Signal-selectivity of parathyroid hormone (PTH)/PTH-related peptide receptor-mediated regulation of differentiation in conditionally immortalized growth-plate chondrocytes

Type-1 PTH/PTH-related peptide receptors (PTH1Rs), which activate both aden ylyl cyclase and phospholipase C (PLC), control endochondral bone developme nt by regulating chondrocyte differentiation. To directly analyze PTH1R fun ction in such cells, we isolated conditionally transformed clonal chondrocy tic cell lines from tibial growth plates of neonatal mice heterozygous for PTH1R gene ablation. Among 104 cell lines isolated, messenger RNAs for PTH1 R, collagen II, and collagen X were detected in 28%, 90%, and 29%, respecti vely. These cell lines were morphologically diverse. Some appeared large, r ounded, and enveloped by abundant extracellular matrix; whereas others were smaller, flattened, and elongated. Two PTH1R-expressing clones showed simi lar PTH1R binding and cAMP responsiveness to PTH and PTH-related peptide bu t disparate morphologic features, characteristic of hypertrophic (hC1-5) or nonhypertrophic (nhC2-27) chondrocytes, respectively. hC1-5 cells expresse d messenger RNAs for collagen II and X, alkaline phosphatase (ALP), and mat rix GLA protein, whereas nhC2-27 cells expressed collagen II and Indian hed gehog but not collagen X or ALP. In hC1-5 cells, PTH and cAMP analog, but not phorbol ester, inhibited both ALP and mineralization. PTH1R-null hC1-5 subclones were isolated by in vitr o selection and then reconstituted by stable transfection with wild-type PT H1Rs or mutant (DSEL) PTH1Rs defective in PLC activation. ALP and mineraliz ation were inhibited similarly via both forms of the receptor. These result s indicate that PLC activation is not required for PTH1R regulation of mine ralization or ALP in hypertrophic chondrocytes and are consistent with a ma jor role for cAMP in regulating differentiation of hypertrophic chondrocyte s.