Regulation of PTH-induced RankL transcription in osteoblasts

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Abstract: Osteoporosis is a prevalent disease of aging characterized by a decrease in the density and quality of bone tissue and is associated with substantial morbidity/mortality. In osteoporosis the homeostatic processes that form new and remove old/damaged bone are dysregulated, promoting excessive resorption. Parathyroid hormone (PTH) is a key regulator of this homeostasis and along with its analogs has been used to treat osteoporosis. Although PTH has positive anabolic effects on bone it can also stimulate catabolism through activity of receptor activator of nuclear factor kappa-β ligand (RankL). Treatment of osteoporosis via PTH is limited by a short “anabolic window,” after which the positive effects are mitigated by the resorption initiated by RankL. Theoretically, it may be possible to retain just the positive effects of PTH-derived treatments if transcription of RankL can be inhibited. PTH and its analogs bind the same receptor on osteoblasts, activating a signaling cascade leading to RankL transcription. Recent work has implicated a cascade of messengers, enzymes, kinases, and phosphatases in regulating the two coactivators of RankL transcription. The exact mechanisms that cause this regulation and the transcription factor(s) the coactivators associate with to initiate RankL transcription are not established. The purpose of this research is to identify the specific regulatory mechanisms and transcription factor(s) that activate RankL transcription. In completing the aims and training plan outlined in this proposal the graduate student, Michael Mosca, will gain substantial experience in new biochemical techniques, will gain specific knowledge in endocrinology and pharmacology, and will expand his expertise in musculoskeletal tissues to include bone. This research training will build on his biomechanical and histological experiences related to inflammatory and fibrotic mechanisms of muscle, tendon/ligament pathologies and will prepare him for a research career as an independent investigator in the musculoskeletal field. Aim 1.1 will determine the specific individual and/or combined regulatory roles that several salt-inducible kinases and protein phosphatases have on cAMP-regulated transcriptional coactivators 2/3 (CRTC2/3). First, a series of siRNA transfections will be performed with primary calvarial osteoblasts to explore the effects of knocking down each factor on RankL transcription via qRT-PCR. Then, the effect the knockdowns have on CRTC2/3 nuclear translocation will be determined with and without PTH-treatment via quantitative immunofluorescence. Aim 1.2 will assess the roles CRTC2/3 play as RankL co-activators throughout osteoblast differentiation using similar methods. Aim 2 seeks to identify the transcription factor(s) that CRTC2 and CRTC3 each associate with to activate RankL transcription using Mass Spectrometry, Chromatin-immunoprecipitation, and siRNA knockdowns. RankL expression is responsible for catabolism seen in treatment of osteoporosis by PTH and its analogs. Elucidation of the mechanisms that regulate and initiate RankL transcription may identify novel therapeutic targets to inhibit RankL and improve the efficacy of PTH in the treatment of osteoporosis.