Involvement of AMP-activated protein kinase in thrombin-stimulated interleukin 6 synthesis in osteoblasts.
We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression of IL6 mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10  μM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. The IL6 mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.
Department of Clinical Laboratory, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan. tokuda@ncgg.go.jp
H
H
Tokuda
K
K
Kato
H
H
Natsume
A
A
Kondo
G
G
Kuroyanagi
R
R
Matsushima-Nishiwaki
Y
Y
Ito
T
T
Otsuka
O
O
Kozawa
D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism; D002460:Cell Line; D020868:Gene Silencing; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D055396:Bone Morphogenetic Protein 2; D015850:Interleukin-6; D011720:Pyrazoles; D011743:Pyrimidines; C516138:dorsomorphin; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048051:p38 Mitogen-Activated Protein Kinases; D055372:AMP-Activated Protein Kinases; D013917:Thrombin; D000103:Acetyl-CoA Carboxylase