Bone remodeling is a carefully regulated mechanism, responsible for maintaining calcium and phosphate homeostasis in the bloodstream. This mechanism also guaranties the structural integrity of bones. The process of remodeling is coupled. First, the osteoclasts degrade bone tissue, which is later rebuilt by the osteoblasts. After the resting phase, the cycle is repeated after a couple of years. Many local and systemic regulators exist in order to keep the bone remodeling balanced, of which PTH, vitamin D and the RANKL/RANK/OPG triad are vital. RANKL protein is a cytokine which is, in bone, mainly produced by the osteoblast lineage cells. RANKL functions by binding to its receptor RANK, expressed on osteoclasts, causing their activation. Besides bone degradation, RANKL also functions as one of coupling molecules between osteoblasts and osteoclasts. OPG protein acts as a decoy receptor for RANKL. It opposes the function of RANKL by binding to it, therefore preventing its effect on osteoclasts via RANKL-RANK binding. The functions of RANKL, its physiological role and its involvement in bone diseases are well understood; however, the regulation of its expression remains mostly unknown. There are some transcription factors known to influence the expression of the RANKL gene via its distal regulatory regions, for example, vitamin D, PTH and Runx2. On the other hand, the regulation of the RANKL gene expression through its proximal promoter remains unexplored. Thus, the aim of our doctoral thesis was to study the proximal promoter of the RANKL gene and identify novel transcription factors that influence the expression of the RANKL gene, and could be involved in the pathogenesis of the most common bone metabolic disease - osteoporosis. Osteoporosis is a multifactorial disease influenced by a number of genetic and environmental factors. Many authors investigated osteoporosis-related polymorphisms, changes in gene expressions, and changes in proteome and metabolome. They discovered that a large number of frequent changes in many different genes affect the onset of the disease. To date, many such changes remain undiscovered and we can only explain a minor part of the bone mineral density variance, which is the most commonly used indicator of the disease. In the first chapter we performed a literature review of genome-wide studies. The results served as foundation for our following work. In the second chapter, we first conducted a computational analysis of the RANKL gene proximal promoter using the program TF Search. The program revealed potential transcription factors binding sites. Based on data from the literature review we decided to focus on the transcription factors Sry and c-Myb. We tested their effect on the activity of the RANKL gene promoter and discovered that c-Myb activates, while Sry reduces the activity of the proximal promoter of the RANKL gene. We continued with overexpression and silencing of the c-Myb and Sry genes. We confirmed that c-Myb increases the expression of the RANKL gene in the cell model in vitro, while Sry decreases it. The involvement of these transcription factors in the regulation of RANKL gene expression and in osteoporosis was also tested in vivo. We measured their expressions and the expression of the RANKL gene in bone tissue samples of donors with osteoporosis and controls. We discovered that in the osteoporotic bone tissue samples the expression of the RANKL gene was increased 17-times, compared to controls. We also discovered that, in male osteoporotic bone tissue, the expression of male specific Sry gene was 105-times decreased compared to controls. These results suggest that, in osteoporotic men, the increased expression of RANKL comes as a result of the immense decrease of Sry expression, and that the gender differences in RANKL gene expression and in quality of bones are caused by the actions of the male specific transcription factor Sry. The expression of c-Myb was also reduced in osteoporotic bone tissue samples. Therefore, we cannot argue that c-Myb is involved in the pathogenesis of osteoporosis. We can, however, confirm its role in bone biology.
In the third chapter we tested if other members from the SOX family of transcription factors (besides Sry) regulate the expression of RANKL gene through its proximal promoter, and if they are involved in the pathogenesis of osteoporosis. Using in vitro cell models we tested if SOX4, SOX5, SOX6, and SOX9 can regulate the activity of the RANKL gene promoter, and if they can regulate its expression. Using human bone tissue, we tested the in vivo relevance of our results. We have demonstrated that SOX5 and SOX9 activate the RANKL gene promoter and increase the expression of RANKL gene in osteoblast cell model. In addition, we have shown that the expression of the RANKL gene in the bone tissue of osteoporotic men is increased and the expression of the SOX9 gene is decreased compared to controls. Also, the expression of the SOX9 gene may have a predictive value for osteoporosis. The osteoporotic and control group of donors were confirmed using micro-CT, currently the best method for bone tissue structure and quality evaluation. In the additional chapter, we examined whether the transcription factors from the SOX family affect the expression of the RANKL gene and are responsible for the bone changes also in osteoarthritis. Authors of many studies describe the bone phenotype of this disease as the opposite of that found in osteoporosis; however, the results are not consistent. Our results revealed degraded structure of the subchondral bone in osteoarthritis, increased expressions of RANKL and SOX5 genes and decreased expression of SOX9 gene, as compared to controls. Furthermore, we discovered that mesenchymal stem cells isolated from bone samples of osteoarthritic patients express more SOX5 and RANKL than the mesenchymal stem cells of the control patients after osteogenic differentiation. This confirmed that the transcription factors from the SOX family could contribute to the bone changes, which we discovered in patients with osteoarthritis, by increasing the expression of RANKL. In the doctoral thesis, we discovered novel transcription factors that impact the expression of the RANKL gene through binding to its proximal promoter, and are involved in bone changes in osteoporosis (c-Myb, Sry, SOX5 and SOX) and in osteoarthritis (SOX5 and SOX9). Transcriptional regulators of the RANKL gene represent novel targets for prediction, diagnosis and treatment of bone diseases.
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