BMP-6 exerts its osteoinductive effect through activation of IGF-I and EGF pathways

Abstract

We have recently shown that human recombinant BMP-6 (rhBMP-6), given systematically, can restore bone in animal models of osteoporosis. To further elucidate the underlying mechanisms of new bone formation following systemic application of BMPs, we conducted gene expression profiling experiments using bone samples of oophrectomised mice treated with BMP-6. Gene set enrichment analysis revealed enrichment of insulin-like growth factor-I and epidermal growth factor related pathways in animals treated with BMP-6. Significant upregulation of IGF-I and EGF expression in bones of BMP-6 treated mice was confirmed by quantitative PCR. To develop an in vitro model for evaluation of the effects of BMP-6 on cells of human origin, we cultured primary human osteoblasts. Treatment with rhBMP-6 accelerated cell differentiation as indicated by the formation of mineralised nodules by day 18 of culture versus 28-30 days in vehicle treated cultures. In addition, alkaline phosphatase gene expression and activity were dramatically increased upon BMP-6 treatment. Expression of IGF-I and EGF was upregulated in human osteoblast cells treated with BMP-6. These results collectively indicate that BMP-6 exerts its osteoinductive effect, at least in part, through IGF-I and EGF pathways, which can be observed both in a murine model of osteopenia and in human osteoblasts.

Fig. 1

Ex vivo BMD values of sham, OVX and BMP-6 treated mice as analysed by PIXImus. Within 9 weeks of therapy BMP-6 restored bone volume of tibiae (a), femurs (b) and lumbar spine (c). *Significantly different from OVX control mice (p < 0.05, ANOVA, Dunnett test)

Fig. 2

Primary human osteoblasts upon BMP-6 treatment. (a) Cell number during the culture period. BMP-6 did not significantly affect cell growth when compared with untreated cultures. (b) An increase in alkaline phosphatase activity is seen beginning at day 10 of culture in BMP-6 treated cultures. This activity appeared to peak at day 23 of culture. *Significantly different from untreated cell cultures (p <0.05, student t-test). (c) In vitro human osteoblast cultures were Von Kossa stained. High magnification views represent BMP-6 treated human osteoblasts at days 19, 23 and 28 days of culture. Areas of mineralisation appear brown. Mineralisation usually appeared at approximately day 19 of culture

Fig. 3

Gene expression of osteogenic markers in mice and human primary osteoblast treated by BMP-6. Total RNA was isolated from femurs of mice (n = 15) (a–d) following 4 weeks of BMP-6 therapy and from human osteoblasts at certain time points (day 2,6, 10, 14, 19, 23 and 28) of culture (e–h). Results are represented as fold change of comparative expression level to OVX group of animals (a–d) or as fold change as compared to the first day of culture period (e–h). *Significantly different from OVX control mice (p < 0.05, ANOVA, Dunnett test) (a–d) or control cultures (p<0.05, student test) (e–h). HOBs were either left untreated (□,▵) or were BMP-6 treated (■,▴)

Fig. 4

Gene expression of IGF-I (a), EGF (b) and IGFbp3 (c) in mice, and IGF-I (d) and EGF (e) in human primary osteoblast treated with BMP-6. Total RNA was isolated from femurs of mice (n = 15) (a–c) following 4 weeks of BMP-6 therapy and from human osteoblasts at certain culturing time points (day 7, 12, 17, 23) (d, e). Results are represented as fold change of comparative expression level to OVX group of animals (a–c) or as fold change as compared to the untreated cultures (d, e). *Significantly different from OVX control mice (p < 0.05, ANOVA, Dunnett test)