Prevention of ERK activation involves melatonin‐induced G1 and G2/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, G1 and G2/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 G1 phase, and of cyclin B1 and CDK1, related to the G2/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 G1 and G2/M phase arrest, and this inhibitory effect is potentially via the ERK, but not p38, JNK, or Akt, pathway.