Microarray analysis reveals PI3K-mediated pathway in mechanotransduction of osteoblasts

Objectives: Mechanical loading is one of the critical stimuli for proliferation and differentiation of osteoblasts. Few studies, however, have been conducted to unravel complex molecular pathways through genome-wide analyses. Using microarray-derived data with mouse osteoblasts, we identified potential signaling pathways underlying the responses to fluid flow and their role in mechanotransduction of bone.

Methods: Mouse osteoblasts (MC3T3 E1 cells, clone 4) were exposed to 1-h flow fluid at intensity of 1 Pa using a Streamer Gold flow device. Total RNA was extracted 1 h, 3 h, and 5 h after the onset of fluid flow. Three independent pairs of samples (flow-treated and control) were prepared for whole mouse genome arrays (G4112A, Agilent). Some of the microarray data points were confirmed by quantitative real-time PCR. We then employed Pathway-Express and determined the most relevant pathways in the context of complex molecular interactions. Immunoblotting was performed with anti-Akt and p-Akt (Ser473) antibodies. To evaluate the involvement of osteopontin in the phosphatidylinositol 3-kinase (PI3K) patway, we employed a PI3K inhibitor, LY294002.

Results: Micoroarray data indicated the involvement of the PI3K and circadian regulatory pathways. Furthermore, protein analyses revealed that fluid flow activated phosphorylation of Akt in the PI3K pathway. Compared to control, the mRNA level of osteopontin under fluid flow was elavated 2.1 times 5h after flow application. Lastly, LY294002 blocked Akt phosphorylation and significantly abolished fluid flow-driven elevation of the osteopontin mRNA level.

Conclusion: Fluid flow in osteoblasts activated the PI3K pathway and induced phosphorylation of Akt. This PI3K pathway mediated flow-driven elevation of the osteopontin mRNA level.

NIH R01 AR50008