Characterisation of a small acidic protein-a novel potential regulator of chondrocyte hypertrophy during endochondral ossification

Al-Amoudi, Waiel M. 2006. Characterisation of a small acidic protein-a novel potential regulator of chondrocyte hypertrophy during endochondral ossification. PhD Thesis, Cardiff University.

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Abstract

Endochondral ossification is the process of cell proliferation and differentiation leading to bone formation. It requires temporal and spatial coordination of the actions of various cell types and is mediated by many signaling molecules amongst which Ihh and PTHrP are included. A novel gene product identified recently as SAP (Small Acidic Protein) has also been proposed to play a major role in endochondral ossification. The main aim of this study is to characterize the functional roles of SAP that are potentially associated with new bone formation. The embryonic chick sternum has been rarely used in the previous studies of the development of cartilage. Thus, both tissues (tibia and sternum) from 17 days old embryonic chicks are included in this project. The areas of investigation are as follows: (1) Localising the expression of SAP, Ihh and type X collagen using an in situ hybridisation technique. (2) To investigate whether the expression of SAP is associated with the Ihh/PTHrP loop. (3) Studying the over expression of SAP in chick sternal chondrocytes using Replication-Competent Retrovirus. Tibia and sternum show a close similarity in terms of their cell growth structure as confirmed by histological investigation. The tissues are composed of three characteristic zones (caudal, middle and cephalic) distinguishable by their respective chondrocyte phenotypes: proliferative, prehypertrophic, and hypertrophic. As revealed by in situ hybridisation, SAP is predominantly localised in the proliferative chondrocytes of both, tibia and sternum, although low intensity of expression is also observed in the prehypertrophic and hypertrophic cells. Ihh is highly expressed in the prehypertrophic chondrocytes in both tissues and its expression continues throughout this transitional zone where the majority of cells are stained strongly, but the reactivity becomes less pronounced throughout the hypertrophic part of the tissues. Unlike SAP and Ihh, type X collagen is found to accumulate exclusively in the hypertrophic zone. Under normal culture conditions where the samples are not treated with cyclopamine, the proliferative chondrocytes show high levels of SAP, PTHrP and Patched expression, but do not express Ihh and type X collagen. When treated with cyclopamine, the proliferative chondrocytes no longer express SAP and PTHrP but unexpectedly show the expression of Ihh and type X collagen. The initiation of the expression of Ihh and type X collagen by the proliferative chondrocytes following cyclopamine treatment is also accompanied by a decline in the production of GAG and hydroxyproline, suggesting the onset of cell differentiation. Cyclopamine is a naturally-occurring chemical that belong to the group of steroidal jerveratrum alkaloids. It has been shown that cyclopamine blocks the hedgehog signalling pathway by influencing the balance between active and inactive forms of the Smoothened protein (Taipale et al, 2002) Cyclopamine inhibits SAP and PTHrP expression in the prehypertrophic and hypertrophic chondrocytes where Ihh and type X collagen remains unaffected. As SAP expression is predominantly found in the proliferative zone and downregulated in prehypertrophic and hypertrophic chondrocytes, this may indicate its involvement in endochondral ossification as a promoter of cell proliferation and as a transcription regulator preventing the cells from reaching hypertrophy or maturation. An attempt to investigate the biological role of SAP by over expressing the SAP gene in cell cultures has not been successful and it remains to be confirmed whether SAP is an integral part of the Ihh/PTHrP feedback control loop.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Biosciences
ISBN:	9781303208256
Date of First Compliant Deposit:	30 March 2016
Last Modified:	09 Jan 2018 17:52
URI:	https://orca.cardiff.ac.uk/id/eprint/56159

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