AMP kinase acts as a negative regulator of RANKL in the differentiation of osteoclasts

Introduction: AMP-activated protein kinase (AMPK) has been reported to stimulate differentiation and proliferation of osteoblasts, but the role of AMPK in the physiology of osteoclasts has not been investigated. Method: Osteoclasts were differentiated from mouse BMM phi s. TRAP-positive multinucleated cells were considered to be osteoclasts using TRAP staining, and resorption area was determined by incubation of cells on dentine discs. Signaling pathways were investigated using Western blotting and RT-PCR. Results: RANKL induced phosphorylation/activation of AMPK-alpha. in BMM phi s and stimulated formation of TRAP-positive multinucleated cells. Pharmacological inhibition of AMPK with compound C and siRNA-mediated knockdown of AMPK-alpha, the predominant alpha-subunit isoform in BMWs, increased RANKLinduced formation of TRAP-positive multinucleated cells and bone resorption via activation of the downstream signaling elements p38, JNK, NF-kappa B, Akt, CREB, kappa-Fos, and NFATcl. STO-609, an inhibitor of CaMKK, completely blocked the RANKL-induced activation of AMPK-alpha, but KN-93, an inhibitor of CaMK, did not. siRNA-mediated TAM knockdown also blocked RANKL-induced activation of AMPK-a. The AMPK activators metformin, (-)-epigallocatechin-3-gallate, berberine, resveratrol, and alpha-lipoic acid dosedependently suppressed formation of TRAP-positive multinucleated cells and bone resorption. Conclusion: AMPK negatively regulates RANKL, possibly by acting through CaMKK and TAK1. Thus, the development of AIVIPK activators may be a useful strategy for inhibiting the resorption of bone that is stimulated under RANKL-activated conditions. (C) 2010 Elsevier Inc. All rights reserved.