monensin and Osteosarcoma

We have previously described a specific, saturable receptor for rat collagenase-3 in the rat osteosarcoma cell line, UMR 106-01. Binding of rat collagenase-3 to this receptor is coupled to the internalization and eventual degradation of the enzyme and correlates with observed extracellular levels of the enzyme. In this study we have shown that decreased binding, internalization, and degradation of 125I-rat collagenase-3 were observed in cells after 24 h of parathyroid hormone treatment; these activities returned to control values after 48 h and were increased substantially (twice control levels) after 96 h of treatment with the hormone. Subcellular fractionation studies to identify the route of uptake and degradation of collagenase-3 localized intracellular accumulation of 125I-rat collagenase-3 initially in Golgi-associated lysosomes and later in secondary lysosomes. Maximal lysosomal accumulation of the radiolabel and stimulation of general lysosomal activity occurred after 72 h of parathyroid hormone treatment. Preventing fusion of endosomes with lysosomes (by temperature shift, colchicine, or monensin) resulted in no internalized 125I-collagenase-3 in either lysosomal fraction. Treatment of UMR cells with the above agents or ammonium chloride decreased excretion of 125I-labeled degradation products of collagenase-3. These experiments demonstrated that degradation of collagenase-3 required receptor-mediated endocytosis and sequential processing by endosomes and lysosomes. Thus, parathyroid hormone regulates the expression and synthesis of collagenase-3 as well as the abundance and functioning of the collagenase-3 receptor and the intracellular degradation of its ligand. The coordinate changes in the secretion of collagenase-3 and expression of the receptor determine the net abundance of the enzyme in the extracellular space.

Topics: Alkaline Phosphatase; alpha-Mannosidase; Ammonium Chloride; Animals; beta-Galactosidase; Biological Transport; Biomarkers; Bone Neoplasms; Colchicine; Collagenases; Endocytosis; Endosomes; Intracellular Fluid; Isoenzymes; Ligands; Lysosomes; Mannosidases; Matrix Metalloproteinase 13; Monensin; Neoplasm Proteins; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Rats; Receptors, Cell Surface; Temperature; Tumor Cells, Cultured

Some integrin receptors have been reported to be functionally distinct in different cell types. In endothelial and melanoma cells, the vitronectin receptor, alpha v beta 3 binds fibrinogen (fg) and von Willebrand factor (vWf) in addition to vitronectin itself, whereas it fails to do so in MG-63 osteosarcoma cells. In this report, it is shown that, in the presence of Mn2+, MG-63 cells attach more efficiently to vitronectin and acquire the de novo capacity to adhere to fg- and vWf-coated surfaces. The latter phenomenon occurs with full cell spreading, F-actin microfilament organization, and alpha v and beta 3 clustering at focal contacts. In contrast, beta 1 and beta 5 do not localize to adhesion plaques under the same experimental conditions. An antiserum to the beta 3 chain and a synthetic peptide containing the sequence Gly-Arg-Gly-Asp-Ser-Pro block MG-63 attachment to fg and vWf in the presence of Mn2+. The minimal active concentration of Mn2+ is in the range of 0.1 to 1 microns. These data suggest that the acquired capacity of MG-63 to attach to fg and vWf in the presence of Mn2+ is mediated by alpha v beta 3 and that differences in alpha v beta 3 receptor specificity may be modulated by exogenous factors.

Topics: Amino Acid Sequence; Cations, Divalent; Cell Adhesion; Fibrinogen; Fibronectins; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Integrins; Manganese; Molecular Sequence Data; Monensin; Oligopeptides; Osteosarcoma; Peptides; Receptors, Immunologic; Receptors, Vitronectin; Tumor Cells, Cultured; von Willebrand Factor

