Effects of Phytoestrogen α-ZAL and Mechanical Stimulation on Proliferation, Osteoblastic Differentiation, and OPG/RANKL Expression in MC3T3-E1 Pre-Osteoblasts

Abstract

Estrogen and mechanical strain are two important regulators of bone remodeling. α-zearalanol (α-ZAL) is a new oomycete phytoestrogen that is similar to 17-beta estradiol, but has less adverse effects. The aim of this study was to investigate cell proliferation, differentiation, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) expression in the pre-osteoblast MC3T3-E1 cell line in response to α- ZAL, mechanical strain, or a combination of both. Cell proliferation was assessed by flow cytometry, and alkaline phosphatase (ALP) activity was measured using a spectrophotometric detection kit. mRNA expression of ALP, runt-related transcriptional factor 2 (Runx2), OPG, and RANKL genes was determined by real-time Reverse Transcription-Polymerase Chain Reaction (real-time RT-PCR). Protein expression of Runx2, OPG, and RANKL was determined by Western blotting. α-ZAL alone (10−6–10−10 M) suppressed proliferation of MC3T3-E1 cells and promoted the activity and gene expression of ALP, an early marker of osteogenesis. It also enhanced Runx2 mRNA expression and the OPG/RANKL ratio. Mechanical strain alone promoted proliferation and differentiation of MC3T3-E1 cells. In addition, Runx2 protein, but not mRNA expression, was upregulated and the OPG/RANKL ratio increased. Although a combination of α-ZAL and mechanical strain inhibited cell proliferation, high concentrations of α-ZAL (10−6 M) combined with a high magnitude of strain (2500 μe), significantly enhanced ALP activity. α-ZAL with a high magnitude of strain increased RUNX2 protein expression, but lowered the OPG/RANKL ratio. The OPG/RANKL ratio significantly increased in response to α-ZAL combined with a low magnitude of strain (1000 μe). Thus, α-ZAL might be a promising candidate for the treatment of osteoporosis. Mechanical strain was effective in inducing osteogenic differentiation. Taken together, our data suggest that α-ZAL combined with a low, but not high magnitude of strain, may provide benefits for osteogenesis.