Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction

Yanghui Xing, Yan Gu, Lichong Xu, Christopher Siedlecki, Henry J. Donahue, Jun You

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y2 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.

Original languageEnglish (US)
Pages (from-to)2350-2359
Number of pages10
JournalJournal of Cellular Physiology
Volume226
Issue number9
DOIs
StatePublished - Sep 1 2011

Fingerprint

Glycosylphosphatidylinositols
Flow of fluids
Cholesterol
Membranes
Calcium
Lipids
Phosphorylation
Calcium Signaling
glycoprotein phospholipase D
Cell membranes
Proteins
Shear stress
Membrane Proteins
Adenosine Triphosphate
Purinergic P2Y2 Receptors
Cell Membrane Structures
Membrane structures
Fluids
Extracellular Fluid
Cyclodextrins

All Science Journal Classification (ASJC) codes

  • Physiology
  • Clinical Biochemistry
  • Cell Biology

Cite this

@article{d7ab1ce69a13489496ddafc6c03b99c5,
title = "Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction",
abstract = "We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y2 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.",
author = "Yanghui Xing and Yan Gu and Lichong Xu and Christopher Siedlecki and Donahue, {Henry J.} and Jun You",
year = "2011",
month = "9",
day = "1",
doi = "10.1002/jcp.22579",
language = "English (US)",
volume = "226",
pages = "2350--2359",
journal = "Journal of Cellular Physiology",
issn = "0021-9541",
publisher = "Wiley-Liss Inc.",
number = "9",

}

Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction. / Xing, Yanghui; Gu, Yan; Xu, Lichong; Siedlecki, Christopher; Donahue, Henry J.; You, Jun.

In: Journal of Cellular Physiology, Vol. 226, No. 9, 01.09.2011, p. 2350-2359.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Effects of membrane cholesterol depletion and GPI-anchored protein reduction on osteoblastic mechanotransduction

AU - Xing, Yanghui

AU - Gu, Yan

AU - Xu, Lichong

AU - Siedlecki, Christopher

AU - Donahue, Henry J.

AU - You, Jun

PY - 2011/9/1

Y1 - 2011/9/1

N2 - We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y2 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.

AB - We previously demonstrated that oscillatory fluid flow activates MC3T3-E1 osteoblastic cell calcium signaling pathways via a mechanism involving ATP releases and P2Y2 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.

UR - http://www.scopus.com/inward/record.url?scp=79958755753&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79958755753&partnerID=8YFLogxK

U2 - 10.1002/jcp.22579

DO - 10.1002/jcp.22579

M3 - Article

C2 - 21660958

AN - SCOPUS:79958755753

VL - 226

SP - 2350

EP - 2359

JO - Journal of Cellular Physiology

JF - Journal of Cellular Physiology

SN - 0021-9541

IS - 9

ER -