ROLES OF P2Y2 PURINERGIC RECEPTOR AND LIPID RAFTS IN BONE CELL MECHANOTRANSDUCTION

Open Access
Author:
Xing, Yanghui
Graduate Program:
Bioengineering
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
December 16, 2009
Committee Members:
  • Dr Jun You, Dissertation Advisor
  • Jun You, Committee Chair
  • Henry Joseph Donahue, Committee Member
  • Ronald R Gomes Jr, Committee Member
  • Cheng Dong, Committee Member
  • Peter J Butler, Committee Member
Keywords:
  • mechanotransduction
  • P2Y2
  • lipid raft
  • osteoblast
Abstract:
ABSTRACT Osteoblasts can sense and respond to their physical environments such as mechanical stimuli, but the molecular mechanisms how the cells receive the signals and transduce them into biochemical responses are not fully understood. Our lab and others have shown that mechanical loading-induced fluid flow results in ATP release from bone cells and subsequently activates one of ATP receptors, P2Y2 receptor. Thus, we hypothesized that P2Y2 receptor plays an important role in osteoblast mechanotransduction. To test our hypothesis, we first examined the role of P2Y2 receptor in osteoblast mechanotransduction in vitro with both MC3T3-E1 osteoblastic cell line and primary mouse long bone cells. We found that overexpression of P2Y2 increases fluid flow-induced ERK1/2 phosphorylation in osteoblasts; whereas knockdown of P2Y2 receptor expression decreases fluid flow-induced ERK1/2 phosphorylation in osteoblasts. Secondly, we observed in vivo that P2Y2 knockout mice have significantly less bone volume, bone thickness, bone stiffness and bone ultimate breaking force compared with those in wild type mice. More importantly, we also found P2Y2 knockout mice have decreased bone formation rate in response to mechanical loading. Additionally, previous studies have demonstrated the importance of lipid rafts in osteoblastic proliferation and differentiation as well as their involvement in the regulation of the vascular shear stress response in endothelial cells, suggesting that lipid raft structure may be responsible for transmitting mechanical signals in osteoblasts. Therefore, we further examined the role of lipid rafts in osteoblast mechanotransduction. Our results showed that disruption of lipid raft structure in osteoblasts significantly inhibits mechano-sensitivity to oscillatory fluid flow in terms of intracellular calcium mobilization and ERK1/2 phosphorylation as well as ATP release. Taken together, our results suggest that both P2Y2 receptor and lipid raft play important roles in osteoblast mechanotransduction.