Fluid shear stress and melatonin in combination activate anabolic proteins in MC3T3-E1 osteoblast cells

In this study, we investigated whether fluid shear stress and melatonin in combination stimulate the anabolic proteins through the phosphorylation of extracellular signal-regulated kinase (p-ERK) in MC3T3-E1 osteoblast cells. First, we researched why fluid shear stress and melatonin in combination influence cell survival. Fluid shear stress (1hr) and melatonin (1mm) in combination reduced autophagic marker LC3-II compared with fluid shear stress (1hr) and/or melatonin (0.1mm). Under the same conditions for fluid shear stress, markers of cell survival signaling pathway p-ERK, phosphorylation of serine-threonine protein kinase (p-Akt), phosphorylation of mammalian target of rapamycin (p-mTOR), and p85-S6K were investigated. p-Akt, p-mTOR (Ser 2481) expressions increased with the addition of 1mm melatonin prior to 0.1mm melatonin treatment. However, p-S6K expression did not change significantly. Next, mitochondria activity including Bcl-2, Bax, catalase, and Mn-superoxide dismutase (Mn-SOD) were studied. Expressions of Bcl-2, Bax, and catalase proteins were low under fluid shear stress plus 1mm melatonin compared with only fluid shear stress alone, whereas Mn-SOD expression was high compared with conditions of no fluid shear stress. Finally, the anabolic proteins of bone, osteoprotegerin, type I collagen (collagen I), and bone sialoprotein II (BSP II) were checked. These proteins increased with combined fluid shear stress (1, 4hr) and melatonin (0.1, 1mm). Together, these results suggest that fluid shear stress and melatonin in combination may increase the expression of anabolic proteins through the p-ERK in MC3T3-E1 osteoblast cells. Therefore, fluid shear stress in combination with melatonin may promote the anabolic response of osteoblasts.