucuronic acid, 14C only in L-iduronic acid units) during biosynthesis of the polysaccharide starting material. Labelled uronic acids were also identified by paper chromatography, after liberation from disaccharides by acid hydrolysis or by glucuronidase digestion. Similar procedures, applied to disaccharides treated with NaB3H4, indicated 2,5-anhydro-D-mannitol as reducing terminal unit. On the basis of these results, and the known positions and configurations of the glycosidic linkages in heparin, the two non-sulphated disaccharides were identified as 4-O-(beta-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. 3. The three monosulphated [1-3H]anhydromannitol-labelled disaccharides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analysed by paper electrophoresis. The results, along with the 1H n.m.r. spectra of the corresponding unlabelled disaccharides, permitted the allocation of O-sulphate groups to various positions in the disaccharides. These were thus identified as 4-O-(beta-D-glucopyranosyl-uronic acid)-2,5-anhydro-D-mannitol 6-sulphate, 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate and 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was found to be a poor substrate for the iduronate sulphatase of human skin fibroblasts, as compared with the disulphated species, 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol 6-sulphate. 4. The identified [1-3H]anhydromannitol-labelled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulphates. Low N-sulphate contents, most pronounced in the heparin sulphates, were associated with high ratios of mono-O-sulphated/di-O-sulphated (N-sulphated) disaccharide units, and in addition, with relatively large amounts of 2-sulphated L-iduronic acid residues bound to C-4 of N-sulpho-D-glucosamine units lacking O-sulphate substituents.

SECMA 1®, a mitogenic hexapeptide from Ulva algeae modulates the production of proteoglycans and glycosaminoglycans in human foreskin fibroblast

1998 ◽  
Vol 17 (1) ◽  
pp. 18-22 ◽  
Author(s):  
R Ennamany ◽  
D Saboureau ◽  
N Mekideche ◽  
E E Creppy
Keyword(s):  
Glucuronic Acid ◽  
De Novo ◽  
Heparan Sulphate ◽  
Guanidine Hydrochloride ◽  
Sodium Deoxycholate ◽  
Effective Concentration ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Dermatan Sulphate ◽  
Salt Extract

SECMA 1® is a polypeptide purified from a green algeae of the Ulva species by several gel chromatographies, showing the following sequence (Glu-Asp-Arg-Leu-Lys-Pro). In order to determine the effect of SECMA 1® on human skin fibroblasts extracellular matrix, proteoglycans (PGs) and glycosaminoglycans (GAGs) were assayed after 24 h incubation of 20 day-old foreskin fibroblasts at the 2nd passage. The results revealed that most of [35S]sulphate was associated with fibroblast membranes, which contained (67%) of the total de novo synthesized sulphated PGs, in two distinct forms: one hydrophilic (39%), and one hydrophobic (28%). The remaining `matrix' retained 5% of proteoglycans. The remaining 35S-label may represent the free label in the cytosol. After 24 h incubation of skin fibroblasts with different concentrations of SECMA 1® (2, 4 and 10 μg/ml), the [35S] sulphate incorporation into PGs of Salt-extract, sodium deoxycholate (DOC) extract and Guanidine hydrochloride (GuA-HCl)-extract was increased significantly ( P<0.005) with 4 μg/ml, as compared to untreated control. The most effective concentration (4 μg/ml) increased the different [35S]sulphate PGs extracts (NaCl, DOC and GuA-HCl) by respectively (66; 17 and 75%). The relative contents of iduronic and glucuronic acid in the GAG produced by skin fibroblasts were estimated. No effect of SECMA 1® on the incorporation of [35S]sulphate into Heparan sulphate was found. The incorporation of [35S]sulphate into (chondroïtine sulphate + heparan sulphate) and (chondroïtine sulphate + dermatan sulphate) was increased by respectively 37% and 11% by SECMA 1® (4 μg/ml).

scholarly journals Changes in the synthesis, distribution and sulphation of glycosaminoglycans of cultured human skin fibroblasts upon ascorbate feeding

1983 ◽  
Vol 64 (1) ◽  
pp. 245-254
Author(s):  
M. Edward ◽  
R.F. Oliver
Keyword(s):  
Ascorbic Acid ◽  
Cell Density ◽  
Human Skin ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Collagen Production ◽  
Sulphated Glycosaminoglycans ◽  
Cell Fractions

The effect of ascorbic acid on the synthesis, distribution and sulphation of glycosaminoglycans by human skin fibroblasts has been examined. Medium was supplemented with ascorbate over several days, and cultures incubated with [3H]glucosamine and Na2(35)SO4 for 48 h, followed by analysis of the glycosaminoglycans in the medium, in collagenase and trypsin extracts, and in cell fractions. Ascorbate feeding resulted in a reduction in hyaluronate synthesis, which was the main 3H-labelled component and was distributed mainly in the medium fractions. Sulphated glycosaminoglycans showed a reduction in incorporation of 3H label, but increased sulphation following ascorbate feeding. In control cultures 53% of 3H-labelled sulphated glycosaminoglycans and 63% of 35S-labelled glycosaminoglycans were present in the medium fraction, while in ascorbate-fed cultures, 41% of 3H label and 38% 35S label were incorporated into medium-sulphated glycosaminoglycans. Ascorbate also caused an increase in cell density and in collagen production and deposition.

scholarly journals Studies on the mechanism of hydrated collagen gel reorganization by human skin fibroblasts

