Projects / Programmes
Identification of new expression regulators of RANKL, a key molecule not only in bone remodelling
| Code |
Science |
Field |
Subfield |
| 3.07.00 |
Medical sciences |
Metabolic and hormonal disorders |
|
| Code |
Science |
Field |
| B580 |
Biomedical sciences |
Skeleton, muscle system, rheumatology locomotion |
| Code |
Science |
Field |
| 3.02 |
Medical and Health Sciences |
Clinical medicine |
Osteoprotegerin, lung cancer, chondrocyte, osteoarthrosis, osteoblast, osteoporosis, cell model, cloning, functional genomics, bioinformatics
Researchers (13)
Organisations (3)
Abstract
Receptor activator of nuclear factor-κB ligand (RANKL) is a part of the RANKL/RANK/OPG system which plays a crucial role in bone resorption and several other physiological processes. RANKL deregulation has been implicated in osteoporosis (OP), osteoarthritis (OA), autoimmune diseases, chronic inflammatory diseases and various cancers, leading to approval of denosumab, a monoclonal antibody to RANKL, for OP treatment and prevention of skeletal-related events in patients with bone metastases. Although a lot was invested in understanding consequences of RANKL and its signalling, much less is known about stimuli and molecular pathways that regulate RANKL expression itself. Most information regarding transcriptional regulation of RANKL gene comes from the studies of mouse osteoblastic/stromal cells, while much less is known about RANKL regulation in human cells. Studies performed so far have demonstrated the relevance of both very distant enhancers as well as proximal promoter region, which may act together in an unknown fashion. Moreover, results from transcriptional regulation of mouse RANKL gene are not directly applicable to human RANKL gene regulation and some regulatory regions show cell type specificity thus neccessitating further studies of human RANKL gene regulation in different cell types. RANKL undoubtedly plays an important role in common degenerative bone and joint diseases, OP and OA, respectively as well as in lung cancer, which is the leading cause of cancer mortality. However, RANKL transcriptional regulation has not been extensively studied neither in human osteoblasts, chondrocytes nor in lung-cancer cells. Therefore, the primary aim of the IREGULAR project- Identification of new expression regulators of RANKL, is to identify transcriptional regulators of RANKL gene expression in the proximal promoter region in human osteoblast, chondrocyte and lung cancer cells. Additionally, we intend to evaluate how these transcription factors (TF) interact with single stranded DNA molecules in order to understand dynamics of DNA-TF binding process.
To achieve these goals, 4 reporter plasmids with inserted RANKL promoter regions of different sizes, covering first 900 bp region upstream of the transcription start site will be constructed. The influence of each construct on RANKL transcription in human chondrocytes, non small cell lung cancer cell lines with different metastatic potential and osteoblast cell lines in different stages of maturation will be evaluated by luciferase activity. Bioinformatic analysis and site-directed mutagenesis will aid in the detection of the exact locations of TF binding sites, while the binding of specific TF will be confirmed by electrophoretic mobility shift assay. These in vitro findings will be translated into in vivo setting by measuring the TF in human bone tissue samples of OP and OA patients as well as in healthy controls using immunohistochemistry and quantitative polymerase chain reaction. Additionally, information about potential secondary structures of RANKL promoter regions and insight into DNA-TF interactions at the molecular level will be provided by structural analysis using nuclear magnetic resonance (NMR) and other complementary spectroscopic methods.
Identification of new TF involved in RANKL transcriptional regulation will increase our knowledge on pathways taking place in deregulation of RANKL in various diseases like OP, OA and lung cancer. These TF and their upstream regulators might be used as potenital biomarkers of RANKL activity and/or denosumab treatment efficacy and also serve as potential new drug targets. Elucidation of the cell type-specific RANKL regulation could provide more targeted therapeutic approaches with minimal side effects in non-target tissues. In addition, results of spectroscopic studies could contribute to the development of aptamer-based therapeutics or aptamer-based plasma protein diagnostics.
Significance for science
The relevance of RANKL signalling in human is well known, but little is known about how RANKL expression is regulated. Understanding of RANKL regulation is though very important, because RANKL influence multiple physiological processes, such as bone remodelling, activity of T lymphocytes and endothelial cells etc. Moreover, RANKL deregulation has been implicated in several diseases in cluding osteoporosis, osteoarthritis and various cancers. The aim of IREGULAR project is to discover the transcription factors that mediate RANKL gene expression. Discovery of new transcription factors will provide better understanding of RANKL expression control and provide solid base for further research of new signalling pathways regulated by the discovered transcription factors which are potentially involved in pathogenesis of three very common diseases in aged population like osteoporosis and osteoarthritis as well as of most frequent type of cancers in humans like lung cancer. Since regulatory mechanisms can be tissue specific, the outcomes of IREGULAR project will provide important information for more targeted therapeutic approaches compared to systemic action of currently available RANKL inhibitor denosumab.
Significance for the country
New transcription factor binding sites in RANKL promotor region, discovered in IREGULAR project, provide the basis for development of novel inhibitors of RANKL. While RANKL can be inhibited systemically by monoclonal antibody denosumab, targeting the transcription factors could provide a new approach for tissue specific regulation of RANKL expression. This would reduce the side effects and increase the efficacy of treatment. Solving the structure of DNA-transcription factor complex would also provide the basis for the novel aptamers-based approach to target RANKL. Aptamers are highly specific and have several advantages over monoclonal antibodies: they can be synthesised in test tubes without requirement for cell cultures; aptamers are more stable than antibodies; and aptamers have low immunogenic properties. Recently, FDA approved the first aptamer-based therapeutic for use in human. Besides therapeutic potential the outcomes of IREGULAR study also have commercial potential in diagnostics. Based on the changes in levels of transcription factors in RANKL proximal promotor we could predict complications caused by increased expression of RANKL and select good responders to treatment with denosumab. The results of IREGULAR study provide scientific basis for the development of such diagnostic test. The therapeutic and diagnostic developed on top of these results would provide a better patient care, particularly for elderly patients with diseases such as osteoporosis, osteoarthritis and lung cancer.
Most important scientific results
Interim report,
final report
Most important socioeconomically and culturally relevant results
Interim report,
final report
