Expression of insulin-like growth factor system constituents in differentiating rat osteoblastic cell populations

The synthetic glucocorticoid dexamethasone (Dex) and insulin-like growth factor-I and -II (IGF-I and -II) stimulate osteoprogenitor proliferation and differentiation in bone cell populations isolated from adult rat vertebrae. Since glucocorticoids have been shown to regulate gene expression of IGFs and IGF binding proteins (IGFBPs) in several experimental models, we investigated whether Dex-stimulated osteoprogenitor proliferation and differentiation was associated with changes in mRNA levels of the IGF system components (i.e., IGF-I and -II, the type 1 and 2 IGF receptor, the insulin receptor and six IGFBPs). Osteoprogenitor-containing bone cell populations were isolated from the outgrowth of vertebral explant cultures of 3-month-old female rats and cultured for 20 days. Total RNA was extracted at day 8, 14, and 20, and mRNA levels of the IGF system constituents were compared between differentiating (Dex-treated) and non-differentiating (control) cultures. Northern hybridization data from 8- and 20-day cultures showed that mRNA levels of IGF-I were markedly lower in Dex-treated cultures than in control cultures at day 8 and 20. At day 20, mRNA levels of IGFBP-3 were also lower in Dex-treated cultures. Signals of IGFBP-5 mRNA were undetectable. To increase the sensitivity of our detection methods and therefore evaluate mRNA levels of all the components of the IGF system, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA extracted at day 8, 14, and 20 of culture. In agreement with the Northern data, IGF-I mRNA levels in Dex-treated cultures were lower than in control cultures at all three time points, and IGFBP-3 levels were lower in Dex-treated cultures at day 20 of culture. However, at day 8 and 14, IGFBP-3 mRNA levels were higher in Dex-treated cultures than in controls. Levels of the 2 IGF receptor mRNA and the insulin receptor mRNA were lower in Dex-treated cultures. Dex-treated cultures also had decreased levels of IGFBP-1 mRNA but increased levels of IGFBP-2 mRNA at all three time points. IGFBP-4 levels were lower at day 14 in Dex-treated cultures than in controls but higher at day 20. IGF-II and IGFBP-5 mRNA levels in control and Dex-treated cultures were similar. Signals for IGFBP-6 were undetectable. Our findings show that glucocorticoid-induced osteoprogenitor proliferation and differentiation in adult rat bone cell populations are associated with significant changes in the mRNA levels of virtually all components of the IGF system. Some of these changes are dependent on the stages of development (e.g., regulation of IGFBP-3 and -4) and some remain similar trends at all stages (e.g., regulation of IGF-I and the three receptors).