MULTIPLE NEGATIVE ELEMENTS IN A GENE THAT CODES FOR AN EXTRACELLULAR-MATRIX PROTEIN, COLLAGEN-X, RESTRICT EXPRESSION TO HYPERTROPHIC CHONDROCYTES

During skeletal development, chondrocytes go through several stages of differentiation. The last stage, chondrocyte hypertrophy, occurs in a reas of endochondral ossification. Mature hypertrophic chondrocytes di ffer from immature chondrocytes in that they become postmitotic, incre ase their cellular volume up to eightfold, and synthesize a unique set of matrix molecules. One such molecule is a short collagenous protein , collagen X. Previous studies have shown that collagen X is not expre ssed by other cell types and that its specific expression in hypertrop hic chondrocytes is controlled by transcriptional mechanisms. To defin e these mechanisms, plasmid constructs containing the chicken collagen X gene promoter and 5' flanking regions fused to a reporter gene (chl oramphenicol acetyl transferase, CAT) were transfected into primary cu ltures of collagen X-expressing and nonexpressing cells. A construct c ontaining a short (558 bp) promoter exhibited high levels of CAT activ ity in all cell types (fibroblasts, immature, and hypertrophic chondro cytes). Adding a 4.2-kb fragment of 5' flanking DNA to this construct resulted in a dramatic reduction of CAT activity in fibroblasts and im mature chondrocytes, but had no effect in hypertrophic chondrocytes. A ddition of three subfragments of the 4.2-kb fragment to the initial co nstruct, either individually or in various combinations, showed that a ll subfragments reduced CAT activity somewhat in non-collagen X-expres sing cells, and that their effects were additive. Unrelated DNA had no effect on CAT activity. The results suggest that multiple, diffuse up stream negative regulatory elements act in an additive manner to restr ict transcription of the collagen X gene to hypertrophic chondrocytes.