Effects of parathyroid hormone-related peptide on chick growth plate chondrocytes.

Parathyroid hormone-related peptide (PTHrP) and parathyroid hormone (PTH) have similar biological effects in vitro that are mediated through the PTH receptor. PTH receptors have been demonstrated in the zone of provisional calcification and the hypertrophic zone of the cartilaginous growth plate. The current study examined the biological effects of PTHrP on chick growth plate chondrocytes. Chondrocytes were exposed to varying doses of PTHrP for 24 h, and the incorporation of radioactive thymidine into DNA was used as an index of proliferation. A dose-dependent stimulation of proliferation was seen, with a maximal 27-fold increase at 50 nM PTHrP. A dose-dependent stimulation of cAMP was seen, with a maximal effect at a dose of 50 nM. Proteoglycan synthesis, measured by incorporation of radioactive sulfate, was stimulated, with a maximal effect of 65% at 1 nM. Collagen synthesis and alkaline phosphatase activity from both cellular and matrix vesicle sources decreased in a dose-dependent fashion, with a maximal inhibition of approximately 50% of the control value. The physiologic significance of the PTH and PTHrP-responsiveness of growth plate chondrocytes is uncertain at the present time. It is possible that PTH or PTHrP, or both, act as a systemic, developmental modulator of cellular proliferation and differentiation in the growth plate.

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 the severe disruption of growth-plate structure and function in parathyroid hormone-related protein-deficient transgenic mice. In the present study, the effects of parathyroid hormone-related protein on the synthesis of collagen mRNA and protein were studied in short-term cultures of isolated chick growth-plate chondrocytes. Parathyroid hormone-related protein selectively inhibits type-X collagen protein synthesis with no significant effect on type-II collagen protein synthesis. These effects were present in all maturationally distinct populations of chondrocytes separated by countercurrent centrifugal elutriation. In cultures of resting chondrocytes, the onset of type-X collagen expression was 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 with synthetic oligonucleotide cDNA probes, and the level of expression was quantified using digital image analysis. Dose-dependent suppression of type-X collagen gene expression by parathyroid hormone-related protein was observed, with no significant effect on type-II collagen mRNA detected. The results were confirmed by analysis of Northern blots of total chondrocyte mRNA. These experiments demonstrated differential transcriptional regulation of type-II and type-X collagen, with selective suppression of type-X collagen expression, by parathyroid hormone-related protein in growth-plate chondrocytes. In addition, excellent agreement was found between traditional protein and mRNA analyses and microscopic digital image analysis techniques, supporting the use of this convenient and sensitive assay method. Parathyroid hormone-related protein inhibits chondrocyte maturation and is known to stimulate proliferation, suggesting that this autocrine factor may function to regulate premature hypertrophy in the growth plate.