Insulin-like growth factor I (IGF-I) is a polypeptide that has the potential to modulate osteoblast cell function. We are conducting clinical studies to determine the effects of rhIGF-I and estrogen on skeletal turnover in postmenopausal women. To better understand the mechanisms involved in IGF-I action on bone, we propose to study the effects of rhIGF-I on the extracellular matrix metabolism of human osteoblasts in vitro. Specifically, we plan to measure the effect of IGF-I on (a) mRNA expression and protein levels of extracellular matrix components; (b) bone cell IGF-I response as a function of the age of the patient and mineralized state of the matrix; (c) stability and competency of the matrix as a scaffolding for mineralization; and (d) the influence of growth hormone, estrogen, and PTH on bone cell response to IGF-I (as approached in a and c). 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 PI or utilized in prior studies of human osteoblast extracellular matrix metabolism. The effect of the temporal sequence of bone cell development in culture (i.e. either a premineral 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 growth hormone, 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 """"""""functionality"""""""" (i.e. 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.
| Dieudonne, S C; Xu, T; Chou, J Y et al. (1998) Immortalization and characterization of bone marrow stromal fibroblasts from a patient with a loss of function mutation in the estrogen receptor-alpha gene. J Bone Miner Res 13:598-608 |
