CHONDROGENIC DIFFERENTIATION OF CLONAL MOUSE EMBRYONIC-CELL LINE ATDC5 IN-VITRO - DIFFERENTIATION-DEPENDENT GENE-EXPRESSION OF PARATHYROID-HORMONE (PTH) PTH-RELATED PEPTIDE RECEPTOR/

The regulatory role of parathyroid hormone (PTH)/PTH-related peptide ( PTHrP) signaling has been implicated in embryonic skeletal development , Here, we studied chondrogenic differentiation of the mouse embryonal carcinoma-derived clonal cell line ATDC5 as a model of chondrogenesis in the early stages of endochondral bone development, ATDC5 cells ret ain the properties of chondroprogenitor cells, and rapidly proliferate in the presence of 5% FBS. Insulin (10 mu g/ml) induced chondrogenic differentiation of the cells in a postconfluent phase through a cellul ar condensation process, resulting in the formation of cartilage nodul es, as evidenced by expression of type II collagen and aggrecan genes, We found that differentiated cultures of ATDC5 cells abundantly expre ssed the high affinity receptor for PTH (M(r) similar to 80 kD; K-d = 3.9 nM; 3.2 X 10(5) sites/cell), The receptors on differentiated cells were functionally active, as evidenced by a PTH-dependent activation of adenylate cyclase, Specific binding of PTH to cells markedly increa sed with the formation of cartilage nodules, while undifferentiated ce lls failed to show specific binding of PTH, Northern blot analysis ind icated that expression of the PTH/PTHrP receptor gene became detectabl e at the early stage of chondrogenesis of ATDC5 cells, preceding induc tion of aggrecan gene expression, Expression of the PTH/PTHrP receptor gene was undetectable in undifferentiated cells, The level of PTH/PTH rP receptor mRNA was markedly elevated parallel to that of type II col lagen mRNA, These lines of evidence suggest that the expression of fun ctional PTH/PTHrP receptor is associated with the onset of chondrogene sis, In addition, activation of the receptor by exogenous PTH or PTHrP significantly interfered with cellular condensation and the subsequen t formation of cartilage nodules, suggesting a novel site of PTHrP act ion.