DIFFERENTIAL REGULATION OF TYPE-II AND TYPE-X COLLAGEN-SYNTHESIS BY PARATHYROID HORMONE-RELATED PROTEIN IN CHICK GROWTH-PLATE CHONDROCYTES

Parathyroid hormone-related protein is a critical autocrine regulator of endochondral ossification in the growth plate, as demonstrated by t he severe disruption of growth-plate structure and function in parathy roid hormone-related protein-deficient transgenic mice. In the present study, the effects of parathyroid hormone-related protein on the synt hesis of collagen mRNA and protein were studied in short-term cultures of isolated chick growth-plate chondrocytes. Parathyroid hormone-rela ted protein selectively inhibits type-X collagen protein synthesis wit h no significant effect on type-II collagen protein synthesis. These e ffects were present in all maturationally distinct populations of chon drocytes separated by countercurrent centrifugal elutriation. In cultu res of resting chondrocytes, the onset of type-X collagen expression w as inhibited, while the synthesis of type-X collagen was decreased in cultures of hypertrophic chondrocytes. Synthesis of type-II and type-X collagen mRNA was examined by nonradioactive in situ hybridization wi th synthetic oligonucleotide cDNA probes, and the level of expression was quantified using digital image analysis. Dose-dependent suppressio n of type-X collagen gene expression by parathyroid hormone-related pr otein was observed, with no significant effect on type-II collagen mRN A detected. The results were confirmed by analysis of Northern blots o f total chondrocyte mRNA. These experiments demonstrated differential transcriptional regulation of type-II and type-X collagen, with select ive suppression of type-X collagen expression, by parathyroid hormone- related protein in growth-plate chondrocytes. In addition, excellent a greement was found between traditional protein and mRNA analyses and m icroscopic digital image analysis techniques, supporting the use of th is convenient and sensitive assay method. Parathyroid hormone-related protein inhibits chondrocyte maturation and is known to stimulate prol iferation, suggesting that this autocrine factor may function to regul ate premature hypertrophy in the growth plate.