Research

Identification of new expression regulators of RANKL, a key molecule not only in bone remodelling

Code:

J3-7245

Range:

01. January 2016 - 31. December 2018

Range:

1,88 FTE

Leader:

Janja Marc

Field:

3.07 Medical sciences/Metabolic and hormonal disorders

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.