Administration of excessive amounts of parathyroid hormone (PTH) in the treatment of osteoporosis can reverse the beneficial effects of a low-dose, intermittent regime. To investigate the direct actions and the possible cellular mechanisms of PTH in inducing desensitization of PTH receptors, we studied the effects of desensitization on rat osteoblastic UMR-106 cells. When the osteoblasts were preincubated with bPTH-(1-34), complete refractoriness to a subsequent challenge with the hormone developed within 1 h and at hormone concentrations as low as 5 nM. When osteoblasts thus desensitized were incubated in hormone-free medium, recovery of the cAMP responses began within 2 h and reached maximum after 16 h. Cycloheximide did not affect the process of desensitization. [Nle8,Nle18,Tyr34]bPTH-(3-34)amide significantly impaired the desensitization process by PTH-(1-34) but did not have stimulatory effect on cAMP responses. No significant heterologous desensitization was obvious after preincubation with isoprenaline (50 microM), prostaglandin E1 (50 microM), or prostaglandin E2 (50 microM) for 2 h. Binding experiments with [125I]PLP-(1-36)amide after desensitization revealed that there was an approximate twofold decrease in receptor affinities as analyzed by Scatchard analysis, showing that the decrease in affinity was prominent in the process of desensitization. When the cells were treated with monensin during desensitization, PTH challenge after desensitization produced significantly lower cyclic AMP responses. Recovery after desensitization occurred over a period of 16 h. Inclusion of monensin, but not cycloheximide, impaired the recovery. The results show that homologous desensitization of rat osteoblasts to PTH is brought about by the occupancy of receptors by PTH-(1-34) but not by cAMP generation itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cyclic AMP; Iodine Radioisotopes; Monensin; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Proteins; Receptors, Cell Surface; Receptors, Parathyroid Hormone; Tumor Cells, Cultured

Chemotaxis is an important step in monocyte recruitment in inflammation, wound healing, and tumor growth. We reported previously that monocyte chemotactic activity secreted by malignant cells and normal smooth muscle cells is associated with a protein or family of proteins that are related to the monocyte-specific smooth muscle cell-derived chemotactic factor (SMC-CF) (Graves, D. T., Jiang, Y. L., Williamson, M. J., and Valente, A. J. (1989) Science 245, 1490-1493). Similar monocyte chemotactic proteins (MCP-1) produced by U-105MG human glioma cells have also been identified (Yoshimura, T., Robinson, E. A., Tanaka, S., Appella, E., Kuratsu, J., and Leonard, E. J. (1989) J. Exp. Med. 169, 1449-1459). We now report that the MCP-1 gene is expressed in MG-63 human osteosarcoma and vascular smooth muscle cells and that SMC-CF antiserum specifically immunoprecipitates proteins synthesized by U-105MG glioma cells. Experiments were undertaken to elucidate the processing pathway of MCP-1/SMC-CF-like proteins in each of these cell types. These experiments demonstrate that larger MCP-1/SMC-CF-like proteins are derived from a Mr = 9000 precursor. Post-translational modification involves the addition of O-linked carbohydrates and sialic acid residues. Differences in carbohydrate processing account for the heterogeneity in MCP-1/SMC-CF-like proteins produced by different cell types. Secretion of these proteins occurs rapidly following processing events in the endoplasmic reticulum-Golgi compartment.

Topics: Blotting, Northern; Chemokine CCL2; Chemotactic Factors; Glioma; Glycosylation; Humans; Molecular Weight; Monensin; Muscle, Smooth, Vascular; Osteosarcoma; Precipitin Tests; Protein Processing, Post-Translational; RNA, Messenger; Tumor Cells, Cultured; Tunicamycin

The human osteosarcoma-derived cell line U-2 OS expresses c-sis mRNA and synthesizes platelet-derived growth factor (PDGF)-like proteins. Pulse-chase experiments indicate that proteins of 23 kDa and 180 kDa are synthesized first. The 23-kDa protein undergoes dimerization and proteolysis, giving rise to the 30-kDa dimeric protein secreted by the cells. The 180-kDa protein is proteolytically cleaved in a complex series of steps that give rise to several intracellular species. It is also the likely precursor of high molecular mass PDGF-like or PDGF-associated proteins secreted by these cells. The processing and secretion of the 180-kDa protein is slower than that of the 23-kDa protein. Subcellular fractionation and studies with the antibiotic monensin indicate that the processing events occur in the Golgi-endoplasmic reticulum compartment of U-2 OS cells.

Topics: Cell Line; Chemical Precipitation; Endoplasmic Reticulum; Golgi Apparatus; Humans; Kinetics; Molecular Weight; Monensin; Osteosarcoma; Platelet-Derived Growth Factor; Protein Processing, Post-Translational