Effect of 12-O-tetradecanoylphorbol-13-acetate on cell-surface glycosaminoglycans in human diploid fibroblasts

1984 ◽  
Vol 62 (12) ◽  
pp. 1354-1357
Author(s):  
Bruce R. Wolff ◽  
Bernard R. Glick ◽  
Niels C. Bols
Keyword(s):  
Hyaluronic Acid ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Human Fibroblasts ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Other ◽  
Human Skin Fibroblasts ◽  
Human Diploid Fibroblasts

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the loss of glycosaminoglycans (GAGs) from the pericellular compartment of human skin fibroblasts was studied. GAGs from this compartment were analyzed by anion-exchange chromatography after cultures had been labelled with both [3H]glucosamine and [35S]sulfate and then chased in either the presence or absence of TPA. In both control and TPA cultures radioactivity was found in glycopeptides, hyaluronic acid, heparan sulfate (HS), and chondroitin sulfate. The amount of radioactivity that was found in HS was reduced in the TPA cultures, whereas the amounts in the other GAGs were essentially unchanged when control and treated cultures were compared. These results suggest that TPA stimulates the loss of HS from the surface of human fibroblasts.

scholarly journals Relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan.

1976 ◽  
Vol 71 (1) ◽  
pp. 280-294 ◽  
Author(s):  
R H Cohn ◽  
J J Cassiman ◽  
M R Bernfield
Keyword(s):  
Hyaluronic Acid ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Cell Density ◽  
Growth Control ◽  
Cellular Distribution ◽  
High Cell ◽  
Density Dependent ◽  
Relationship Of

Mouse 3T3 cells and their Simian Virus 40-transformed derivatives (3T3SV) were used to assess the relationship of transfromation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan (GAG). Glucosamine- and galactosamine-containing GAG were labeled equivalently by [3H=A1-glucose regardless of culture type, allowing incorporation into the various GAG to be compared under all conditions studied. Three components of each culture type were examined: the cells, which contain the bulk of newly synthesized GAG and are enriched in chondroitin sulfate and heparan sulfate; cell surface materials released by trypsin, which contain predominantly hyaluronic acid; and the media , which contain predominantly hyaluronic acid and undersulfated chondroitin sulfate. Increased cell density and viral transformation reduce incorporation into GAG relative to the incorporation into other polysaccharides. Transformation, however, does not substantially alter the type or distribution of newly synthesized GAG; the relative amounts and cellular distributions were very similar in 3T3 and 3T3SV cultures growing at similar rates at low densities. On the other hand, increased cell density as well as density-dependent growth inhibition modified the type and distribution of newly synthesized GAG. At high cell densities both cell types showed reduced incorporation into hyaluronate and an increase in cellular GAG due to enhanced labeling of chondroitin sulfate and heparan sulfate. These changes were more marked in confluent 3T3 cultures which also differed in showing substantially more GAG label in the medium and in chondroitin-6-sulfate and heparan sulfate at the cell surface. Since cell density and possibly density-dependent inhibition of growth but not viral transformation are major factors controlling the cellular distribution and type of newly synthesized GAG, differences due to GAG's in the culture behavior of normal and transformed cells may occur only at high cell density. The density-induced GAG alterations most likely involved are increased condroitin-6-sulfate and heparan sulfate and decreased hyaluronic acid at the cell surface.

Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts. Implications for their roles in cell-substratum adhesion

1993 ◽  
Vol 106 (1) ◽  
pp. 55-65 ◽  
Author(s):  
M. Yamagata ◽  
S. Saga ◽  
M. Kato ◽  
M. Bernfield ◽  
K. Kimata
Keyword(s):  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
The Other ◽  
Stress Fibers ◽  
Chondroitin Sulfate Proteoglycan ◽  
Pericellular Matrix ◽  
Sulfate Proteoglycan ◽  
Cultured Fibroblasts ◽  
Focal Contacts ◽  
The Difference

