Biophysical factors generated during loading inhibit osteoclastogenesis through regulation of two molecules, RANKL and eNOS. This response requires activation of ERK1/2, indicating that strain must initiate proximal events in the MARK signaling cascade. Our study of upstream molecules has revealed that low-magnitude strains activate a single isoform of Ras, H-Ras; and further we show that RNA silencing of H-Ras prevents strain regulated gene expression. The selective Ras activation offers clues to the nature of the mechanotransducer: Ras isoforms have specific spatial distributions within the membrane. H-Ras is located within 'signaling centers' associated with organized membrane (lipid rafts or caveolae). Thus, for H-Ras, the membrane serves as a platform where MARK signaling events are regulated. Our data further shows that disruption of lipid rafts prevents strain activation of H-Ras. The requirement for an intact organized membrane leads to our hypothsis that membrane organization of signaling molecules is required for mechanical regulation of RANKL and eNOS expression and that the organized membrane may serve as the mechanotransducer. We propose to focus on the proximal requirements for converting a mechanical signal into an intracellular chemical signal. In SA#1 we will examine the requirement for H-Ras in the cellular strain response resulting in distal changes in RANKL and eNOS. SA#2 examines the association of H-Ras with the organized membrane, including the requirement for caveolin-1 in mechanotransduction.
These aims utilize 1 degree stromal murine cells challenged with substrate strain and measurements of specific Ras activation, RANKL and eNOS response, and silencing of key molecules through siRNA. SA#3 asks whether membrane organization is relevant for application of load to mice in vivo. We will apply load to tibia of wt C57/B6 as well as to H-Ras null and caveolin-1 null mice. Local expression of genes (RANKL, eNOS, Ras isoforms, caveolin) and bone histomorphometry will be analyzed after loading. With this work we will not only define the mechanisms involved in strain regulation of bone remodeling but also generate a new paradigm for a general mechanotransducer that converts mechanical information into intracellular signals through perturbation of the organized plasma membrane.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR052014-04
Application #
7282450
Study Section
Skeletal Biology Development and Disease Study Section (SBDD)
Program Officer
Sharrock, William J
Project Start
1993-06-01
Project End
2009-08-31
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
4
Fiscal Year
2007
Total Cost
$274,100
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Sen, Buer; Xie, Zhihui; Case, Natasha et al. (2014) mTORC2 regulates mechanically induced cytoskeletal reorganization and lineage selection in marrow-derived mesenchymal stem cells. J Bone Miner Res 29:78-89
Case, N; Sen, B; Thomas, J A et al. (2011) Steady and oscillatory fluid flows produce a similar osteogenic phenotype. Calcif Tissue Int 88:189-97
Case, Natasha; Rubin, Janet (2010) Beta-catenin--a supporting role in the skeleton. J Cell Biochem 110:545-53
Rubin, Janet; Rubin, Clinton (2010) Stand UP! J Clin Endocrinol Metab 95:2050-3
Case, Natasha; Xie, Zhihui; Sen, Buer et al. (2010) Mechanical activation of ýý-catenin regulates phenotype in adult murine marrow-derived mesenchymal stem cells. J Orthop Res 28:1531-8
Styner, Maya; Sen, Buer; Xie, Zhihui et al. (2010) Indomethacin promotes adipogenesis of mesenchymal stem cells through a cyclooxygenase independent mechanism. J Cell Biochem 111:1042-50
Sen, Buer; Styner, Maya; Xie, Zhihui et al. (2009) Mechanical loading regulates NFATc1 and beta-catenin signaling through a GSK3beta control node. J Biol Chem 284:34607-17
Case, Natasha; Ma, Meiyun; Sen, Buer et al. (2008) Beta-catenin levels influence rapid mechanical responses in osteoblasts. J Biol Chem 283:29196-205
Rahnert, Jill; Fan, Xian; Case, Natasha et al. (2008) The role of nitric oxide in the mechanical repression of RANKL in bone stromal cells. Bone 43:48-54
Sen, Buer; Xie, Zhihui; Case, Natasha et al. (2008) Mechanical strain inhibits adipogenesis in mesenchymal stem cells by stimulating a durable beta-catenin signal. Endocrinology 149:6065-75

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