Inhibition of Protein Prenylation by Bisphosphonates Causes Sustained Activation of Rac, Cdc42, and Rho GTPases

Abstract

N-BPs, which inhibit bone resorption by preventing prenylation of small GTPases, unexpectedly cause the accumulation of GTP-bound, unprenylated Rho family GTPases in macrophages and osteoclasts. In macrophages, this also leads to sustained, Rac-mediated activation of p38. The antiresorptive activity of N-BPs may therefore be caused at least in part, by the accumulation of unprenylated small GTPases, causing inappropriate activation of downstream signaling pathways.Introduction: Nitrogen-containing bisphosphonates (N-BPs) are potent inhibitors of bone resorption that act by inhibiting farnesyl diphosphate synthase, thereby indirectly preventing the prenylation of Rho family GTPases that are required for the function and survival of bone-resorbing osteoclasts. However, the effect that these drugs have on the activity of Rho family GTPases has not been determined. Materials and Methods:The effect of N-BPs on the activity of Rho family GTPases in J774 macrophages and osteoclasts was measured using a pull-down assay to isolate the GTP-bound forms. The effect of N-BPs, or decreasing Rac expression using siRNA, on downstream p38 activity was evaluated by Western blotting and apoptosis assessed by measurement of caspase 3/7 activity. Results: Rather than inhibiting GTPase function, loss of prenylation after treatment with N-BPs caused an increase in the GTP-bound form of Rac, Cdc42, and Rho in J774 cells and osteoclast-like cells, which paralleled the rate of accumulation of unprenylated small GTPases. Activation of Rac also occurred with other inhibitors of prenylation of Rho-family proteins, such as mevastatin and the geranylgeranyl transferase I inhibitor GGTI-298. The Rac-GTP that increased after N-BP treatment was newly translated, cytoplasmic unprenylated protein, because it was not labeled with [

Binding Kinetics of a Fluorescently Labeled Bisphosphonate as a Tool for Dynamic Monitoring of Bone Mineral Deposition In Vivo

“…Anesthetized mice were injected intravenously with 100 mL phosphate-buffered saline (PBS) containing 2 nmol of dissolved OsteoSense750EX (PerkinElmer, Waltham, MA, USA), a fluorescently conjugated pamidronate derivative, (21) and imaged 2,4,6,8,10,15,20, and 30 minutes after injection using the NightOwl planar imaging system (Berthold Technologies, Bad Wildbad, Germany) to qualitatively determine kinetic distribution. A phantom was placed over the mouse bladder to help position the limbs and prevent obscuring of the limb signal from the urinary pool of bisphosphonate.…”

“…(8,9) This results in the accumulation of unprenylated proteins with aberrant functions within the cell. (10) With the conjugation of infrared and near-infrared fluorescent dyes, bisphosphonates are now being assessed for their utility as a tool for monitoring bone dynamics in preclinical models.…”

Protective effect of hypoxia on bisphosphonate‑related bone cell damage

“…osteoblast and osteoclast exposure to aln and pamidronate was found to result in the activation of caspase-3 and the activation of the p38 MaPK and JnK signaling pathways (40). Dunford et al recently reported that BPs cause apoptosis and activate the MaPK and JnK signaling pathways in osteoclasts and J774 macrophages in vitro (19). JnK induces cell apoptosis by phosphorylation and activation of the transcription factor p53 (20,21).…”

“…Inhibition of protein prenylation may affect many proteins found in an osteoclast, influencing osteoclastogenesis, cell survival and cytoskeletal dynamics (15)(16)(17)(18). BPs also cause apoptosis and activate the MaPK and c-Jun n-terminal kinase (JnK) signaling pathways in osteoclasts and J774 macrophages in vitro (19). Various studies have shown that JnK induces cell apoptosis by phosphorylation and activation of the transcription factor p53 (20,21) or by phosphorylation (and inactivation) of the anti-apoptotic proteins of the Bcl-2 family (22)(23)(24).…”

Bisphosphonates in the treatment of metabolic bone diseases

“…Non-amino bisphosphonates disrupt the ATP metabolic pathway in osteoclasts that leads to osteoclast apoptosis, while the amino bisphosphonates impair FPPS. This inhibitory effect on FPPS leads to reduced osteoclast capacity to prenylate specific intracellular proteins necessary for normal osteoclastic cell function (5). The end result is a reduced ability of osteoclasts to induce bone resorption and to induce programmed cell death (apoptosis) of osteoclasts ( Figure 1).…”