We showed previously that a large chondroitin sulfate proteoglycan, PG-M (also known as versican), inhibits cell-substratum adhesion, while basement membrane heparan sulfate proteoglycan (recently named perlecan) does not (Yamagata et al. (1989) J. Biol. Chem. 264, 8012–8018). To extend our understanding of the adhesive function of these proteoglycans, we examined the pericellular localization of the proteoglycans and their ligands and also that of some matrix receptors and cytoskeletal molecules in various fibroblast culture systems. PG-M was abundant in the subcellular space of fibroblasts, but was excluded selectively from focal contacts where vinculin, integrins and fibronectin were localized. Hyaluronan, CD44 and tenascin were distributed similarly as PG-M. In contrast, perlecan was associated with fibronectin and was included in focal contacts. Syndecan-1, a membrane heparan sulfate/chondroitin sulfate proteoglycan, was associated with fibronectin at the cell surface, partly at focal contacts and in association with stress fibers. Thus, complexes of PG-M with hyaluronan, tenascin and CD44, are not involved in focal contacts. On the other hand, perlecan and syndecan-1 together with fibronectin may participate in focal contacts. The difference in localization between these proteoglycans may be related to their glycosaminoglycan content and to their distinctive roles in cell-substratum adhesion.

scholarly journals Codistribution of heparan sulfate proteoglycan, laminin, and fibronectin in the extracellular matrix of normal rat kidney cells and their coordinate absence in transformed cells.

1982 ◽  
Vol 94 (1) ◽  
pp. 28-35 ◽  
Author(s):  
E G Hayman ◽  
A Oldberg ◽  
G R Martin ◽  
E Ruoslahti
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Rat Kidney ◽  
Heparan Sulfate Proteoglycan ◽  
Chondroitin Sulfate Proteoglycan ◽  
Transformed Cells ◽  
Kidney Cells ◽  
Sulfate Proteoglycan ◽  
Normal Rat

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.

scholarly journals Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases.

1993 ◽  
Vol 4 (7) ◽  
pp. 669-677 ◽  
Author(s):  
P Ferrigno ◽  
T A Langan ◽  
P Cohen
Keyword(s):  
Rat Liver ◽  
Protein Phosphatase ◽  
Human Fibroblasts ◽  
High Mobility ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
High Mobility Group Protein ◽  
Cell Extracts ◽  
Dependent Protein

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.

scholarly journals Regulation of haemopoiesis in long-term bone marrow cultures. IV. Glycosaminoglycan synthesis and the stimulation of haemopoiesis by beta-D-xylosides.

1983 ◽  
Vol 96 (2) ◽  
pp. 510-514 ◽  
Author(s):  
E Spooncer ◽  
J T Gallagher ◽  
F Krizsa ◽  
T M Dexter
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Glycosaminoglycan Synthesis ◽  
Fivefold Increase ◽  
Bone Marrow Cultures ◽  
Marrow Cultures ◽  
Stimulation Of

Sulfated glycosaminoglycans (GAGs) are distributed in consistent and distinctive patterns between the cell surface and the growth medium of haemopoietically active long-term bone marrow cultures. Heparan sulfate is the main cell surface component and chondroitin sulfate is the major sulfated species in the medium. When the cultures are supplemented with beta-D-xylosides a significant increase in chondroitin sulfate synthesis is observed but no stimulation of heparan sulfate synthesis occurs. The chondroitin sulfate accumulates in the culture medium in beta-D-xyloside-treated cultures but the composition of sulfated GAGs in cell-surface derived material is unaffected. beta-D-xylosides also stimulate the production of haemopoietic cells without any apparent alteration in the adherent stromal cells of the marrow cultures. Equivalent increases are obtained in cells at all stages of development so that a fivefold increase in pluripotent stem cells (CFU-S) is matched by fivefold increase in the granulocyte-macrophage progenitors (GM-CFC) and in mature granulocytes. The stimulation persists for many weeks in beta-D-xyloside-treated cultures. These results indicate that the sulfated GAGs may play an important role in the regulation of haemopoiesis.

scholarly journals Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia.

1989 ◽  
Vol 109 (5) ◽  
pp. 2509-2518 ◽  
Author(s):  
A Rapraeger
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Chondroitin Sulfate ◽  
Transforming Growth Factor ◽  
Core Protein ◽  
Protein Characterization ◽  
Tgf Beta ◽  
The Core ◽  
Cell Surface Proteoglycan

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.

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