Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42, and Rho GTPases.
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.


BACKGROUND
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.


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 [(14)C] mevalonate, and the increase in Rac-GTP was prevented by cycloheximide. Furthermore, this unprenylated Rac-GTP retained at least part of its functional activity in J774 cells, because it mediated N-BP-induced activation of p38. Paradoxically, although risedronate induces apoptosis of J774 macrophages by inhibiting protein prenylation, the p38 inhibitor SB203580 enhanced N-BP-induced apoptosis, suggesting that Rac-induced p38 activation partially suppresses the pro-apoptotic effect of N-BPs in these cells.


CONCLUSIONS
N-BP drugs may disrupt the function of osteoclasts in vivo and affect other cell types in vitro by inhibiting protein prenylation, thereby causing inappropriate and sustained activation, rather than inhibition, of some small GTPases and their downstream signaling pathways.
Dunford
James E
JE
Bone Research Group, Institute of Medical Sciences, University of Aberdeen, Foresterhill, UK.
Rogers
Michael J
MJ
Ebetino
Frank H
FH
Phipps
Roger J
RJ
Coxon
Fraser P
FP
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004164:Diphosphonates / Q000494:pharmacology*; D004789:Enzyme Activation; D008264:Macrophages / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017368:Protein Prenylation / Q000187:drug effects*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism*
D004164:Diphosphonates; D048051:p38 Mitogen-Activated Protein Kinases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins