Mechanical loading of bone induces fluid shear stress (FSS), which stimulates Src and the mitogen- activated protein kinases Erk1/2 leading to increased osteoblast/cyte proliferation and survival. We found that FSS activates the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) pathway, and that PKG activation is necessary for shear-induced Src and Erk activation. NO donors and cGMP analogs mimicked the effect of FSS on Src/Erk in osteoblasts/cytes, while siRNA knock-down of membrane-bound PKG II abolished it. Src activation by FSS or cGMP occurred through de-phosphorylation of Src Tyr529 (an inhibitory site), which required the protein tyrosine phosphatases (PTP) Shp-1 and -2, and cell attachment through $3 integrins. PKG II, Src, and Shp-2 co-localized with $3 in focal adhesion complexes in FSS-stimulated osteoblasts, and PKG II phosphorylated Shp-1 and -2, but not Src. We hypothesize that PKG II activates Src through activation or recruitment of Shp-1/2, which de-phosphorylate Src Tyr529, and/or through inhibition or displacement of C- terminal Src kinase (CSK), which phosphorylates Tyr529. Consistent with the skeletal phenotype of NO synthase-deficient mice, and based on defective FSS-induced signaling in PKG II-null osteoblasts, PKG II deficiency may lead to decreased bone formation during growth and/or in response to skeletal loading.
The Specific Aims are: (i) to determine the mechanism(s) of Src activation by NO/cGMP/PKGII in FSS-stimulated osteoblasts/cytes;(ii) to characterize the Src-containing signaling complex activated by FSS and NO/cGMP/PKG II;and (iii) to define the role of PKG II in bone (re)modeling in mice. We will map Shp-1/2 phosphorylation by PKG II, and test the effects of PKG II on PTP and CSK activity and subcellular localization. We will assess PKG II, SHP-1/2, and $3 integrin functions in shear-induced Src activation using siRNA approaches with reconstitution of wild type and mutant proteins. We will use co-immunoprecipitation, immuno- fluorescence staining, and bimolecular fluorescence complementation to characterize the PKG-regulated Src signaling complex, and use a proteomics approach to identify novel PKG II substrates and interacting partners in osteoblast membranes. We will examine the skeletal phenotype of osteoblast/cyte-specific PKG II knockout mice during skeletal growth and aging, and under conditions of unloading and reloading, using micro-CT, histo- morphometry, and gene expression analysis. We will analyze FSS responses and differentiation of primary PKG-/- osteoblasts. These studies will provide new insights into NO/cGMP/PKG actions in bone, and could lead to improved therapies for osteoporosis.

Public Health Relevance

Mechanical stimulation is a potent stimulus for bone cell (osteoblast) growth and differentiation, improving bone strength and preventing osteoporosis;however, the molecular mechanisms by which osteoblasts convert mechanical stimuli into biochemical changes (a process known as mechanotransduction) remain poorly understood. We recently defined a novel function of the nitric oxide/cGMP/cGMP-dependent protein kinase (PKG) signal transduction pathway in osteoblast mechanotransduction, and we now propose to study the mechanisms whereby PKG controls important down-stream signaling proteins in mechanically-stimulated osteoblasts and determine the role of PKG II in bone remodeling in genetically-modified mice. These studies will provide the foundation for novel and improved treatment strategies in osteoporosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR051300-15
Application #
8701861
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Chen, Faye H
Project Start
1998-09-01
Project End
2015-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
15
Fiscal Year
2014
Total Cost
$328,104
Indirect Cost
$116,424
Name
University of California San Diego
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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