monensin and Bone-Neoplasms

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

Human giant cell tumor (GCT) consists of multinucleated giant cells and mononuclear stromal cells, and is characterized by frequent vascular invasion without distant metastases. To study the role of matrix metalloproteinases (MMPs) in the vascular invasion, we examined production of MMP-1 (tissue collagenase), -2 (gelatinase A), -3 (stromelysin-1), -9 (gelatinase B), and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in GCT. MMP-9 was highly and predominantly expressed in giant cells by both immunohistochemistry and in situ hybridization. Expression of other MMPs was also observed in some cases but was inconstant. Sandwich enzyme immunoassays demonstrated that MMP-9 is the predominant MMP secreted by GCT. There was a definite imbalance between the amounts of MMP-9 and those of TIMPs in the culture media of GCT, leading to detectable gelatinolytic activity in an assay using 14C-gelatin. Gelatin zymography demonstrated the main activity at about 90 kd, which was identified as the zymogen of MMP-9 by immunoblotting. Immunohistochemistry for type IV collagen and laminin, major basement membrane components, showed that disappearance of the proteins is closely associated with MMP-9-positive giant cells. These results indicate the production of MMP-9 by multinucleated giant cells and suggest that the metalloproteinase may contribute to proteolysis associated with vascular invasion and local bone resorption in human GCT.

Topics: Adolescent; Adult; Basement Membrane; Bone Neoplasms; Collagenases; Electrophoresis, Polyacrylamide Gel; Female; Follow-Up Studies; Giant Cell Tumor of Bone; Giant Cells; Glycoproteins; Humans; Immunoblotting; Immunoenzyme Techniques; In Situ Hybridization; Male; Matrix Metalloproteinase 9; Metalloendopeptidases; Middle Aged; Monensin; Neoplasm Invasiveness; Protease Inhibitors; Proteins; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinases

Osteoclast interaction with extracellular matrix drives the sequential events that end with bone resorption. However, the role of matrix proteins is not yet fully understood. We studied this problem on human osteoclast-like cells derived from giant cell tumors of bone (GCT cells). On GCT cells we considered cytoskeletal organization, adhesion properties, and integrin expression upon plating in serum-free medium onto fibronectin (FN), collagen (COL), thrombospondin (TSP), bone sialoprotein (BSPII), and osteopontin (OPN). GCT cells promptly adhered and spread on FN, BSPII, and OPN, while only 50% adhered on COL and none on TSP. The integrin beta 1 chain was always associated to focal adhesions, while the alpha v beta 3 heterodimer was detected in focal contacts only upon plating on BSPII, OPN, and FN. The focal clustering of beta 1 was impaired by monensin treatment, indicating that endogenous FN secretion was required to drive beta 1 into focal contacts. Conversely, alpha v beta 3 clustering was also not affected by monensin when cells were plated onto plasma FN. Immunoprecipitation of metabolically labeled GCT cell lysates showed that three different heterodimers (alpha v beta 3, alpha 3 beta 1, and alpha 5 beta 1) were assembled. Adhesion to FN was completely inhibited by beta 1 antibodies at dilutions up to 1:400, while beta 3 antibodies, at similar dilutions, impaired spreading but not adhesion. We conclude that alpha v beta 3 is the main integrin used by GCT cells in bone recognition. We also suggest that selected substrata may induce the release and the organization of endogenous FN that eventually drives the recruitment of a beta 1 integrin receptor into focal contacts.

Topics: Amino Acid Sequence; Bone Neoplasms; Cell Adhesion; Extracellular Matrix Proteins; Fibronectins; Fluorescent Antibody Technique; Giant Cell Tumors; Humans; In Vitro Techniques; Integrins; Molecular Sequence Data; Monensin; Oligopeptides; Osteoclasts; Tumor Cells, Cultured