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Connective Tissue Oncology Society |
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| 2001 CTOS Annual Meeting Posters— Biology ENCHONDROMATOSIS
CAUSED BY A MUTANT TYPE I PTH/PTHRP RECEPTOR OBJECTIVE: Enchondromas are common benign cartilage tumours. When they occur in multiple locations in enchondromatosis (Ollier’s disease), the risk of skeletal deformity and of malignant change to chondrosarcoma is high. Enchondromas are usually in close proximity to, or in continuity with, growth plate cartilage. Consequently, they may result from abnormal regulation of proliferation and terminal differentiation of chondrocytes in the adjoining growth plate. In normal growth plates, differentiation of proliferating chondrocytes to post-mitotic hypertrophic chondrocytes is regulated in part by a tightly coupled signalling relay involving Indian hedgehog (IHH) Parathyroid hormone related protein (PTHrP). We speculated that inappropriate regulation of the IHH-PTHrP pathway contributes to the genesis of enchondromas. METHODS: We utilized semiquantitative RT-PCR and Western blot analysis to test for expression of IHH-PTHrP pathway members, and a short term primary cartilage tumour explant culture system to test the functional effects of Hedgehog and PTHrP agonists and antagonist in vitro. Proliferation was assessed by tritiated thymidine uptake, and differentiation by type X collagen expression level (an exclusive product of hypertrophic chondrocytes). Single strand conformation polymorphism analysis and manual sequencing were used for mutational screening. In vitro Cyclic adenosine monophospate (cAMP) and Inositol triphosphate (IP3) assays were performed using wild type (WT) and mutant constructs generated via site-directed mutagenesis, which were transiently transfected into COS-7 cells and embryonic stem cells lacking native Type 1 PTH/PTHrP receptor (PTHR1). Transgenic mice were generated by pronuclear microinjection of WT or mutant PTHR1 cDNA flanked by the regulatory elements of Type II collagen (ColII) for expression in cartilage. Genomic DNA was extracted from tails and screened by Southern blot for integration of the transgene. Paraffin embedded sections from transgenic mice were used for immunohistochemistry, safranin-O histology and tartrate resistant acid phosphatase (TRAP) staining. RESULTS: We showed that key IHH-PTHrP pathway members are expressed in enchondromas and chondrosarcomas. The IHH and PTHrP signalling pathways were functional, but the feedback loop regulating IHH was dysregulated in these lesions. We identified a mutant PTHR1 in two patients with enchondromatosis. This mutant lowered baseline cAMP level and abolished IP3 accumulation in vitro. Expression of the mutant, but not WT, PTHR1 in the growth plates of transgenic mice resulted in the appearance of multiple enchondromas. These enchondromas were likely caused by abnormal proliferation and not abnormal resorption, since growth plate zonal architecture was altered, but the number of TRAP positive cells, which resorb the growth plate, were not. CONCLUSION: These data suggest that enchondromas can arise due to abnormal growth plate development. In particular, some cases of enchondromatosis are likely caused by a mutant PTHR1. The persistence of growth plate tissue in the form of enchondromas beyond adolescence, when growth plate tissue has normally disappeared in humans, may allow accumulation of secondary genetic events which cause chondrosarcoma to arise within a preexisting enchondroma. Agents that block IHH-PTHrP signalling might be of therapeutic benefit in preventing the deleterious consequences of enchondromas.
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