Differential regulation of type‐II and type‐X collagen synthesis by parathyroid hormone‐related protein in chick growth‐plate chondrocytes

Abstract

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.

PTH Stimulated Growth and Decreased Col‐X Deposition are Phosphotidylinositol‐3,4,5 Triphosphate Kinase and Mitogen Activating Protein Kinase Dependent in Avian Sterna

“…In avian sterna, we have shown that PTHrP delayed differentiation, prolonged chondrocyte proliferation, and decreased cellular apoptosis (Harrington et al, 2004). Similar results were obtained with cultured growth plate chondrocytes; Col-X was decreased, proliferation increased and type II collagen was not changed (O'Keefe et al, 1997). The continued proliferation enables further synthesis of Ihh, thereby, creating a feedback loop that controls the pace of chondrocyte differentiation.…”

Histomorphological and proliferative characterization of developing periosteal neochondrocytes in vitro

“…This might be accomplished using PTHrP. Recent work using gene 'knockout' mice has shown that PTHrP is essential for the normal differentiation of growth plate chondrocytes and PTHrP downregulates and delays terminal differentiation of growth plate chondrocytes [37,32,33,48,55,57]. Third, it might even be possible to induce dedifferentiation of the mature large round cell chondrocytes back into the immature small round cell chondrocytes, which might then reacquire their proliferative potential.…”

Restoration of growth plate function following radiotherapy is driven by increased proliferative and synthetic activity of expansions of chondrocytic clones

“…We have previously demonstrated that a single 17.5 Gy irradiation dose produces a reproducible significant uniform irradiation induced derangement of the growth plate. We have also shown that this dose is well beyond the approximate 1.5 Gy LD 50 radiation dose necessary to kill individual chondrocytes, even after considering the negligible but significant absorption of the X radiation by bone and adjacent soft tissues, such that all the cells of the growth plate are likely to be equally affected by the irradiation.…”

“…9,54,55 The action of PTHrP, via the PTHr1 receptor, has been shown to decrease differentiation, thereby reducing the growth contribution of hypertrophic enlargement [56][57][58] and decreasing hypertrophic cell production of collagen X. 50 We concede that the design of this study does not directly translate to the clinical problem described, as single-dose radiotherapy is not the clinical norm. However, from an experimental standpoint, a clinically relevant multiple fraction radiation exposure algorithm presents a number of complications that would make this experiment difficult to interpret in the context of understanding the spatial and temporal emergence of clones.…”

“…The accumulation of matrix produced by cells undergoing clonal expansion in the proliferative zone following irradiation appears to be the primary force driving physeal elongation in this model. Other investigators have also shown that production of the matrix proteins aggrecan and collagen II is independent of PTHrP levels 50 but may be regulated by prolyl-4-hydroxylase (PHD) activity in the proliferative cell zone of the growth plate during a period of radiation-induced hypoxia. 51 Indeed, we have presented data previously suggesting that hypoxia there may play a significant role in modulating this signal pathway, though this was not examined in this experiment.…”