Spatial, chemical, and mechanical signals all contribute to lineage allocation of pluripotent mesenchymal stem cells (MSCs). When the fate of MSCs tips in favor of adipogenesis and away from osteogenesis in conditions such as unloading, aging, or estrogen deficiency, bone quality is diminished and risk of fracture increases. Dynamic skeletal loading inhibits adipogenesis in vitro and in vivo by enhancing ?-catenin activity in MSCs. MSC potential is preserved by a signaling pathway, which is initiated at focal adhesions (FAs) setting off a cascade of ?Fyn/FAK to ?mTORC2 to ?Akt to ?GSK3? and ??-catenin. We have shown that mechanical strain recruits mTORC2 and Akt to FAs; however, the mechanisms responsible for this intracellular signal partitioning and the specific activation sites of mTORC2 subunits are unknown. Preliminary work suggests that strain induces an association of mTORC2 with myosin motors. Just as other intracellular cargo, including ?- integrins, attach to myosins to be transported to FAs, myosins may enable recruitment of mTORC2 to FAs in response to mechanical force. We have also shown that Fyn and mTORC2/Akt participate in mechanically regulated activation of RhoA; however, preliminary work suggests that GEF or GAP intermediaries may be required for RhoA-induced cytoskeletal reorganization and adipogenic repression. The focus of this proposal will be to examine the regulatory modifications of mTORC2-specific subunits and to determine how strain recruits mTORC2 to FAs to be activated. Additionally, we will ask how mTORC2/Akt regulate GEF and GAP RhoA effectors to auto-regulate the cytoskeleton in response to physical force. The proposed hypotheses will be examined through the following specific aims: 1) determine how mTORC2 is activated by mechanical strain; 2) identify mechanisms by which mTORC2 regulates cytoskeletal reorganization. Pharmacological inhibition/knockdown studies will be performed using primary marrow-derived MSCs to examine myosin- mediated mTORC2 FA recruitment, to identify mechanically responsive phosphorylation sites on mTORC2 subunits, and to determine how these modifications influence cytoskeletal remodeling. Additionally, RhoA, GEF, and GAP pull down assays will be employed to study the effects of force on modulators of cytoskeletal adaptation. These studies have implications for understanding the mechanisms by which mechanical loading regulates cytoskeletal assembly and reinforcement, a process essential for proper regulation of mechanosensation and MSC lineage fate. As such, this work will inform the design of future strategies for using exercise to affect development of fat and bone.
Osteoporosis and obesity are two of the most debilitating conditions in the United States, and although osteoporosis primarily affects the elderly, obesity is a major health concern among children and adolescents. Estimates indicate that 25% of American children are overweight, while 11% are obese; an enormous concern in that obese children are predisposed to type 2 diabetes and have an increased lifetime risk of cardiovascular disease and cancer. The studies proposed here will examine how mechanical forces, similar to those during walking or exercise, regulate the fate of mesenchymal stems cells to develop into bone cells or fat cells, and will be essential to the design of effective exercise interventions to promote bone health and prevent excess fat formation.
| Thompson, William R; Yen, Sherwin S; Uzer, Gunes et al. (2018) LARG GEF and ARHGAP18 orchestrate RhoA activity to control mesenchymal stem cell lineage. Bone 107:172-180 |
| Warden, Stuart J; Thompson, William R (2017) Become one with the force: optimising mechanotherapy through an understanding of mechanobiology. Br J Sports Med 51:989-990 |
| Uzer, Gunes; Fuchs, Robyn K; Rubin, Janet et al. (2016) Concise Review: Plasma and Nuclear Membranes Convey Mechanical Information to Regulate Mesenchymal Stem Cell Lineage. Stem Cells 34:1455-63 |
