scholarly journals Plasma-membrane-intercalated heparan sulphate proteoglycans in an osteogenic cell line (UMR 106-01 BSP)

1992 ◽  
Vol 285 (1) ◽  
pp. 25-33 ◽  
Author(s):  
D J McQuillan ◽  
R J Midura ◽  
V C Hascall ◽  
M Yanagishita
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Cell Line ◽  
Granulosa Cells ◽  
Core Protein ◽  
Close Association ◽  
Heparan Sulphate ◽  
Osteosarcoma Cell ◽  
Ovarian Granulosa Cells ◽  
Umr 106

The heparan sulphate (HS) proteoglycans associated with the cell layer of a rat osteosarcoma cell line [UMR 106-01 (BSP)] were compared with similar cell-associated proteoglycans from other cells, and their interaction with the plasma membrane was studied. HS proteoglycans were metabolically labelled by incubation of cell cultures with [3H]glucosamine or [3H]leucine and [35S]sulphate. HS proteoglycan core protein preparation generated by heparitinase digestion of the major species from UMR 106-01 (BSP) cells co-migrated on PAGE with identical preparations from ovarian granulosa cells and parathyroid cells (at approximately 70 kDa). The hydrophobic nature of the major HS proteoglycans from these diverse cell lines, based on elution position from octyl-Sepharose, were also comparable. Linkages of the HS proteoglycan to the cell membrane were investigated by labelling plasma-membrane preparations with a lipid soluble photoactivatable reagent, 3-(trifluoromethyl)-3- (m-[125I]iodophenyl)diazirine (TID), which selectively labels plasma-membrane-spanning peptide domains. Purified HS proteoglycan from UMR 106-01 (BSP) cells was shown to be accessible to the [125I]TID, and the core protein portion of the molecule was labelled, confirming its close association with the plasma membrane. Approx. 36% of 35S-labelled HS proteoglycans were released from the cell surface by phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol-linked proteins. In the presence of insulin, the metabolism of the phospholipase C-sensitive population was unaltered; however, release of the phospholipase C-insensitive population into the medium was increased. These data indicate that a subpopulation of HS proteoglycans are covalently bound to the plasma membrane by a glycosylphosphatidylinositol structure, with the remainder representing those species directly inserted into the plasma membrane via a hydrophobic peptide domain. These observations are similar to those reported for ovarian granulosa cells [Yanagishita & McQuillan (1989) J. Biol. Chem. 264 17551-17558], and thus may represent a general phenomenon for many cell types.

scholarly journals Brefeldin A inhibits the endocytosis of plasma-membrane-associated heparan sulphate proteoglycans of cultured rat ovarian granulosa cells

1995 ◽  
Vol 310 (1) ◽  
pp. 271-278 ◽  
Author(s):  
L Uhlin-Hansen ◽  
M Yanagishita
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Granulosa Cells ◽  
Protein Transport ◽  
Heparan Sulphate ◽  
Brefeldin A ◽  
Inhibitory Effect ◽  
Surface Proteins ◽  
Gel Chromatography ◽  
Ovarian Granulosa Cells

Rat ovarian granulosa cells were labelled with [35S]sulphate for 0.5-20 h and chased in the presence or absence of 1-2 micrograms/ml of brefeldin A (BFA) for up to 21 h. Heparan [35S]sulphate (HS) proteoglycans from the culture medium, plasma membrane and intracellular fractions were then analysed by gel chromatography. In the absence of BFA, about 85% of the plasma membrane-associated HS proteoglycans were endocytosed and subsequently degraded intracellularly. Recirculation of the HS proteoglycans between the intracellular pool and the cell surface was not observed. Exposing the cells to BFA for less than 1 h did not influence the turnover of the HS proteoglycans, whereas the effect of the drug on the Golgi functions reached a maximum in approx. 10 min. When the cells were treated with BFA for more than 1-2 h, the rate of endocytosis of HS proteoglycans was reduced to about 50% of the control. The delivery of endocytosed HS proteoglycans to lysosomes were not affected by the drug. Cycloheximide also reduced the endocytosis of HS proteoglycans, but not as much as BFA, indicating that the inhibitory effect of BFA can be only partly accounted for by a block of protein transport from the endoplasmic reticulum to the plasma membrane. In contrast with the endocytosis of HS proteoglycans, neither that of 125I-transferrin, known to be mediated by clathrin-coated vesicles, nor that of 125I-ricin, a marker molecule for bulk endocytosis, was affected by BFA. The half-life of 125I-transferrin and 125I-ricin in the plasma membrane was about 10 and 25 min respectively compared with about 5 h for the HS proteoglycans. Altogether, these results indicate that the endocytosis of plasma-membrane-associated HS proteoglycans is mediated by different mechanisms than the endocytosis of most other cell-surface proteins. Further, the mechanisms involved in the endocytosis of HS proteoglycans are sensitive to BFA.

scholarly journals Proteoglycans synthesized by an osteoblast-like cell line (UMR 106-01)

1991 ◽  
Vol 277 (1) ◽  
pp. 199-206 ◽  
Author(s):  
D J McQuillan ◽  
D M Findlay ◽  
A M Hocking ◽  
M Yanagishita ◽  
R J Midura ◽  
...  
Keyword(s):  
Cell Line ◽  
Core Protein ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Cell Types ◽  
Heparan Sulphate Proteoglycan ◽  
Sulphate Proteoglycan ◽  
Chondroitin Sulphate Proteoglycan ◽  
Umr 106

