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02Whole.Pdf (2.478Mb) Parallel Analysis of Gene Expression: Bone Cells as a Model System By Wendy Elizabeth Simcock B. Sc. (Hons) (Adelaide) Presented in fulfillment of the requirements for the degree of Doctorate of Philosophy of Medical Research Griffith University, Gold Coast Campus, Queensland 2003 ABSTRACT The use of comparative gene expression techniques has expanded considerably in recent years, especially with advances in microarray technology. In this project, a number of these techniques have been used to identify genes worthy of further research as potential mediators of bone cell differentiation and function. Bone is a tissue with many potential as yet unidentified regulatory molecules. The skeleton is constantly undergoing replacement, with old bone being degraded by osteoclasts, bone resorbing cells derived from the haematopoietic lineage, and replaced with new bone by osteoblasts, bone synthesizing cells derived from the mesenchymal stem cell lineage. When the rate of bone resorption exceeds the rate of bone synthesis, osteoporosis can occur. Osteoporosis is the most common form of disease affecting the skeleton, and one of the most common age-related diseases, and is a major social and economic burden. Recent studies have shown that cells of mesenchymal lineage are capable of adopting alternate differentiation fates, suggesting that cell-based therapies may be a useful therapeutic approach for this disease. Therefore, identification of molecular mechanisms involved in regulating the behavior and development of bone forming and bone resorbing cells is essential. The aim of this project, therefore, was to identify genes involved in various stages of bone cell differentiation using comparative gene expression techniques. The specific objectives of this project became: 1) to identify molecules expressed by osteoblasts which may increase or decrease bone synthesis; these may have potential for exploitation to treat bone loss in osteoporosis, or excess bone deposition in 1 osteopetrosis; and 2) to identify molecules expressed by osteoclasts which may increase or decrease bone resorption; these may have potential for exploitation to treat excess bone deposition in osteopetrosis, or bone loss in osteoporosis. The first objective, identification of molecules expressed by osteoblasts involved in bone deposition, was addressed using three techniques: subtractive hybridization, DNA microarray analysis, and DNA macroarray analysis. These techniques were used to identify genes transcribed at different levels between foetal osteoblasts and fibroblasts. The key difference between DNA arrays, and subtractive hybridization, as techniques is that DNA arrays utilize a cDNA population fixed to a rigid medium as starting material. This means, therefore, that in order to identify a gene as being expressed, the gene must be present on the array, as a member of the cDNA library the original starting array was made from. This inhibits identification of truly novel transcripts, a bias which is removed in techniques such as subtractive hybridization. The technique of subtractive hybridization is used to identify genes transcribed at higher levels in one DNA sample compared with another. The subtractive hybridization technique described here was modified to enrich a foetal osteoblast phagemid library by removing phagemid which contain transcripts common to foetal osteoblasts and dermal fibroblasts, thus resulting in identification of genes expressed uniquely or at higher levels in osteoblasts. The technique identified 65 genes that were expressed either highly or specifically in osteoblasts when compared with fibroblasts. Some of the genes identified were found in multiple library clones, such as collagen 2 and fibronectin, both of which are key structural components of bone, abundantly expressed by osteoblasts. Expression of some other identified genes had not previously been detected in osteoblasts, making them interesting targets for further investigation. Interesting genes revealed using this technique included prohibitin, leptin-receptor gene related protein, ornithine decarboxylase antizyme, amyloid precursor peptide and connective tissue growth factor. The usefulness of the technique was verified by performing real-time PCR to confirm the expression of these genes either specifically or abundantly in osteoblast cells. DNA array analysis was undertaken to identify transcripts previously identified in other tissues, but not investigated in bone cells to date. Micro and macroarray analysis was used to identify known genes that were over or underexpressed. The microarray comparison of fibroblasts and osteoblasts using cDNA differentially labeled with fluorescent dyes hybridized to glass microarrays, showed that the two cell types were