Quantitative Proteomic Analysis of Bone Cells
EMSL Project ID
32593
Abstract
The ability of the skeleton to respond to physical forces is essential for the maintenance of proper bone mass. Perturbations in physical stimulation, such as by body immobilization, paralysis, or long-term exposure to microgravity during extended spaceflight, lead to pathological losses of skeletal integrity. The mechanisms by which bone transduces physical stimuli are poorly understood but it is widely accepted that osteocytes are the bone cells responsible for mechanosensation. Osteocytes are characterized by long membrane extensions (dendrites) with which these cells communicate with neighboring osteocytes and bone-forming osteoblasts. The proteins responsible for the formation and function of membrane dendrites are unknown, and the current lack of information regarding the mechanisms by which osteocytes communicate with each other and control osteoblast activity represents a critical gap in the understanding of bone physiology. We postulate that the segregation of key functional proteins between the cell body and dendrites plays a major role in the control of the osteocyte mechanosensory network. We recently found that the lipid growth factor lysophosphatidic acid (LPA) stimulated the formation of osteocyte dendrites, and we devised a method for the physical separation of dendrites from cell bodies of cells grown on microporous substrates. In a previous EMSL User Proposal project whole cell lysates and dendrite extracts were subjected to LC-MS/MS to establish Potential Mass and Time tag databases. This led to the identification of more than 13,000 peptides from osteocytes cultured in the presence or absence of LPA. Filtering of very low abundance proteins resulted in a dataset of over 7,000 peptides. Based on peptide counts we tentatively identified 284 and 844 proteins that appeared to exhibit significant changes in abundance after cells were treated with LPA for 6 hr and 24 hr, respectively. Most of the proteins identified as being regulated by LPA were not previously known to be expressed by osteocytes. We now propose to quantify more precisely the LPA-induced changes in osteocyte protein amount and subcellular distribution using LC-FTICR-MS. The response of bone cells to LPA includes the activation of signaling pathways coupled to protein phosphorylation, and we also propose to compare the phosphoproteomes of control and LPA-treated osteocytes to test the hypothesis that LPA-induced dendrite formation requires the phosphorylation of specific regulatory proteins. These studies will provide valuable new insights into the function of osteocytes and contribute to the formulation of mechanistic models of osteocyte dendrite formation and cell-cell communication. This Open Call proposal has direct relevance to the EMSL Science Theme: Biological Interactions and Dynamics.
Project Details
Start Date
2008-12-22
End Date
2009-12-27
Status
Closed
Released Data Link
Team
Principal Investigator
Related Publications
Govey PM, JM Jacobs, SC Tilton, AE Loiselle, Y Zhang, WM Freeman, KM Waters, NJ Karin, and HJ Donahue. 2014. "Integrative Transcriptomic and Proteomic Analysis of Osteocytic Cells Exposed to Fluid Flow Reveals Novel Mechano-Sensitive Signaling Pathways." Journal of Biomechanics 47(8):1838-1845. doi:10.1016/j.jbiomech.2014.03.022
Waters KM, JM Jacobs, MA Gritsenko, and NJ Karin. 2010. "Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid." PNNL-SA-74808, Pacific Northwest National Laboratory, Richland, WA.