Year: 2011
Type: dissertation
Abstract: Craniosynostosis is the premature fusion of cranial sutures and results in the compensatory malformation of the skull to accommodate the rapid growth of the brain during early childhood. This PhD thesis aims to look at the molecular mechanisms at play during this premature fusion; in particular it follows on from a recent microarray study of craniosynostosis tissue conducted in this laboratory. This study showed a 37x down regulation of RBP4 in fused sutures in humans. RBP4 is a retinol binding protein whose function is to transport retinol in the blood to target tissue, where it is metabolised to retinoic acid. This is of interest as retinoic acid is known to have an influence on bone growth. In this PhD project we have used animal and cell culture models to assess the levels of RBP4 during suture fusion and osteogenesis and its possible role in this process. Expression of Rbp4, Stra6 and other markers of osteogenesis were assessed using quantitive PCR in a mouse model of Saethre-Chotzen craniosynostosis syndrome (Twist1⁺/⁻). This demonstrated an initial correlation between suture fusion and Rbp4 down regulation as well as an inverse relationship between Rbp4 and Stra6 expression. However, sutures that did not fuse and parietal bone also displayed downregulation of Rbp4 at later timepoints. Histology showed that this might be related to parietal bone thickening. Multiple cell culture models were trialed, but proved unsuitable for RBP4 studies in osteogenesis. The commonly used mouse pre-osteoblastic cell line, MC3T3-E1, mineralised but did not express Rbp4. Primary coronal suture cells were isolated from mice, which expressed Rbp4, but failed to mineralise. Subsequently, primary cell cultures from human sutures were tested in osteogenesis assays and showed a decrease in RBP4 levels during mineralisation. Immunocytochemistry was used to determine the localisation of RBP4 in suture cells compared to Huh7 cells, a liver carcinoma cell line with known secretion of RBP4. Results showed that RBP4 is localised to the endoplasmic reticulum in suture cells, differing to the localization seen in Huh7 cells. Western blot analysis also demonstrated that unlike liver cells, human suture cells do not secrete detectable levels of RBP4. Finally, functional studies to analyse the role of RBP4 in osteogenesis using a lentiviral delivery system for over expression of RBP4 showed no effect on the ability of human suture cells to mineralise. A high level of overexpression was achieved however there were issues with infection efficiency which may have affected the outcome of these experiments. These studies demonstrate some unique characteristics of RBP4 in suture cells and extend its role beyond a simple serum transporter of retinol. In addition to a role in suture fusion, these results could be a reflection of a broader function of RBP4 in normal bone growth and osteogenesis.
Author: Victoria D. Leitch
Language: English
Subfield: Surgery
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2757656510