The proteoglycans synthesized by an osteoblast-like cell line of rat origin (UMR 106-01) were defined after biosynthetic labelling with [35S]sulphate and [3H]glucosamine. Newly synthesized labelled proteoglycans were characterized by differential enzymic digestion in combination with analytical gel filtration and SDS/PAGE. UMR 106-01 cells were found to synthesize three major species of proteoglycan: a large chondroitin sulphate proteoglycan of Mr approximately 1 x 10(6), with a core protein of Mr approximately 350,000-400,000; a small chondroitin sulphate-containing species of Mr approximately 120,000 with a core protein of Mr 43,000; and a heparan sulphate proteoglycan of Mr approximately 150,000, with a core protein of Mr approximately 80,000. Over 70% of the newly synthesized intact proteoglycan species are associated with the cell layer of near-confluent cells; however, accessibility to trypsin digestion suggests an extracellular location. Chemical characteristics of the proteoglycans and preliminary mRNA hybridization indicate that the small chondroitin sulphate proteoglycan is probably PG II (decorin). The large chondroitin sulphate proteoglycan is most likely related to a hyaluronate-aggregating species from fibroblasts (versican), and the heparan sulphate proteoglycan bears striking similarities to cell-membrane-intercalated species described for a number of cell types.

Characterization of Phospholipase C Activity of the Plasma Membrane and Cytosol of an Osteoblast-like Cell Line

1989 ◽  
Vol 297 (3) ◽  
pp. 135-144 ◽  
Author(s):  
Yasuo Suzuki ◽  
Keith A. Hruska ◽  
Ian Reid ◽  
Ulises M. Alvarez ◽  
Louis V. Avioli
Keyword(s):  
Plasma Membrane ◽  
Phospholipase C ◽  
Cell Line

A Monoclonal Antibody to a Mammary Cell Line Recognizes Two Distinct Subtypes of Ovarian Granulosa Cells*

Endocrinology ◽  
1985 ◽  
Vol 117 (4) ◽  
pp. 1490-1499 ◽  
Author(s):  
GREGORY F. ERICKSON ◽  
CHRIS HOFEDITZ ◽  
MICHAEL UNGER ◽  
W. ROSS ALLEN ◽  
RENATO DULBECCO
Keyword(s):  
Monoclonal Antibody ◽  
Cell Line ◽  
Granulosa Cells ◽  
Ovarian Granulosa Cells ◽  
Mammary Cell

scholarly journals Annexins A2, A6 and Fetuin-A Affect the Process of Mineralization in Vesicles Derived from Human Osteoblastic hFOB 1.19 and Osteosarcoma Saos-2 Cells

2021 ◽  
Vol 22 (8) ◽  
pp. 3993
Author(s):  
Lukasz Bozycki ◽  
Joanna Mroczek ◽  
Laurence Bessueille ◽  
Saida Mebarek ◽  
René Buchet ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Matrix Vesicles ◽  
Osteoblastic Cell ◽  
Apatite Formation ◽  
Osteosarcoma Cell ◽  
Osteoblastic Cell Line ◽  
Inorganic Pyrophosphate ◽  
Fetuin A ◽  
Cytoplasmic Vesicles

The mineralization process is initiated by osteoblasts and chondrocytes during intramembranous and endochondral ossifications, respectively. Both types of cells release matrix vesicles (MVs), which accumulate Pi and Ca2+ and form apatites in their lumen. Tissue non-specific alkaline phosphatase (TNAP), a mineralization marker, is highly enriched in MVs, in which it removes inorganic pyrophosphate (PPi), an inhibitor of apatite formation. MVs then bud from the microvilli of mature osteoblasts or hypertrophic chondrocytes and, thanks to the action of the acto-myosin cortex, become released to the extracellular matrix (ECM), where they bind to collagen fibers and propagate mineral growth. In this report, we compared the mineralization ability of human fetal osteoblastic cell line (hFOB 1.19 cells) with that of osteosarcoma cell line (Saos-2 cells). Both types of cells were able to mineralize in an osteogenic medium containing ascorbic acid and beta glycerophosphate. The composition of calcium and phosphate compounds in cytoplasmic vesicles was distinct from that in extracellular vesicles (mostly MVs) released after collagenase-digestion. Apatites were identified only in MVs derived from Saos-2 cells, while MVs from hFOB 1.19 cells contained amorphous calcium phosphate complexes. In addition, AnxA6 and AnxA2 (nucleators of mineralization) increased mineralization in the sub-membrane region in strongly mineralizing Saos-2 osteosarcoma, where they co-localized with TNAP, whereas in less mineralizing hFOB 1.19 osteoblasts, AnxA6, and AnxA2 co-localizations with TNAP were less visible in the membrane. We also observed a reduction in the level of fetuin-A (FetuA), an inhibitor of mineralization in ECM, following treatment with TNAP and Ca channels inhibitors, especially in osteosarcoma cells. Moreover, a fraction of FetuA was translocated from the cytoplasm towards the plasma membrane during the stimulation of Saos-2 cells, while this displacement was less pronounced in stimulated hFOB 19 cells. In summary, osteosarcoma Saos-2 cells had a better ability to mineralize than osteoblastic hFOB 1.19 cells. The formation of apatites was observed in Saos-2 cells, while only complexes of calcium and phosphate were identified in hFOB 1.19 cells. This was also evidenced by a more pronounced accumulation of AnxA2, AnxA6, FetuA in the plasma membrane, where they were partly co-localized with TNAP in Saos-2 cells, in comparison to hFOB 1.19 cells. This suggests that both activators (AnxA2, AnxA6) and inhibitors (FetuA) of mineralization were recruited to the membrane and co-localized with TNAP to take part in the process of mineralization.

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