• Molecular Mechanisms of Osteocyte Mechanotransduction

      Lyons, James Stephen; Stains, Joseph P. (2017)
      Diseases of skeletal fragility affect >200 million people worldwide and contribute to ~9 million factures annually. Preventing bone loss and/or restoring lost bone mass is of vital importance to limiting the personal and economic impact of these diseases. The adaptation of the skeleton to its mechanical environment is orchestrated by mechanosensitive osteocytes, largely through regulating the secretion of sclerostin, an inhibitor of bone formation. Osteocytes sense mechanical load in the form of fluid shear stress (FSS), and respond by reducing expression of sclerostin leading to "de-repression" of osteoblastogenesis and stimulation of de novo bone formation. However, key mechanistic details of how osteocytes sense mechanical load, transduce these signals to biologic effectors, the identity of these effectors and how sclerostin bioavailability is regulated remain unclear. A widely accepted technique for mechanically stimulating cells in culture is the introduction of FSS on cell monolayers. Here, we describe a novel, multifunctional fluid flow device for exposing cells to FSS. We validated the device using the biologic response of UMR-106 cells in comparison to a commercially available system of FSS. Utilizing this FSS device we show that the microtubule (MT) network plays a critical role in how osteocytes sense and respond to FSS. We define a microtubule-dependent mechanotransduction pathway that links FSS to the generation of react ROS and Ca2+ signals, leading to reductions in sclerostin in osteocytes. In Ocy454 osteocyte-like cells, we demonstrate that an intact MT network is required for FSS-induced Ca2+-influx, calcium calmodulin-dependent protein kinase II phosphorylation, and reduction in sclerostin. Further, the abundance of detyrosinated Glu-tubulin dictates the cytoskeletal stiffness of these cells. By tuning the abundance of Glu-tubulin/cytoskeletal stiffness, we demonstrate that Glu-tubulin regulates the mechano-responsive range at which FSS elicits a Ca2+ response in osteocytes. Further, we determined that the FSS-induced reduction in sclerostin requires activation of a signaling cascade that includes production of Nox2-activated ROS, which stimulates Ca2+-influx through the cation-permeable channel TRPV4 and the subsequent activation of CamKII. By developing a better understanding of this fundamental aspect of skeletal physiology, we will raise the possibility of outlining new drug targets to combat diseases of skeletal fragility.