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Effects of membrane cholesterol depletion and GPI‐anchored protein reduction on osteoblastic mechanotransduction
Author(s) -
Xing Yanghui,
Gu Yan,
Xu LiChong,
Siedlecki Christopher A.,
Donahue Henry J.,
You Jun
Publication year - 2011
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.22579
Subject(s) - mechanotransduction , lipid raft , microbiology and biotechnology , intracellular , calcium in biology , phosphorylation , cell membrane , signal transduction , chemistry , extracellular , cell , biology , biochemistry
We previously demonstrated that oscillatory fluid flow activates MC3T3‐E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y 2 puringeric receptors. However, the molecular mechanisms by which fluid flow initiates cellular responses are still unclear. Accumulating evidence suggests that lipid rafts, one of the important membrane structural components, may play an important role in transducing extracellular fluid shear stress to intracellular responses. Due to the limitations of current techniques, there is no direct approach to study the role of lipid rafts in transmitting fluid shear stress. In this study, we targeted two important membrane components associated with lipid rafts, cholesterol, and glycosylphosphatidylinositol‐anchored proteins (GPI‐anchored proteins), to disrupt the integrity of cell membrane structures. We first demonstrated that membrane cholesterol depletion with the treatment of methyl‐β‐cyclodextrin inhibits oscillatory fluid flow induced intracellular calcium mobilization and ERK1/2 phosphorylation in MC3T3‐E1 osteoblastic cells. Secondly, we used a novel approach to decrease the levels of GPI‐anchored proteins on cell membranes by overexpressing glycosylphosphatidylinositol‐specific phospholipase D in MC3T3‐E1 osteoblastic cells. This resulted in significant inhibition of intracellular calcium mobilization and ERK1/2 phosphorylation in response to oscillatory fluid flow. Finally, we demonstrated that cholesterol depletion inhibited oscillatory fluid flow induced ATP releases, which were responsible for the activation of calcium signaling pathways in MC3T3‐E1 osteoblastic cells. Our findings suggest that cholesterol and GPI‐anchored proteins, two membrane structural components related to lipid rafts, may play an important role in osteoblastic cell mechanotransduction. J. Cell. Physiol. 226: 2350–2359, 2011. © 2010 Wiley‐Liss, Inc.