CSF-1, released by osteoblasts and stromal cells, stimulates the proliferation and differentiation of osteoclast progenitors and enhances osteoclast survival. In the op/op mouse model, a thymidine insertion in the coding osteopetrosis. The long-term goal of this proposal is to determine the effect of soluble(s) and membrane-bound (m) CSF-1 isoforms on osteoclastogenesis and define cell-specific cis-acting elements in the 5' flanking region of the CSF-1 promoter that regulate their expression. Our first hypothesis is that sCSF-1 and mCSF-1 are differentially synthesized and stimulate osteoclast formation due, in part, to their interaction with the c-fms receptor. To address this issue in vitro, op/op stromal cells will be transfected with sCSF-1 or mCSF-1 cDNA and stable transfectants examined for CSF-1 synthesis, bioactivity, osteoclast support and c-fms tyrosine kinase activation in target cells. The effect of each isoform on osteoclast formation in vivo will be explored by targeting isoform to ameliorate osteopetrosis in op/op mice, genetic crosses between sCSF-1 and mCSF-1 transgenic mice and heterozygous op/wt mice, genetic crosses between sCSF-1 and mCSF-1 transgenic mice and heterozygous op/wt mice will be carried out to establish op/op mutants expressing each transgene. Mice will be examined for serum CSF-1 bone growth, incisor eruption and resolution of osteopetrosis. Bone sections will be assessed for osteoclast activity; histomorphometric analysis will evaluate both static and dynamic indices of bone remodeling. The in vivo therapeutic effect of targeting each isoform to the bone in op/op mice will be assessed using adenoviral vectors designed to limit CSF-1 expression in osteoblasts. Our second hypothesis is that specific regulatory elements in the 5' flanking region of the CSF-1 promoter direct cell-specific gene expression in vitro and in vivo during murine development. The temporal and spacial expression of CSF-1 during murine skeletal development will be assessed by in situ hybridization and immunohistochemistry. To determine potential cis-acting elements that control cell-specific expression of CSF-1, deletion constructs generated from the 5' flanking region of CSF- 1 will be tested for their ability to direct transcription in osteoblast, stromal, liver, muscle, epithelial and B cell lines. Relevant CSF-1 cell specific promoter sequences will be assessed in vivo by generating transgenic mice harboring cell-specific CSF-1 promoter segment(s) linked to the bacterial lacZ reporter gene. These studies should increase our understanding of the molecular mechanisms that activate CSF-1 during development and may suggest novel therapeutic strategies designed to regulate osteoclast formation in a variety of bone disorders such as osteoporosis and bone fracture.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR042306-07
Application #
6341780
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Program Officer
Sharrock, William J
Project Start
1994-01-01
Project End
2002-12-31
Budget Start
2001-01-01
Budget End
2001-12-31
Support Year
7
Fiscal Year
2001
Total Cost
$221,803
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
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