very similar, with just 64 genes found to be regulated 5-fold or more- 37 were down-regulated in osteoblasts, and 27 were upregulated in osteoblasts, out of the 19,000 genes represented on the array. Genes shown to be significantly upregulated in osteoblasts by the 19k microarray included several neural proteins and transcription factors, while genes downregulated in osteoblasts included cell signal transducers and other transcriptional activity modifiers. The set of Atlas cDNA arrays consists of three pairs of arrays, with each pair containing 1176 genes. Two pairs of filters, or 2352 genes, were probed in the 3 osteoblast/fibroblast comparison, while all three pairs, or 3528 genes, were probed in the osteoclast/macrophage comparison. The data from the DNA macroarray experiment, in which radioactively-labelled cDNA from osteoblasts and fibroblasts was hybridized to two separate sets of nylon arrays (ATLAS human cDNA arrays) showed that 12 out of the 2352 genes assayed were significantly regulated, with eight upregulated in osteoblasts, and four downregulated in osteoblasts. Genes upregulated in osteoblasts included transcription factor Dp-2, cAMP response element binding protein 1, and transcription factor ATF2. Genes down-regulated in osteoblasts included teratocarinoma derived growth factor, thymosin beta-10, and transcription factor 3. To address the second objective, and identify molecules expressed by osteoclasts involved in bone resorption, the DNA macroarray analysis approach was repeated. cDNA isolated from osteoclasts was compared with cDNA isolated from macrophages to identify genes differentially transcribed between these two cell types which differentiate from the same developmental lineage in vitro. DNA macroarray analysis, performed using radioactively labeled cDNA from osteoclasts and macrophages hybridized to ATLAS human cDNA arrays identified 53 genes as upregulated in osteoclasts, including GM-CSF Receptor, signalling molecule calmodulin 1, and transcription regulatory molecule Nuclear Factor of Activated T- cells (NFAT). Seventeen genes were shown to be downregulated in osteoclasts, including transcription factor zpf36, Integrin 5, and X-ray repair complementing protein. 4 The studies described here suggested that osteoclasts and macrophages have more differentially expressed genes than osteoblasts and fibroblasts, with 1.98% of genes arrayed showing differential expression between osteoclasts and macrophages, but only 0.3% of genes arrayed showing differential expression between fibroblasts and osteoblasts. This suggests that the morphological differences between cell types may be a direct reflection of the molecular differences between them as well, as osteoblasts and fibroblasts are quite similar, while osteoclasts and macrophages are morphologically quite distinct. From this project, it would appear that comparisons of different populations of cells require the use of different techniques to yield the best results, and that the techniques of array analysis and subtractive hybridization may be best utilized in combination, rather than exclusively of each other. Many of the genes identified as differentially expressed between fibroblasts and osteoblasts using DNA arrays were fairly well- characterised ‘house-keeping’ type genes- metabolic, structural, and not specific or likely to play a significant role in the differentiation of osteoblasts, or the development of bone disease. One possible reason for this is that the arrays are too limited by the number of genes featured, to be able to detect many differences between similar cell types, where there are fewer differences to detect. In contrast, using the same macroarrays to compare the more distinct osteoclasts and macrophages resulted in identification of several interesting candidate genes, showing that some cell type comparisons can be performed adequately using this technology. By using the subtractive hybridization method to enrich a pre-existing phagemid library, any bias related to the genes able to be detected was removed. Although this 5 technique requires manufacture of a cDNA library of the cell type of interest, it may be worthwhile for the comparison of similar cell types where sensitivity is an issue. In summary, this project used the techniques of subtractive hybridization and DNA macroarray and microarray analysis to detect genes showing differential expression between osteoblasts and fibroblasts, and used DNA macroarray analysis to detect genes differentially expressed between osteoclasts and macrophages. Of particular note were results for two interesting genes, amyloid precursor protein and ornithine decarboxylase antizyme. Amyloid Precursor Protein was identified as expressed in high levels in osteoblasts by subtractive hybridization,
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