Our parent R01 project broadly explores age-related deficits in activation of the Ca2+/NFAT pathway and interventions to enhance bone adaptation at senescence. We use in vivo knock-out and senescent mouse models to assay tissue adaptation to mechanical stimuli (via dynamic histomorphometry) and single time point in vivo cellular function assays (via immunohistochemistry and RT-PCR). In vivo studies are complemented with extensive in silica modeling of tissue adaptation emerging from cell-cell interactions (via agent-based computational modeling). The background of the investigators in the R01 project reflect these bioengineering and statistical requirements and included expertise in bone mechanotransduction, in vivo animal models, histomorphometry and immunohistochemistry (T.S. Gross, S. Srinivasan), agent-based models (S. Srinivasan), and statistics (T.S. Richardson). Our application will require development of a new interdisciplinary collaboration with scientists whose expertise includes the study of signaling pathways and transcriptional control of these pathways in a variety of contexts. In this regard, senior scientist E.M. Gardiner (Ph.D. in Human Genetics), and post-doctoral fellow Leah Worton (Ph.D. in Medicine), directly address this need given their combined expertise in utilizing molecular techniques to study a variety of signaling pathways involved in cancer biology, and in endocrine and neuronal skeletal regulation. As neither the PI nor co-Investigators have previously formally collaborated (e.g., conference proceedings, journal articles, grants and contracts), this nascent effort complies with the criteria outlined in the BIRT RFA. The collaboration arose from informal conversations during Dr. Gardiner's sabbatical the University of Washington. Success of this BIRT application will substantially catalyze opportunities for long-term collaboration with Dr. Gardiner to explore other research areas of mutual interest such as the role of protein networks and memory in bone mechanotransduction. The concept for our application to the BIRT RFA arose from our most exciting initial result in our R01 project. Specifically, we identified a novel strategy (that of supplementing mild mechanical stimuli with low-dose Cyclosporin A;CsA) that completely rescues bone adaptation at senescence. Additional studies and synthesis of literature have identified a critical need to more directly understand the cellular mechanisms by which mechanical stimuli interacts with this low-cost drug in order to optimize the intervention and improve the potential for direct translation into a clinical study. Specifically, we believe that determining how CsA interacts with mechanical stimuli at senescence to enhance Ca2+/NFAT signaling at the level of cytoplasmic and nuclear proteins and transcription factor - DNA binding will permit a unique opportunity to optimize interventions for a wide variety of mechanical stimuli. Such a characterization will require experiments with primary cultures and involve assays including Westerns, ChIP, and siRNA approaches that are beyond the expertise of the original investigator group of the R01 project. Our new collaboration therefore includes scientists with molecular biology training and specific expertise in studying a variety of signaling pathways at this level (E.M. Gardiner, Ph.D., and Leah Worton, Ph.D.). This expansion of our initial investigative team will clearly augment our expertise and greatly improve our ability to exploit our exciting results. There is little doubt that a mechanistic understanding of our tissue level observations will substantially enhance prospects for timely clinical translation of this promising concept. Further, we believe that these additional molecular studies will enhance our modeling efforts, which will lead to identification of other potential interventions for bone loss pathologies that would otherwise remain unreachable in the context of the parent R01.

Public Health Relevance

The focus of this project is to mechanistically explore and optimize interactions between a low-cost drug and mechanical stimuli to restore age-related deficits in bone mechanotransduction. Clinically, success of this project would provide the rationale for examination of this concept to augment bone mass in the aged population.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Special Emphasis Panel (ZAR1-KM (M1))
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Sharrock, William J
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University of Washington
Schools of Medicine
United States
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Srinivasan, Sundar; Ausk, Brandon J; Bain, Steven D et al. (2015) Rest intervals reduce the number of loading bouts required to enhance bone formation. Med Sci Sports Exerc 47:1095-103
Recidoro, Anthony M; Roof, Amanda C; Schmitt, Michael et al. (2014) Botulinum toxin induces muscle paralysis and inhibits bone regeneration in zebrafish. J Bone Miner Res 29:2346-56
Srinivasan, Sundar; Threet, Dewayne; Worton, Leah E et al. (2014) Distinct cyclosporin a doses are required to enhance bone formation induced by cyclic and rest-inserted loading in the senescent skeleton. PLoS One 9:e84868
Worton, Leah E; Kwon, Ronald Y; Gardiner, Edith M et al. (2014) Enhancement of Flow-Induced AP-1 Gene Expression by Cyclosporin A Requires NFAT-Independent Signaling in Bone Cells. Cell Mol Bioeng 7:254-265
Ausk, Brandon J; Huber, Philippe; Srinivasan, Sundar et al. (2013) Metaphyseal and diaphyseal bone loss in the tibia following transient muscle paralysis are spatiotemporally distinct resorption events. Bone 57:413-22
Worton, Leah E; Ausk, Brandon J; Downey, Leah M et al. (2013) Systems-based identification of temporal processing pathways during bone cell mechanotransduction. PLoS One 8:e74205
Srinivasan, Sundar; Gross, Ted S; Bain, Steven D (2012) Bone mechanotransduction may require augmentation in order to strengthen the senescent skeleton. Ageing Res Rev 11:353-60
Srinivasan, Sundar; Ausk, Brandon J; Prasad, Jitendra et al. (2010) Rescuing loading induced bone formation at senescence. PLoS Comput Biol 6: