2132

EXT2

SOTV;exostosin glycosyltransferase 2;EXT2;exostosin glycosyltransferase 2

N-acetylglucosaminyl-proteoglycan 4-beta-glucuronosyltransferase|exostosin-2|glucuronosyl-N-acetylglucosaminyl-proteoglycan/N-acetylglucosaminyl-proteoglycan 4-alpha-N-acetylglucosaminyltransferase|multiple exostoses protein 2|putative tumor suppressor pr

11p12-p11

protein-coding

The EXT family of putative tumor suppressor genes affect endochondral bone growth, and mutations in EXT1 and EXT2 genes cause the autosomal dominant disorder Hereditary Multiple Exostoses (HME). Loss of heterozygosity (LOH) of these genes plays a role in the development of exostoses and chondrosarcomas. In this study, we characterized EXT genes in 11 exostosis chondrocyte strains using LOH and mutational analyses. We also determined subcellular localization and quantitation of EXT1 and EXT2 proteins by immunocytochemistry using antibodies raised against unique peptide epitopes. In an isolated non-HME exostosis, we detected three genetic hits: deletion of one EXT1 gene, a net 21-bp deletion within the other EXT1 gene and a deletion in intron 1 causing loss of gene product. Diminished levels of EXT1 and EXT2 protein were found in 9 (82%) and 5 (45%) exostosis chondrocyte strains, respectively, and 4 (36%) were deficient in levels of both proteins. Although we found mutations in exostosis chondrocytes, mutational analysis alone did not predict all the observed decreases in EXT gene products in exostosis chondrocytes, suggesting additional genetic mutations. Moreover, exostosis chondrocytes exhibit an unusual cellular phenotype characterized by abnormal actin bundles in the cytoplasm. These results suggest that multiple mutational steps are involved in exostosis development and that EXT genes play a role in cell signaling related to chondrocyte cytoskeleton regulation.

The tumor suppressor genes EXT1 and EXT2 are involved in the formation of multiple osteochondromas, which can progress to become secondary peripheral chondrosarcomas. The most common chondrosarcoma subtype is primary central chondrosarcoma, which occurs in the medullar cavity of bone. The EXT1/EXT2 protein complex is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, which is important for signal transduction of Indian hedgehog (IHH), WNT, and transforming growth factor (TGF)-beta. The role of EXT and its downstream targets in central chondrosarcomas is currently unknown. EXT1 and EXT2 were therefore evaluated in central chondrosarcomas at both the DNA and mRNA levels. Immunohistochemistry was used to assess HSPG (CD44v3 and SDC2), WNT (beta-catenin), and TGF-beta (PAI-1 and phosphorylated Smad2) signaling, whereas IHH signaling was studied both by quantitative polymerase chain reaction and in vitro. mRNA levels of both EXT1 and EXT2 were normal in central chondrosarcomas; genomic alterations were absent in these regions and in 30 other HSPG-related genes. Although HSPGs were aberrantly located (CD44v3 in the Golgi and SDC2 in cytoplasm and nucleus), this was not caused by mutation. WNT signaling negatively correlated with increasing histological grade, whereas TGF-beta positively correlated with increasing histological grade. IHH signaling was active, and inhibition decreased cell viability in one of six cell lines. Our data suggest that, despite normal EXT in central chondrosarcomas, HSPGs and HSPG-dependent signaling are affected in both central and peripheral chondrosarcomas.