Mechanical forces enhance bone mass and strength, whereas glucocorticoid excess (GC) decreases bone formation and increases bone fragility. Mechanical stimuli increase proliferation of pre-osteoblasts, accelerate osteoblast differentiation, and inhibit osteoblast and osteocyte apoptosis;and directly activates Wnt-dependent transcription and downregulates the Wnt antagonists sclerostin and Dkk1. In contrast, GC inhibit osteoblast differentiation and induce osteoblast and osteocyte apoptosis;and inhibit Wnt-dependent transcription and increase Dkk1 expression. Work leading to this application indicates that these converse effects might stem from opposing actions on the focal adhesion kinases FAK and Pyk2, which regulate interactions between cellular integrins and the extracellular matrix. Thus, mechanical stimuli prevent osteoblast/osteoblast apoptosis by outside-in signaling mediated by integrins resulting in activation of FAK and ERKs;and GC oppose these survival signals by activating Pyk2 and its target JNK, leading to inside-out signaling and cell detachment- induced apoptosis. Remarkably, FAK/ERK activation and anti-apoptosis induced by mechanical stimulation is abolished by Dkk1 or ?-catenin degradation. Conversely, Pyk2-dependent apoptosis by GC is inhibited by Wnts;and Pyk2 activates GSK3?, the enzyme responsible for degrading ?-catenin. Based on these lines of evidence, it is hypothesized that there is an antagonistic interplay between mechanical forces and GC governed by FAK/Pyk2 signaling, which regulates the Wnt/?-catenin pathway, bone formation, and osteoblast/osteocyte survival. This hypothesis will be tested by a combination of in vitro studies using established cell lines and primary osteoblasts and osteocytes, and in vivo approaches using transgenic and knockout mice.
Aim 1 will determine the role of FAK-mediated outside-in signaling and Wnt activation in mechanotransduction. It will be investigated whether loading-induced anabolism is impaired in mice lacking FAK in osteoblasts and/or osteocytes, and whether this response is rescued by ?-catenin stabilization;and whether there is a cell autonomous requirement of FAK for mechano-responsiveness using osteocytes and osteoblasts in which FAK was knocked-down or knocked-out.
Aim 2 will determine the role of Pyk2-mediated inside-out signaling and Wnt inhibition in GC effects. It will be investigated whether inhibition of Pyk2 or downstream targets JNK and RhoA/Rock prevents osteoblast/osteocyte apoptosis, the decrease in bone formation, and the loss of strength induced by GC, by using Pyk2 and FAK null mice and mice treated with Pyk2, JNK, or Rock inhibitors;whether ?-catenin stabilization or enhanced Wnt signaling prevents GC deleterious effects, using mice treated with GSK3? inhibitors or Sost null mice;and whether activation of Pyk2 is responsible for Wnt inhibition by GC in vitro, using cells in which Pyk2 is knocked-out or knocked-down, or cells treated with Pyk2 inhibitors.
Aim 3 will investigate whether mechanical forces and GC antagonize in vivo and the role of FAK in the protective action of loading when applied simultaneously, before, or after initiation of GC treatment.

Public Health Relevance

These studies will advance our understanding of the mechanism of action of mechanical forces and GC on bone and have the long-term goal of developing new therapeutic strategies for preservation or restoration of skeletal health in patients treated with GC or exhibiting endogenous elevation of the steroids, such as during aging.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Skeletal Biology Development and Disease Study Section (SBDD)
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Chen, Faye H
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Indiana University-Purdue University at Indianapolis
Anatomy/Cell Biology
Schools of Medicine
United States
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Rhee, Yumie; Lee, Eun-Young; Lezcano, Virginia et al. (2013) Resorption controls bone anabolism driven by parathyroid hormone (PTH) receptor signaling in osteocytes. J Biol Chem 288:29809-20
Gortazar, Arancha R; Martin-Millan, Marta; Bravo, Beatriz et al. (2013) Crosstalk between caveolin-1/extracellular signal-regulated kinase (ERK) and ýý-catenin survival pathways in osteocyte mechanotransduction. J Biol Chem 288:8168-75