Osteoporosis (OP) is a bone metabolic disease which is considered a global health problem. Literature data have shown that patients taking antiresorptive drugs, such as bisphosphonates (BP), have an increased risk of developing atypical femoral fracture (AFF). The pathogenesis of AFF remains unknown, but studies in patients with OP who suffered AFFs have revealed that there is a mutation in the enzyme geranylgeranyl pyrophosphate synthase (Ggps1), a component of mevalonate pathway, which is the target of nitrogen-containing bisphosphonates (NBPs). Our overall hypothesis is that Ggps1 mutation predispose patients to diaphyseal fragility, spontaneously or when treated with NBPs. Here we will determine in vitro whether this mutated gene in the AFF patients affects bone cell functions and determines in vivo whether these genetic alterations promote diaphyseal fragility in ovariectomized mice exposed to long term NBPs. Thus, the aims of this study are: 1) to understand the effect of Ggps1 knockdown on differentiation and/or function of bone cells and identify which proteins are affected by altered geranylation in these cells, and 2) to determine whether Ggps1 haploinsufficiency, a model of partial loss of function, increases diaphyseal fragility in ovariectomized (OVX) mice and/or after treatment with NBPs, compared to non-nitrogen containing BPs. For this, Ggps1+/- and Ggps1+/+ mice will be generated and osteoclast (OC) differentiation will be tested in primary bone marrow macrophages (BMMs) isolated from the long bones of Ggps1+/+ and Ggps1+/- mice. Cells treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-ß ligand (RANKL) will be evaluated for expression of OC markers; multinucleated tartrate-resistant acid phosphatase (TRAP) positive cells will be counted and resorbing function analyzed by classical pit assay. Because the mutation in the patients is present in all cells, osteoblast (OB) and osteocyte (OCY) differentiation will also be assessed in bone marrow stromal cells (BMSCs), primary calvarial OBs, and OCY isolated from the long bones of Ggps1+/- and Ggps1+/+ mice by evaluating the expression of OB and OCY markers, as well as alkaline phosphatase (ALP) activity and mineral deposition. Ggps1+/- mice (on a C57/BL6 background) will be crossed with wild type C57/BL6 mice (Ggps1+/+) to generate Ggps1+/- and Ggps1+/+ littermates, in order to later characterize the effects of Ggps1 haploinsufficiency on the skeletal response to NBPs using X-rays, µCT, histomorphometry, analysis of serum bone markers, expression of OCY-specific genes by qRT-PCR and biomechanical testing. Treatment effects will be evaluated using ANOVA for all continuous variables, with post-hoc testing by Fischer's and/or Tukey's tests. We will use a two-factor ANOVA to determine whether the effect of treatment depends on the genotype. Differences will be considered significant at p<0.05.
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