title: Prevention of ERK activation involves melatonin-induced G(1)  and G(2) /M phase arrest in the human osteoblastic cell line hFOB 1.19.
abstract: Melatonin regulates mitogen-activated protein kinase (MAPK) and Akt signaling pathways. The MAPK family mainly includes extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Our previous study documented that melatonin delays osteoblast proliferation; however, the mechanism of action of melatonin remains unclear. Here, we demonstrate that melatonin significantly inhibited phosphorylation of ERK but not p38, JNK, or Akt in a human osteoblastic cell line 1.19 (hFOB), as measured by western blot. The expression of ERK, p38, JNK, and Akt was not altered. PD98059 (a selective inhibitor of MEK that disrupts downstream activation of ERK) and melatonin alone, and especially in combination, significantly induced an antiproliferative effect, G(1)  and G(2) /M phase arrest of the cell cycle, and downregulation of the expression at both the protein and mRNA levels of cyclin D1 and CDK4, related to the G(1)  phase, and of cyclin B1 and CDK1, related to the G(2) /M phase, as measured by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method, flow cytometry after propidium iodide staining, and both western blot and real-time PCR, respectively. Moreover, the combination of PD98059 and melatonin synergistically and markedly augmented the action of either agent alone. Coimmunoprecipitation further confirmed that there was an interaction between phosphorylation of ERK and cyclin D1, CDK4, cyclin B1, or CDK1, which was weaken in the presence of melatonin or PD98059. These results suggest that the prevention of ERK activation is involved in melatonin-induced G(1)  and G(2) /M phase arrest, and this inhibitory effect is potentially via the ERK, but not p38, JNK, or Akt, pathway.
authors: Department of Orthopaedics, First Hospital, China Medical University, Shenyang, Liaoning, China.
authors: Lifeng
authors: L
authors: Liu
authors: Yue
authors: Y
authors: Zhu
authors: Ying
authors: Y
authors: Xu
authors: Russel J
authors: RJ
authors: Reiter
mesh_terms: D000975:Antioxidants / Q000494:pharmacology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects*; D002460:Cell Line; D056744:Cyclin B1 / Q000378:metabolism; D019938:Cyclin D1 / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4 / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000096:biosynthesis*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008550:Melatonin / Q000494:pharmacology*; D010006:Osteoblasts / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000096:biosynthesis*
chemical_list: D000975:Antioxidants; C534699:CCNB1 protein, human; C530104:CCND1 protein, human; D056744:Cyclin B1; D005419:Flavonoids; D019938:Cyclin D1; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D008550:Melatonin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one