Abstract

Mechanical stimulation is critical for development and maintenance of skeletal bone mass. However, the mechanosensitivity of bone cells is rapidly reduced in response to continued loading, probably through adaptation, or habituation, of the cellular mechanisms involved in the transduction of this signal. The earliest measured response to mechanical stimulation in osteoblasts is a rapid increase in intracellular Ca2+ (Ca2+i) that is dependent on both channel-mediated Ca2+ entry and intracellular Ca2+ release (iCaR). We have previously characterized a mechanosensitive channel (MSCC) in osteoblasts that is essential for this early Ca2+i response. The underlying hypothesis of this application is that the desensitization of osteoblasts to mechanical stimulation is the result of downregulation of the MSCC and that this downregulation occurs due to the mechanically-induced increase in organization of the actin cytoskeleton. Due to recent in vivo data showing increased bone formation when loading cycles are separated by intervals of rest, we plan to examine the activation, habituation and restoration of mechanosensitivity in osteoblasts using pulsatile fluid shear. Using cell biologic and molecular techniques coupled with Ca2+i imaging and patch clamp analysis, we will: (1) determine the changes in Ca2+i, MSCC and L-type voltage-sensitive Ca2+ channel (CASCC) kinetics, iCaR and gene expression in MC3T3-E1 osteoblasts in response to pulsatile shear when the interval between loading cycles is increased; (2) examine the Ca2+i response, channel kinetics and iCaR during habituation of MC3T3-E1 cells to shear and determine the interaction of these channels with polymerization of the actin cytoskeleton in this loss of mechanosensitivity, and (3) establish the role of the MSCC and the actin cytoskeleton in restoration of MC3T3-E1 osteoblasts to a mechanosensitive cell. Completion of these studies should provide significant insight into the role of these cellular mechanisms play in the response of osteoblasts to mechanical loading and the loss of mechanosensitivity after repeated stimulation.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
7R01AR043222-08
Application #
6924534
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Program Officer
Sharrock, William J
Project Start
1996-08-01
Project End
2007-07-31
Budget Start
2005-08-01
Budget End
2006-07-31
Support Year
8
Fiscal Year
2005
Total Cost
$227,818
Indirect Cost

  Institution

Name
University of Delaware
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
059007500
City
Newark
State
DE
Country
United States
Zip Code
19716

  Publications

Gangadharan, Vimal; Nohe, Anja; Caplan, Jeffrey et al. (2015) Caveolin-1 regulates P2X7 receptor signaling in osteoblasts. Am J Physiol Cell Physiol 308:C41-50
Gardinier, Joseph D; Townend, Chris W; Jen, Kei-Peng et al. (2010) In situ permeability measurement of the mammalian lacunar-canalicular system. Bone 46:1075-81
Shao, Ying; Czymmek, Kirk J; Jones, Patricia A et al. (2009) Dynamic interactions between L-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells. Am J Physiol Cell Physiol 296:C1067-78
Liu, Dawei; Genetos, Damian C; Shao, Ying et al. (2008) Activation of extracellular-signal regulated kinase (ERK1/2) by fluid shear is Ca(2+)- and ATP-dependent in MC3T3-E1 osteoblasts. Bone 42:644-52
Zhang, Jinsong; Ryder, Kimberly D; Bethel, Jody A et al. (2006) PTH-induced actin depolymerization increases mechanosensitive channel activity to enhance mechanically stimulated Ca2+ signaling in osteoblasts. J Bone Miner Res 21:1729-37
Li, Jiliang; Liu, Dawei; Ke, Hua Zhu et al. (2005) The P2X7 nucleotide receptor mediates skeletal mechanotransduction. J Biol Chem 280:42952-9
Genetos, Damian C; Geist, Derik J; Liu, Dawei et al. (2005) Fluid shear-induced ATP secretion mediates prostaglandin release in MC3T3-E1 osteoblasts. J Bone Miner Res 20:41-9
Ryder, K D; Duncan, R L (2000) Parathyroid hormone modulates the response of osteoblast-like cells to mechanical stimulation. Calcif Tissue Int 67:241-6
Chen, N X; Ryder, K D; Pavalko, F M et al. (2000) Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am J Physiol Cell Physiol 278:C989-97
Pavalko, F M; Chen, N X; Turner, C H et al. (1998) Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. Am J Physiol 275:C1591-601

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