Osthole exerts osteoprotective effect by activating ER, PI3K/AKT, MEK/ERK, and Wnt/β-catenin signaling pathways

Figure 4 Estrogen receptor, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/protein kinase B, and Wnt/β-catenin pathways mediate osthole-induced modulation of MG-63 proliferation, alkaline phosphatase activity, and osteoprotegerin/receptor activator of nuclear factor-κB ligand/Osterix expression. ^aP < 0.05 vs control; ^bP < 0.05 vs osthole alone (one-way analysis of variance with Tukey’s post-hoc test). MG-63 cells were pretreated with 100 μM estrogen receptor inhibitor fulvestrant (ICI) 182780, 50 μM PD98059, 200 nM wortmannin, 200 ng/mL dickkopf-1 (DKK-1), or vehicle dimethyl sulfoxide in medium containing 1% fetal bovine serum for 30 minutes, followed by treatment with 1 nM osthole for 2 days. Data are mean ± SD from three independent experiments (n = 3). A: Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; B: Alkaline phosphatase activity was detected using an alkaline phosphatase assay kit. Experiments were independently repeated three times, and data are presented as mean ± SD; C: MG-63 cells were divided into six treatment groups: Control, DKK-1 (200 ng/mL, Wnt/β-catenin pathway inhibitor), ICI 182780 (100 μM, estrogen receptor inhibitor), osthole (1 nM), osthole + DKK-1, and osthole + ICI 182780. After respective treatments, relative mRNA expression levels of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), and Osterix were analyzed by reverse transcription-quantitative polymerase chain reaction; D: Relative protein expression levels of OPG, RANKL, and Osterix in MG-63 cells subjected to the treatment conditions described in panel C, detected by western blot. β-actin served as the loading control; E: MG-63 cells were assigned to six treatment groups: Control, PD98059 (50 μM, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway inhibitor), wortmannin (200 nM, phosphoinositide 3-kinase/protein kinase B pathway inhibitor), osthole (1 nM), osthole + PD98059, and osthole + wortmannin. Following treatment, relative mRNA expression levels of OPG, RANKL, and Osterix were measured by reverse transcription-quantitative polymerase chain reaction; F: Relative protein expression levels of OPG, RANKL, and Osterix in MG-63 cells treated as specified in panel E, determined by western blot analysis with β-actin as the loading control. OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor-κB ligand; ICI: Fulvestrant; DKK: Dickkopf; PD: 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one.