title: Activation of AMP kinase and inhibition of Rho kinase induce the mineralization of osteoblastic MC3T3-E1 cells through endothelial NOS and BMP-2 expression.
abstract: AMP-activated protein kinase (AMPK) and Rho kinase (ROK) are known to modulate the mevalonate pathway. Activation of AMPK suppresses 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase. ROK acts downstream of HMG-CoA reductase, and its inhibition exerts antiatherosclerosis effects. However, whether or not these enzymes are involved in bone metabolism is unclear. The present study was undertaken to investigate the effects of an AMPK activator, 5-aminoimidazole-4-carboxamide1-beta-d-ribonucleoside (AICAR), and a ROK inhibitor, fasudil hydrochrolide, on the mineralization of osteoblastic MC3T3-E1 cells. Real-time PCR and mineralization stainings revealed that both AICAR and fasudil significantly stimulated endothelial nitric oxide synthase (eNOS), bone morphogenetic protein-2 (BMP-2), and osteocalcin mRNA expression as well as mineralization in the cells. Supplementation of either mevalonate or geranyl-geranyl pyrophosphate, the downstream molecules of HMG-CoA reductase, or coincubation with either a nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester, or a BMP-2 antagonist, noggin, significantly reversed these AICAR-induced reactions. Western blot analysis showed that AICAR activated protein kinase B and extracellular signal-regulated kinase (ERK). ERK inhibitor significantly reversed the AICAR-induced increase in eNOS and BMP-2 mRNA expression. Measurement of ROK activities by enzyme-linked immunosorbent assay revealed that both AICAR and fasudil significantly suppressed the phosphorylation of the myosin-binding subunit of myosin phosphate, a ROK substrate. These findings suggest that the AMPK activator and the ROK inhibitor are able to stimulate the mineralization of osteoblasts through modulating the mevalonate pathway. These agents could be candidate drugs that promote bone formation for the treatment of osteoporosis.
authors.affiliation: Dept. of Internal Medicine 1, Shimane Univ. Faculty of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan.
authors.forename: Ippei
authors.initials: I
authors.lastname: Kanazawa
authors.forename: Toru
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Shozo
authors.initials: S
authors.lastname: Yano
authors.forename: Mika
authors.initials: M
authors.lastname: Yamauchi
authors.forename: Toshitsugu
authors.initials: T
authors.lastname: Sugimoto
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000263:Adenylate Kinase / Q000378:metabolism*; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000096:biosynthesis* / Q000235:genetics; D016723:Bone Remodeling / Q000187:drug effects; D002352:Carrier Proteins / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D004789:Enzyme Activation; D006651:Histocytochemistry; D051379:Mice; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis / Q000235:genetics; D010006:Osteoblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012265:Ribonucleotides / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000099176:Noggin Protein
chemical_list: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000263:Adenylate Kinase; D000620:Aminoimidazole Carboxamide; D055396:Bone Morphogenetic Protein 2; D002352:Carrier Proteins; D019331:NG-Nitroarginine Methyl Ester; D052250:Nitric Oxide Synthase Type III; D015675:Osteocalcin; D012333:RNA, Messenger; D012265:Ribonucleotides; D054460:rho-Associated Kinases; D000099176:Noggin Protein; C525719:Bmp2 protein, mouse; C031143:AICA ribonucleotide; C049347:fasudil