Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. By inhibiting HMG-CoA reductase (HMGCR) statins deplete the cellular isoprenoid biosynthetic pathway products farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Current thought in the field is that statins stimulate bone formation through the depletion of GGPP, since exogenous GGPP prevents the effects of statins on osteoblasts in vitro.
We hypothesized that direct inhibition of GGPP synthase (GGPPS) would similarly stimulate osteoblast differentiation. Digeranyl bisphosphonate (DGBP), a specific inhibitor of GGPPS, decreased GGPP levels in MC3T3-E1 pre-osteoblasts and calvarial osteoblasts leading to impaired protein geranylgeranylation. In contrast to our hypothesis, DGBP inhibited the matrix mineralization of MC3T3-E1 cells and the expression of osteoblast differentiation markers in calvarial osteoblasts. The effect on mineralization was not prevented by exogenous GGPP. By inhibiting GGPPS, DGBP led to an accumulation of the GGPPS substrate FPP. We show that FPP and GGPP levels decreased during MC3T3-E1 and calvarial osteoblast differentiation, which correlated with decreased expression of HMGCR and FPP synthase. The decrease in FPP during differentiation was prevented by DGBP treatment. The accumulation of FPP following 24 h DGBP treatment correlated with activation of the glucocorticoid receptor, suggesting a potential mechanism by which DGBP-induced FPP accumulation may inhibit osteoblast differentiation.
To further investigate whether FPP inhibits osteoblast differentiation, we utilized the squalene synthase (SQS) inhibitor zaragozic acid (ZGA), which causes a greater accumulation of FPP than can be achieved with GGPPS inhibition. ZGA treatment decreased osteoblast proliferation, gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization of calvarial osteoblasts. Prevention of ZGA-induced FPP accumulation with HMGCR inhibition prevented the effects of ZGA on osteoblast differentiation. Treatment of osteoblasts with exogenous FPP similarly inhibited matrix mineralization. These results suggest that the accumulation of FPP negatively regulates osteoblast differentiation.
While we did not find that specific depletion of GGPP stimulates osteoblast differentiation, we obtained evidence that GGPP does negatively regulate the differentiation of these cells. Exogenous GGPP treatment inhibited primary calvarial osteoblast gene expression and matrix mineralization. Interestingly, GGPP pre-treatment increased markers of insulin signaling, despite reduced phosphorylation of the insulin receptor (InsR). Inhibition of osteoblast differentiation by GGPP led to the induction of PPARγ and enhanced adipogenesis in osteoblastic cultures, suggesting that GGPP may play a role in the osteoblast versus adipocyte fate decision. Adipogenic differentiation of primary bone marrow stromal cell (BMSC) cultures was prevented by DGBP treatment.
Altogether these data present novel roles for the isoprenoids FPP and GGPP in the regulation of osteoblast differentiation and have intriguing implications for the isoprenoid biosynthetic pathway in the regulation of skeletal homeostasis.