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

ABSTRACT: 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.

SUBMITTER: McQuillan DJ

PROVIDER: S-EPMC1151210 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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Project description: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.

Project description:Parathyroid hormone (PTH) and PTH-related protein (PTHrP) act via PTH receptors in bone to stimulate bone resorption. Bone resorption is also stimulated by certain cytokines, which are produced in bone and bone marrow. The effects of such cytokines on the PTH-receptor system were studied in the osteoblast-like osteosarcoma cell line UMR 106-06. 125I-labelled PTHrP-(1-84)-peptide bound specifically to the cells, and PTHrP-(1-34) and -(1-84) competed with equimolar affinity for binding to UMR 106-06 cells. The specific binding of 125I-PTHrP-(1-84) could be completely blocked by PTH. Therefore 125I-PTHrP-(1-84) bound to a classical receptor in UMR 106-06 cells. Preincubation for 3 days with either tumour necrosis factor alpha (TNF alpha) or retinoic acid (RA) both decreased the specific binding of 125I-PTHrP-(1-84) to about 40% of control levels. These effects were specific for PTH binding, since there was little effect on 125I-salmon-calcitonin binding. Both TNF alpha and RA required 24 h exposure to cells to produce a measurable effect. The decrease in 125I-PTHrP-(1-84) binding was due to a reduced number of binding sites, with little apparent change in affinity. Half-maximal effects were seen with 1 ng of TNF alpha/ml, whereas 1 microM-RA was needed to observe the loss of PTH receptors. Combinations of RA and TNF alpha produced a greater effect than that of either agonist alone. The loss of PTH receptors was accompanied by a specific loss of PTH-stimulated cyclic AMP production. Preincubation with TNF alpha increased the basal plasminogen activator (PA) activity in the cells and decreased the amplitude of the response of PA activity to PTH compared with control cells. Furthermore TNF alpha decreased sensitivity to PTH (50% stimulation of PA activity with 0.1 nM-PTH in control cells versus 50% stimulation with 0.3 nM-PTH in TNF alpha-treated cells). In contrast, TNF alpha pretreatment increased the amplitude of the response of PA activity to calcitonin, whereas sensitivity to calcitonin was not altered. These data are consistent with a specific down-regulation of PTH receptors in osteoblast-like UMR 106-06 cells after exposure to TNF alpha or RA. The loss of PTH receptors is accompanied by a decreased responsiveness to PTH, as measured with the PA system in these cells. A loss of PTH receptors could modulate PTH responses in osteoblasts, either in the local control of bone formation and resorption, or in pathological conditions such as humoral hypercalcaemia of malignancy.

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

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