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


METHODS
Osteoclasts were differentiated from mouse BMMϕs. 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-α in BMMϕs and stimulated formation of TRAP-positive multinucleated cells. Pharmacological inhibition of AMPK with compound C and siRNA-mediated knockdown of AMPK-α1, the predominant α-subunit isoform in BMMϕs, increased RANKL-induced formation of TRAP-positive multinucleated cells and bone resorption via activation of the downstream signaling elements p38, JNK, NF-κB, Akt, CREB, c-Fos, and NFATc1. STO-609, an inhibitor of CaMKK, completely blocked the RANKL-induced activation of AMPK-α, but KN-93, an inhibitor of CaMK, did not. siRNA-mediated TAK1 knockdown also blocked RANKL-induced activation of AMPK-α. The AMPK activators metformin, (-)-epigallocatechin-3-gallate, berberine, resveratrol, and α-lipoic acid dose-dependently suppressed formation of TRAP-positive multinucleated cells and bone resorption.


CONCLUSIONS
AMPK negatively regulates RANKL, possibly by acting through CaMKK and TAK1. Thus, the development of AMPK activators may be a useful strategy for inhibiting the resorption of bone that is stimulated under RANKL-activated conditions.
Lee
Young-Sun
YS
Asan Institute for Life Sciences, Seoul 138-736, Republic of Korea.
Kim
Yang-Soon
YS
Lee
Sun-Young
SY
Kim
Geun-Hyang
GH
Kim
Beom-Jun
BJ
Lee
Seung-Hun
SH
Lee
Ki-Up
KU
Kim
Ghi-Su
GS
Kim
Seung-Whan
SW
Koh
Jung-Min
JM
D000263:Adenylate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001862:Bone Resorption / Q000139:chemically induced; D002392:Catechin / Q000031:analogs & derivatives / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008687:Metformin / Q000494:pharmacology; D051379:Mice; D008813:Mice, Inbred ICR; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D053245:RANK Ligand / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D000077185:Resveratrol; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013267:Stilbenes / Q000494:pharmacology; D000099151:MAP Kinase Kinase Kinase 7
D000263:Adenylate Kinase; D002392:Catechin; D020930:MAP Kinase Kinase Kinases; D016173:Macrophage Colony-Stimulating Factor; D008687:Metformin; D011720:Pyrazoles; D011743:Pyrimidines; D053245:RANK Ligand; D034741:RNA, Small Interfering; D000077185:Resveratrol; D013267:Stilbenes; D000099151:MAP Kinase Kinase Kinase 7; C516138:dorsomorphin; C045651:epigallocatechin gallate