epidermal-growth-factor and Chondrosarcoma

To identify regulators of the cartilaginous phenotype, on the basis of their differential expression in human conventional chondrogenic tumors compared with articular cartilage.. Differential proteomics analysis revealed matrilin-3 (MATN3) as a candidate regulator of the cartilaginous phenotype. Its capacity to modulate gene expression was investigated in human HCS-2/8 chondrosarcoma cells and transfected chondrocytes, using cell culture fractionation, reverse transcription-polymerase chain reaction, and Western blot analyses.. Increased expression of the cartilage-specific matrix protein MATN3 was specifically observed in enchondromas and conventional chondrosarcomas. A substantial fraction of MATN3 was found in cytoplasmic structures of tumor cells, as demonstrated by immunohistochemistry. Analyses of intracellular MATN3 revealed that it corresponded to an imperfectly maturated MATN3 polypeptide, both in HCS-2/8 human chondrosarcoma cells and in transfected human chondrocytes. Moderately increased expression of MATN3 resulted in its intracellular retention. Antibody-mediated blockade of soluble, extracellular MATN3 in HCS-2/8 cell cultures resulted in increased expression of MATN3 and the chondrogenic transcription factor SOX9. Conversely, increased ectopic expression of MATN3 resulted in decreased expression of MATN3 and SOX9 in primary chondrocytes, while a mutant MATN3 lacking its first epidermal growth factor (EGF)-like domain failed to down-regulate SOX9.. Aberrant expression and processing of MATN3 are hallmarks of conventional cartilaginous neoplasms. A particular step in the maturation of MATN3 limits its processing through the secretion machinery, resulting in its intracellular accumulation upon increased expression. Soluble, secreted MATN3, however, down-regulates SOX9 at the messenger RNA and protein levels. The first EGF-like domain of MATN3 is a critical determinant of its regulatory activity toward SOX9. These activities of MATN3 suggest that its increased expression in osteoarthritis might contribute to the degeneration of articular cartilage.

Topics: Adolescent; Adult; Aged; Blotting, Western; Bone Neoplasms; Cartilage, Articular; Cell Differentiation; Cells, Cultured; Child; Child, Preschool; Chondrocytes; Chondroma; Chondrosarcoma; Down-Regulation; Epidermal Growth Factor; Extracellular Matrix Proteins; Female; Flow Cytometry; Humans; Immunohistochemistry; Male; Matrilin Proteins; Middle Aged; Osteoarthritis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; SOX9 Transcription Factor

Size-exclusion and ion-exchange high-performance liquid chromatography were used to purify biologically active growth factors as measured by the ability of the factors to stimulate DNA synthesis in 3T3 cells. Chromatography was performed in aqueous buffer and at neutral pH to avoid possible inactivation of biological activity. The growth factors analyzed were chondrosarcoma growth factor (CHSA-GF), human milk growth factor (HMGF), retinal-derived growth factor (RDGF) and mouse epidermal growth factor (EGF). CHSA-GF, HMGF, and RDGF were eluted from TSK 2000 columns as well-defined peaks of activity with molecular weights of 12,000-15,000, 5000-6000, and 16,000-18,000, respectively. EGF was found to have an abnormally low molecular weight after chromatography on TSK 2000. However, incorporation of guanidine . HCl into the TSK column resulted in the elution of EGF at its known molecular weight of ca. 6000. Anion-exchange high-performance liquid chromatography on AX 300 was used for the purification of HMGF and RDGF, and cation-exchange high-performance liquid chromatography on CM 300 was used for the purification of CHSA-GF. The results show that size-exclusion and ion-exchange chromatography can be used without organic solvents or extremes in pH to purify a number of different growth factors successfully with retention of biological activity.

Topics: Animals; Chondrosarcoma; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; DNA; Epidermal Growth Factor; Growth Substances; Humans; Milk, Human; Peptides