ROLE OF CSF-1 IN OSTEOCLAST FUNCTION

Yale University, New Haven CT

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Abstract

DESCRIPTION (provided by applicant): This Competitive Revision of my currently funded RO1 (DE DE012459-12) is submitted in response to, and in accordance with Notice Number NOT-OD-09-058, titled, NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. One aim of the currently funded award is to define the role of the serine/threonine kinase, LIM kinase-1 in mediating the actions of Colony Stimulating Factor 1 (CSF1) on osteoclasts. Based on preliminary data, we hypothesized that the genetic absence of LIM kinase-1 would lead to an abnormal cytoskeletal response to CSF1 in osteoclasts and abnormal osteoclast function. Both these hypotheses proved to be correct as detailed below. However, quite unexpectedly, we also found a profound disturbance in osteoblast function in LIM kinase-1 knockout mice with a nearly 40% reduction in osteoblast activity as judged by histomorphometry and in vitro mineralization assays. Additionally, there was a significant reduction in the number of CFU-Ob in the marrow of LIM kinase-1 knockout mice, indicating a possible defect in osteoprogenitor lineage allocation. This competitive revision will expand the scope of Specific Aim 2i in the current award, which focuses on LIM kinase-1 in osteoclasts, to also explore the cellular basis for the striking osteoblast abnormalities in LIM kinase-1 knockout mice. To accomplish this we will: 1. determine the cytoskeletal phenotype of osteoblasts isolated from LIM kinase-1 knock out and control mice, using confocal and electron microscopy to analyze the actin and microtubule cytoskeletons and the structure of focal adhesions. 2. Determine the osteoblastogenic and adipogenic potential of mesenchymal stem cells (MSCs) isolated from LIM kinase-1 knockout mice and control mice. 3. Determine if suppressing LIM kinase-1 in a cell model of murine MSCs (C3H T101/2 cells) recapitulates the findings in the LIM kinase-1 knock out MSCs. 4. Express a LIM kinase-1 kinase-dead mutant in LIM kinase knock out osteoblasts and determine if this rescues the phenotype of these cells. These studies will help define how the cytoskeleton regulates osteoblast function, an important but understudied area in bone biology. This in turn, may provide insights into new metabolic pathways that regulate bone growth. PUBLIC HEALTH RELEVANCE: Dental diseases such as periodontal disease, tooth decay and tooth loss, account for billions of dollars in health expenditures annually in the United States. Bone loss is a common component of these diseases and is due both to reduced bone formation and increased bone breakdown. In the course of our work, we have identified a novel and hitherto unrecognized regulator of bone formation, the serine/threonine kinase, LIM kinase 1. This proposal seeks to understand the mechanisms by which the loss of LIM kinase 1 function impairs bone formation. This may lead to new approaches to correcting bone loss in the oral cavity.