heparitin-sulfate and Osteosarcoma

Insulin plays a wide variety of physiological actions in osteoblast cells such as differentiation and gene expression. Integrins are transmembrane heterodimeric proteins consisting of α and β subunits which transduce signals from extracellular matrix into the cell. The integrin-mediated signals regulate gene expression, differentiation and survival of osteoblast. In the present study, we explored to determine if insulin could regulate integrin-linked kinase (ILK) signaling in osteoblast like UMR-106 cells. Insulin rapidly stimulated ILK activity in a time-dependent manner with maximal activity observed at 60 min. The insulin's ability to stimulate ILK was almost completely abolished when the cells were pre-incubated with heparinase III (HepIII), suggesting the heparan sulfates attached to syndecan-1 play an important role in the activation of ILK in response to insulin. Interestingly, insulin also activated Akt activity by phosphorylation, whereas pre-treatment of HepIII failed to interfere Akt activation by insulin. In contrast, HepIII pre-treatment inhibited alkaline phosphatase stimulation and collagen synthesis in response to insulin. These results strongly suggest that heparan sulfates on the syndecan-1 and/or shedding of syndecan-1 play a significant role in regulating ILK by insulin, and thereby regulating alkaline phosphatase and collagen synthesis in osteoblast cells.

Topics: Animals; Blotting, Western; Bone Neoplasms; Cell Differentiation; Heparitin Sulfate; Hypoglycemic Agents; Insulin; Osteosarcoma; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Syndecan-1; Tumor Cells, Cultured

To know the involvement of glycosaminoglycans (GAGs) in the metastasis of mouse FBJ osteosarcoma cells, N(α)-lauroyl-O-(β-D-xylopyranosyl)-L-serinamide (Xyl-Ser-C12), which initiates elongation of GAG chains using the glycan biosynthesis system in cells, was administered to FBJ cells with different metastatic capacities. Production of glycosylated products derived from Xyl-Ser-C12, especially heparan sulfate (HS) GAG-type oligosaccharides such as GalNAc-GlcA-GlcNAc-GlcA-Gal-Gal-Xyl-Ser-C12, was indicated in poorly metastatic FBJ-S1 cells more than in highly metastatic FBJ-LL cells by LC-MS. The results of RT-PCR revealed that HS synthases, Ext1 and Ext2, were expressed in FBJ-S1 cells more than in FBJ-LL cells. Furthermore, siRNA against Ext1 suppressed the expression of HS and enhanced the motility of FBJ-S1 cells. In addition, the expression of heparanase (HPSE) was enhanced in Ext-1-knockdown FBJ-S1 cells, and responsible for the increase in cell motility caused by the down-regulation of Ext1 expression. Our data provide the first evidence that Ext1 regulates the expression of HPSE and also indicated that levels of Ext1 and HPSE influenced the motility of FBJ cells.

Topics: Animals; Carbohydrate Sequence; Cell Line, Tumor; Cell Movement; Gene Expression; Gene Knockdown Techniques; Glucuronidase; Heparitin Sulfate; Mice; Molecular Sequence Data; N-Acetylglucosaminyltransferases; Osteosarcoma; RNA, Small Interfering

Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses.

Topics: Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Glycoproteins; Heparin; Heparitin Sulfate; Humans; Inhibitor of Differentiation Protein 1; Intercellular Signaling Peptides and Proteins; Mucopolysaccharidosis I; Multipotent Stem Cells; Osteosarcoma; Phosphorylation; Promoter Regions, Genetic; Recombinant Proteins; Signal Transduction; Smad1 Protein; Time Factors

An activity isolated from bovine bone was previously shown to stimulate proteoglycan synthesis by several connective tissue cell lines from normal tissues (Matrigenin activity). The effect of this activity on glycoconjugate synthesis by two osteoblastic cell lines, ROS 17/2 and UMR-106, derived from rat osteogenic sarcoma, was examined after labelling of the cells with [3H]glucosamine and [35S]sulfate. The glycoconjugates from the cell layers and the media were separated by DEAE-Sephacel chromatography and the anionic glycoconjugates of the media were further analyzed by chromatography on Sepharose CL-2B and enzymatic digestion of the papain-released glycosaminoglycans. The ROS 17/2 cells secreted at least two distinct species of proteoglycan (one heparan sulfate rich and the other chondroitin sulfate rich), whereas the UMR-106 secreted primarily an anionic glycoprotein. The addition of Matrigenin activity to the ROS 17/2 cells resulted in stimulation of incorporation of radioactivity into the proteoglycan and hyaluronic acid, but in UMR-106 cultures it resulted in decreased incorporation into the anionic glycoprotein. The decrease in incorporation into the anionic glycoprotein from the medium was shown, by alkaline beta-elimination, to have occurred mainly in the oligosaccharide fraction, relative to control cultures.

Topics: Animals; Bone and Bones; Chondroitin Sulfates; Chromatography; Glucosamine; Glycoconjugates; Glycoproteins; Glycosaminoglycans; Heparitin Sulfate; Osteoblasts; Osteosarcoma; Papain; Rats; Sulfates; Sulfur Radioisotopes; Tissue Extracts; Tritium; Tumor Cells, Cultured