Insulin augments mechanical strain-induced ERK activation and cyclooxygenase-2 expression in MG63 cells through integrins

Abstract

Insulin has been proposed to be a positive regulator of osteoblast proliferation and bone formation. In vivo mechanical loading is essential for maintaining skeletal integrity and bone mass. Since insulin and mechanical force activate similar signaling pathways in osteoblasts, it was hypothesized that insulin may affect mechanical stimulation in osteoblasts. The present study tested the hypothesis that insulin augments mechanical strain-induced signaling and early gene expression in MG63 cells via activation of the extracellular signal-regulated kinase (ERK) pathway and cyclooxygenase-2 (Cox-2) expression. Western blot analysis and quantitative polymerase chain reaction demonstrated respectively that insulin enhanced mechanical strain-induced ERK phosphorylation and Cox-2 expression levels in a dose-dependent manner. The effect of insulin on mechanical strain-induced Cox-2 expression was inhibited by blockade of the ERK pathway. In addition, echistatin, an inhibitor of integrin function, prevented the effects of insulin on mechanical strain-induced ERK phosphorylation and Cox-2 expression. The data obtained from this study suggested that insulin augments mechanical strain-induced Cox-2 expression levels via integrin-dependent activation of the ERK pathway in osteoblasts.

Keywords: cyclooxygenase-2; extracellular signal-regulated kinase; insulin; mechanical strain; osteoblast.

Figure 1

Insulin augments tensile stress-induced ERK phosphorylation in MG63 cells. MG63 cells were kept as controls or pretreated with varied doses of insulin (0, 1, 10 and 100 nM) for 4 h and then exposed to tensile stress for 10 min. ERK phosphorylation was determined using western blot analysis. The results are expressed as the mean ± standard deviation. ^*P<0.05 vs. unstimulated control cells; ^#P<0.05 vs. stressed cells without insulin pretreatment. ERK, extracellular signal-regulated kinase; C, control; I, insulin; S, tensile stress.

Figure 2

Insulin augments tensile stress-induced Cox-2 expression levels via the ERK pathway in MG63 cells. (A) MG63 cells were kept as controls or pretreated with varied doses of insulin (0, 1, 10 and 100 nM) for 4 h and then exposed to tensile stress for 1 h. (B) MG63 cells were pretreated with vehicle control DMSO or PD98059 (30 μM) for 1 h then stimulated with insulin (10 nM) for 4 h, followed by exposure to tensile stress for 1 h. Cox-2 expression levels were determined using qPCR. The results are expressed as the mean ± standard deviation. ^*P<0.05 vs. unstimulated control cells; ^#P<0.05 vs. stressed cells without insulin pretreatment; ^&P<0.05 vs. stressed cells without insulin and PD pretreatment. Cox-2, cyclooxygenase-2; ERK, extracellular signal-regulated kinase; DMSO, dimethylsulfoxide; qPCR, quantitative polymerase chain reaction; C, control; I, insulin; S, tensile stress; PD, PD98059.

Figure 3

Insulin augmentation of tensile stress-induced ERK phosphorylation and Cox-2 expression levels in MG63 cells is mediated by integrins. MG63 cells were kept as controls or pretreated with echistatin (50 nM) for 24 h, stimulated with insulin (10 nM) for 4 h and exposed to tensile stress for 10 min (for ERK) or 1 h (for Cox-2). (A) ERK phosphorylation was determined using western blot analysis. (B) Cox-2 expression levels were determined using qPCR. The results are expressed as the mean ± standard deviation. ^*P<0.05 vs. unstimulated control cells; ^#P<0.05 vs. stressed cells without echistatin and insulin pretreatment. Cox-2, cyclooxygenase-2; ERK, extracellular signal-regulated kinase; qPCR, quantitative polymerase chain reaction; C, control; E, echistatin; I, insulin; S, tensile stress.