We seek to understand the molecular mechanisms that direct bone formation and resorption in response to mechanical loading. Pharmacologic manipulation of these mechanotransduction (MTD) signaling processes in bone cells has therapeutic potential. We propose a novel strategy that investigates signaling mechanisms that suppress the stimulatory effects of loading (rather than focusing on signaling pathways that stimulate new bone formation). The fundamental goal is to manipulate MTD pathways so that even modest levels of exercise can have outsized anabolic effects if mechanisms that inhibit load-induced bone formation are pharmacologically suppressed. Osteocytes (OCY), the most abundant cell type in bone, coordinate the response of bone to mechanical load. We propose that the tyrosine kinase Src functions in OCY as a novel suppressor of load-induced bone formation. Global Src null mice have high bone mass (HBM). This is due in part to Src-dependent defects in osteoclast- mediated bone resorption. However, we risk missing an important role that tyrosine kinases may play in the anabolic arm of skeletal MTD if we attribute the HBM phenotype of Src KO mice entirely to an osteoclast defect in bone resorption. We suggest there is an additional underappreciated role for Src in the osteoblast/osteocyte (OB/OCY) population that inhibits mechanically-induced anabolic signals. Specifically, we propose that upon activation by mechanical stimuli, Src dissociates from integrins (membrane mechanosensors) and translocates to the nucleus as part of a multi-protein complex with Proline-rich Kinase-2 (Pyk2) and the methylated DNA binding protein Methyl-CpG Binding Domain Protein-2 (MBD2), to regulate epigenetics of key bone genes. Thus, OCY may utilize a SrcPyk2-MBD2 ?mechanosome? to promote or suppress anabolic or anti-catabolic bone genes by altering promoter- associated CpG islands. We propose to experimentally dissect the molecular mechanisms through which Src inhibits bone formation using in vivo and in vitro approaches with the long term goal of better understanding the clinical and translational potential of Src inhibitors to enhance bone density and fracture susceptibility.
Three aims are proposed:
Aim 1 will determine the effect of targeted Src deletion from osteocytes on basal and load-induced bone formation and on disuse-induced bone loss in mice.
Aim 2 will determine the role of Src in epigenetic regulation of mechanically sensitive bone genes.
Aim 3 will determine the molecular interactions of Src in the cytoplasm and nucleus of osteoblasts and osteocytes subjected to fluid shear stress in vitro using FRET-FLIM microscopy.

Public Health Relevance

Mechanical stimulation to the skeleton, such as occurs during exercise, promotes bone gain and suppresses bone loss, ultimately resulting in improved bone strength and fracture resistance. The molecular signaling pathways within bone cells that sense mechanical stimulation and translate it into improved bone formation are unclear. Through experimental dissection of these pathways we hope to identify target molecules that can be pharmacologically manipulated to enhance bone strength and fracture resistance thus realizing the translational potential of basic skeletal mechanobiology research to benefit human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR069029-02
Application #
9431179
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Alekel, D Lee
Project Start
2017-03-01
Project End
2022-02-28
Budget Start
2018-03-01
Budget End
2019-02-28
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Physiology
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202