IDENTIFICATION AND CHARACTERIZATION OF UP-REGULATED GENES DURING CHONDROCYTE HYPERTROPHY

Chondrocyte hypertrophy involves de novo acquisition and/or increased expression of certain gene products including, among others, type X co llagen, alkaline phosphatase, and matrix metalloproteinases. To analyz e further the genetic program associated with chondrocyte hypertrophy, we have employed a modification of the polymerase chain reaction-medi ated subtractive hybridization method of Wang and Brown (Wang and Brow n [1991] Proc. Natl. Acad. Sci 88:11505). Cultures of hypertrophic tib ial chondrocytes and nonhypertrophic sternal cells were used for poly A+ RNA isolation. Among 50 individual cDNA fragments isolated for up-r egulated hypertrophic genes, 18 were tentatively identified by their s imilarities to entries in the GenBank database, whereas the other 32 s howed no significant similarity. The identified genes included transla tional and transcriptional regulatory factors, ribosomal proteins, the enzymes transglutaminase and glycogen phosphorylase, type X collagen (highly specific for hypertrophic cartilage matrix), gelsolin, and the carbohydrate-binding protein galectin. Two of these, transglutaminase and galectin, were cloned and were further characterized. The chondro cyte transglutaminase revealed previously in hypertrophic cartilage by immunochemical methods appears to be the chicken equivalent of mammal ian factor XIIIa (showing 75% overall protein similarity). The chicken chondrocyte galectin is a variant of mammalian galectin-3. Galectins are known to bind to components found in hypertrophic cartilage, and f actor XIIIa is known to crosslink some of the same components, possibl y modifying them for calcification and/or removal. (C) 1996 Wiley-Liss , Inc.