Osteoblasts are the precursors to osteocytes. A specific genetic program and gene expression profile underlies this differentiation process to a mature osteocyte, but the detailed components of that program are unknown. We have begun to define a subset of gene expression patterns that represent the state of the osteoblast as it progresses towards an osteocyte surrounded by osteoid that is actively mineralizing. Using osteoblast and osteocyte models composed of two cell lines, 2T3, an osteoblast-like cell, and MLO-Y4, an osteocyte-like cell, we have defined a subset of genes that are low in osteoblasts and become highly expressed in the osteocytes model. Additionally, a subset of genes in the osteocyte that respond to fluid flow have been identified Additionally, a subset of genes in the osteocyte that respond to fluid flow have been identified. One genes that encodes for Monocyte Chemoattractant Protein, MCP-3, is 13 TO 19 fold higher in osteocytes compared to osteoblast becomes an osteocyte and identify genes important for osteocyte function. The short- term goal is to determine the role of MCP-3 in osteocyte function. Our hypothesis is that MCP-3 is a marker for differentiation of osteoblasts into osteocytes and a critical molecule in recruitment of osteoclast precursors to bone. In this proposal we will address 1) the role of MCP-3 in osteocyte function and 2) the regulation of MCP-3 gene expression under mechanical strain both in vitro and in vivo. To accomplish these goals, the MPO-Y4 cell line, primary osteocytes, primary osteoblasts and the 2T3 cell line that forms mineralized nodules containing osteocyte-like cells in culture will be used. Increasing magnitude and frequency of fluid flow will be tested for effect on MCP-3 production, gene expression patterns and function in these models. Two rodent in vivo models of mechanical strain, the tooth movement model and ulnae-fatigue loading model will be used to access and validate gene expression changes found in the in vitro models. We will also use transgenic mice with a null allele of the MCP receptor, CCR2 mice will have defects in osteoclast precursor recruitment. Results from these studies will dramatically expand our database on genes important for osteocyte function and should also lead to insights into the mechanism of osteoclast recruitment to sties of bone microfracture.
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