This proposal describes the construction and use of cell culture and murine models of OI to study the mechanisms of abnormal bone formation in OI. The studies, a direct consequence of investigations funded by the principal investigator's FIRST award (R29-AR38474, 8/86-7/91) on the molecular basis of mild OI in cultured dermal fibroblasts from human subjects, will refine those earlier studies by measuring functional effects of abnormal type I collagen structure on the amount and quality of extracellular matrix in mineralizing systems. Studies will include (1) site-specific mutagenesis of a murine pro alpha 2 (I) expression gene to create substitutions for triple-helical glycine residues in several domains, transfection of mutant pro alpha 2 (I) expression genes into MC3T3-E1 preosteoblasts which can undergo in vitro differentiation and mineralization, quantification of mutant gene expression, and biochemical demonstration that type I collagen molecules incorporate mutant pro alpha 2 (I) chains; (2) measurement of the effects of mutant type I collagen molecules on osteocyte proliferation by thymidine incorporation, on the amount and composition of extracellular matrix accumulated by collagen CNBr peptide analysis, amino acid analysis, and antibody staining, and on rates of mineralization by 45Ca uptake into extracellular matrix; (3) extension of in vitro studies by creation of a limited number of transgenic mice with selected pro alpha 2 (I) mutations, and (4) analysis of bone from transgenic animals by histology, histomorphometric measurements, and antibody staining. These experiments, it is hoped, will elucidate the function of different domains in the type I collagen triple helix in the formation of bone matrix and will provide better understanding of the abnormal osteoblast and matrix physiology in OI. They may also provide a means to investigate pharmacologic or genetic therapies for OI and potentially for other disorders of osteopenia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
1R01AR040586-01A1
Application #
3161006
Study Section
General Medicine A Subcommittee 2 (GMA)
Project Start
1991-08-01
Project End
1995-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705