Abstract
The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner.
Highlights
It is well evidenced that mechanical stimulation can increase bone mass in vivo [1] through an increase in the number of osteoblasts, the bone-forming cells
Mechanotranduction plays a key role in the biological processes of a variety of cell types, including smooth muscle cells [54], endothelial cells (56 –58), and bone cells [59]
It involves the conversion of a biophysical force into a cellular/molecular response leading to both rapid changes in kinase-mediated gene expression as well as slower adaptive changes in cytoskeletal arrangement
Summary
Materials—Y402F-c-Myc-tagged PYK2 and Y397F-HA-tagged FAK were kindly provided by Dr Shelton Earp (University of North Carolina Chapel Hill, NC). Cells were plated in six-well tissue culture dishes with type I collagen-coated, flexible silicone bottoms (Bioflex; Flexcell Corp.) at 51⁄7104 cells per well density for proliferation studies and 11⁄7105 cells per well for signal transduction studies. The Bioflex baseplate contain the Bioflex loading station that consists of six buttons per plate that insert into each plate, allowing a uniform magnitude of the strain across 85% of the surface of the flexible well For these experiments a negative pressure of 80 kilopascals was applied through an air pump to the culture plate, bottoms were deformed to a known percentage of elongation, and the membranes were released to their original conformation. Cells were centrifuged at 400 ϫ g for 10 min and washed with complete medium to remove cells from the staining solution. The PI, a measure of the proliferation activity of total cell population, was calculated by ModFit 2.0 using Equation 1,
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