Mechanical loads regulate bone metabolism but the mechanism by which this occurs is poorly understood. The applicant proposes that mechanical load is transduced to bone cells via load-induced physical signals such as fluid-flow-induced shear stress. Furthermore, flow stimulates expression of phenotypic characteristics of differentiating osteoblastic cells via a mechanism involving mobilization of cytosolic calcium and gap junctional intercellular communication (GJIC) in osteoblastic or osteocytic cells. The long-term goals of this project are to examine the effect of flow, applied to osteocytic or osteoblastic cells, on osteoblastic differentiation and the roles cytosolic calcium and GJIC play in this process. Additionally, the ability of parathyroid hormone (PTH) to potentiate, through activation of GJIC, the effect of flow on osteoblastic activity will be examined. These goals will be accomplished through the completion of four specific aims.
Aim 1 is to quantify the effect of flow on cytosolic calcium mobilization and expression of markers of osteoblastic differentiation in a human fetal osteoblastic cell line (hFOB 1. 19) and mouse calvarial osteoblastic cells (MCOB).
Aim 2 proposes to quantify the effect of flow on gap junction expression and function in hFOB 1. 19 and MCOB cells and on expression of markers of osteoblastic differentiation in these same cells with and without functional gap junctions.
In Aim 3, studies will examine the role of GJIC in the effect of flow applied to osteocytic cells on activity of osteoblastic cells not exposed to flow.
Aim 4 proposes studies to quantify the effect of flow in the presence of PTH applied to either osteoblastic or osteocytic cells. Cytosolic calcium concentration will be quantified by microspectrofluorometry with fura-2. As markers of osteoblastic differentiation, studies are proposed to assay alkaline phosphatase activity by spectrophotometry, osteocalcin synthesis, by immunoradiometric assay, and steady-state levels of type I collagen, osteopontin and CBFA1 mRNA, by real time RT-PCR. GJIC will be assessed by dye transfer techniques. The recently established MLO-Y4 cell line will be utilized as an osteocytic cell model. hFOB 1. 19 and MLO-Y4 cells will be rendered GJIC deficient by pharmacological blockers, antisense strategies and inhibiting peptides. MCOB cells isolated from Cx43 null mice will also be utilized. Fluid flow will be applied in a parallel plate chamber and rotating disk chamber. The results of this project will provide insights into the mechanisms by which bone cells adapt to their extracellular environment.
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