The long term objective of the proposed research is to determine the biological function and molecular mechanism of recombinant human insulin like growth factor-I (rhIGF-I). Insulin like growth factor-I (IGF-I) is a polypeptide that has the potential to modulate osteoblast cell function. The proposed studies are an outgrowth of clinical studies designed to determine the effects of rhIGF-I and estrogen on skeletal turnover in postmenopausal women. In order to understand the mechanisms involved in IGF-I action on bone, the Principal Investigator proposes to study the effects of rhIGF-I on the extracellular matrix metabolism of human osteoblasts in vitro. Specifically, to measure the effect of IGF-I on mRNA expression and protein levels of extracellular matrix components, bone cell IGF-I response as a function of the age of the patient and mineralized state of the matrix, stability and competency of the matrix as a scaffolding for mineralization, and the influence of growth hormone (GH), estrogen, and parathyroid hormone (PTH) on bone cell response to IGF-I. The steady-state levels of bone matrix protein and mRNA for type I collagen, the proteoglycans biglycan and decorin, osteonectin and alkaline phosphatase will be analyzed to determine the effect of IGF-I on the composition of the bone cell matrix. Protein and mRNA levels will be determined by techniques developed by the Principal Investigator or utilized in prior studies of human osteoblast extracellular matrix metabolism. The effect of the temporal sequence of bone cell development in culture, either a pre-mineral or a mineralizing extracellular matrix, on IGF-I responsiveness will be assessed by comparing steady-state matrix composition for young adult (10-18 years) and adult (over 50 years) human trabecular bone cell cultures. The stability of the extracellular matrix will be assessed by radiolabeling and pulse-chase analysis of these specific bone matrix components. The effect of IGF-I on the competency of the matrix as a site for mineral deposition will be assessed by determining both the in vitro time frame of mineralization and by analyzing the mineral content and protein (including osteocalcin) of the extracellular matrix. The endocrine modulation of human bone cell responsiveness to IGF-I will be investigated by characterizing the effect of GH, estrogen, and PTH on matrix content (steady-state radiolabeling), stability (pulse-chase radiolabeling), and competency for mineralization. The approach of assaying extracellular matrix composition and stability and mineralization in response to IGF-I treatment and systemic factor modulation will define an integrated pattern of in vitro bone protein and proteoglycan metabolism and bone mineralization.
