Insulin-like growth factors (IGFs) are important stimulators of bone formation. Past findings that IGFBP-4 is a potent inhibitor of IGF action in bone cells and that IGFBP-4 production is regulated by osteoregulatory agents support our premise that IGFBP-4 is a key component of the IGF system in bone. In addition, there is evidence that IGFBP-4 concentration is regulated at the local level by mechanisms involving both synthesis and degradation. In this study, we chose to evaluate the role of IGF-II dependent IGFBP-4 protease (IGFBP-4 protease) in regulating the availability, and thus the activity of IGFBP-4 in human osteoblasts (hOBs) for the following reasons: 1) IGFBP-4 protease produced by hOBs cleaves IGFBP-4 into forms with low IGF binding affinity and thus may control IGF-II release from the IGFBP-4/IGF-II complex, 2) The activity of IGFBP-4 protease is regulated by osteoregulatory agents such as IGF-II and TGF- and is thus relevant to bone formation. 3) IGFBP-4 protease is the newest component of the IGF system and its role in hOB metabolism has not been established.
The aims of this study are to evaluate the role of IGFBP-4 protease in modulating IGF action in hOBs and elucidate the mechanism involved in IGF-II enhancement of IGFBP-4 degradation. Two hypotheses will be tested: 1) IGFBP-4 protease is an important regulatory component of the IGF system in hOBs. 2) Binding of IGF-II to IGFBP-4 alters the conformation such that the protease recognition sequence in IGFBP-4 is more accessible to IGFBP-4 protease. To test hypothesis 1, we will prepare partially purified IGFBP-4 protease to identify the primary cleavage site and the protease recognition sequence. This information together with our knowledge of the localization of the IGF binding domain will be used to design IGFBP-4 analogs which are fully protease resistant (PR) but bind to IGF with similar affinity as that of the wild type (WT). Such analogs will be used to evaluate the role of IGFBP-4 protease in releasing IGF-II from IGF-II/IGFBP-4 complex thereby increasing the local mitogenic activity of the IGFs. To test hypothesis 2, we will prepare analogs that have reduced IGF-II binding affinity but retain the proteolytic domain. If IGF-II fails to enhance degradation of such analogs, such a finding would suggest that the binding of IGF-II to IGFBP-4 is essential to IGFBP-4 proteolysis. Then whether binding of IGF-II to IGFBP-4 increases the association between IGFBP-4 and IGFBP-4 protease will be determined. We anticipate that completion of these studies will lead to our greater understanding of the role of IGFBP-4 protease in the regulation of hOB proliferation and provides a basis for the design of peptide-based IGFBP-4 protease activators for osteoporosis treatment. Moreover, PR IGFBP-4 analog (if more potent than WT IGFBP-4) may have therapeutic potential in the treatment of IGF dependent cancers.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Orthopedics and Musculoskeletal Study Section (ORTH)
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Sharrock, William J
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Loma Linda University
Internal Medicine/Medicine
Schools of Medicine
Loma Linda
United States
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