1985 ◽  
Vol 79 (1) ◽  
pp. 67-81 ◽  
Author(s):  
C. Guidry ◽  
F. Grinnell
Keyword(s):  
Human Skin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Collagen Fibrils ◽  
Skin Fibroblasts ◽  
Electron Microscopic Level ◽  
Total Protein Content ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Electron Microscopic

During reorganization of collagen gels by human skin fibroblasts the total protein content of the gels remained approximately constant. Only 5% of the collagen was degraded, although the volume of the gels decreased by 85% or more. It could be concluded, therefore, that gel reorganization required physical rearrangement of pre-existing collagen fibrils rather than degradation of the original collagen and resynthesis of a new matrix. Collagen molecules in the gels were not covalently crosslinked or otherwise modified enzymically during gel reorganization, as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and collagen repolymerization studies. Serum was required for gel reorganization and, in the absence of serum, cell spreading was predominantly filipodial, i.e. there was little cytoplasmic reorganization. At the electron-microscopic level it was found that many more collagen fibrils became associated with the cells in the presence of serum than in its absence. Serum was also found to promote the synthesis and secretion of proteins by the cells, and conditioned medium could take the place of serum in promoting gel reorganization. The involvement of cell-secreted factors was also demonstrated by the ability of cycloheximide to inhibit gel reorganization. Finally, when gel reorganization was stopped by adding cytochalasin D to the incubations or removing cells by detergent treatment, a small but significant re-expansion of the collagen fibrils was observed. Consequently, a portion of the collagen that had been physically reorganized by the gels was unstable and could not hold its position without continued force exerted by the cells.

scholarly journals Inventory of human skin fibroblast proteoglycans. Identification of multiple heparan and chondroitin/dermatan sulphate proteoglycans

1990 ◽  
Vol 265 (1) ◽  
pp. 289-300 ◽  
Author(s):  
A Schmidtchen ◽  
I Carlstedt ◽  
A Malmström ◽  
L Å Fransson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Heparan Sulphate ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblast ◽  
Heparan Sulphate Proteoglycan ◽  
Dermatan Sulphate ◽  
The Core ◽  
Sulphate Proteoglycan ◽  
Core Proteins

Heparan sulphate and chondroitin/dermatan sulphate proteoglycans of human skin fibroblasts were isolated and separated after metabolic labelling for 48 h with 35SO4(2-) and/or [3H]leucine. The proteoglycans were obtained from the culture medium, from a detergent extract of the cells and from the remaining ‘matrix’, and purified by using density-gradient centrifugation, gel and ion-exchange chromatography. The core proteins of the various proteoglycans were identified by electrophoresis in SDS after enzymic removal of the glycosaminoglycan side chains. Skin fibroblasts produce a number of heparan sulphate proteoglycans, with core proteins of apparent molecular masses 350, 250, 130, 90, 70, 45 and possibly 35 kDa. The major proteoglycan is that with the largest core, and it is principally located in the matrix. A novel proteoglycan with a 250 kDa core is almost entirely secreted or shed into the culture medium. Two exclusively cell-associated proteoglycans with 90 kDa core proteins, one with heparan sulphate and another novel one with chondroitin/dermatan sulphate, were also identified. The heparan sulphate proteoglycan with the 70 kDa core was found both in the cell layer and in the medium. In a previous study [Fransson, Carlstedt, Cöster & Malmström (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5657-5661] it was suggested that skin fibroblasts produce a proteoglycan form of the transferrin receptor. However, the core protein of the major heparan sulphate proteoglycan now purified does not resemble this receptor, nor does it bind transferrin. The principal secreted proteoglycans are the previously described large chondroitin sulphate proteoglycan (PG-L) and the small dermatan sulphate proteoglycans (PG-S1 and PG-S2).

scholarly journals Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways

2001 ◽  
Vol 154 (3) ◽  
pp. 535-548 ◽  
Author(s):  
Vishwajeet Puri ◽  
Rikio Watanabe ◽  
Raman Deep Singh ◽  
Michel Dominguez ◽  
Jennifer C. Brown ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Skin ◽  
Growth Factor Receptor ◽  
Dominant Negative ◽  
Skin Fibroblasts ◽  
Human Skin Fibroblasts ◽  
Cellular Functions ◽  
Intracellular Targeting ◽  
Direct Demonstration ◽  
Early Endosomes

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent (“caveolar-like”) mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a “vital marker” for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

scholarly journals The effects of fibronectin on the migration of human foreskin fibroblasts and Syrian hamster melanoma cells into three-dimensional gels of native collagen fibres

1981 ◽  
Vol 48 (1) ◽  
pp. 301-314
Author(s):  
S.L. Schor ◽  
A.M. Schor ◽  
G.W. Bazill
Keyword(s):  
Human Skin ◽  
Syrian Hamster ◽  
Melanoma Cells ◽  
Skin Fibroblasts ◽  
Collagen Gels ◽  
Human Skin Fibroblasts ◽  
Collagen Fibres ◽  
3 Dimensional ◽  
Native Collagen ◽  
Hamster Melanoma

The effects of fibronectin on the migration of human skin fibroblasts and Syrian hamster melanoma cells into 3-dimensional gels of native collagen fibres have been examined. Cell migration into the 3-dimensional gel was measured by plating cells on the gel surface and then determining the percentage of cells within the gel at various times thereafter by direct microscopic examination. We find that fibronectin bound to collagen inhibits the migration of human skin fibroblasts and stimulates the migration of melanoma cells into the gel matrix. Fibronectin had no apparent effect on cell adhesion to the collagen gels, proliferation or morphology under the conditions studied.

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