Icaritin induces differentiation of MC3T3-E1 subclone 14 cells via estrogen receptor and BMP/Smad signaling pathway in vitro

AIM: To observe the effects of icaritin (ICT) on the proliferation and differentiation of MC3T3-E1 subclone 14 cells (a pre-osteoblast cell line) and to observe the role of estrogen receptor (ER) and bone morphogenetic protein(BMP)/Smads signaling pathways in the differentiation of the cells. METHODS: The methods of WST-8 and BrdU were used to observe the viability and proliferation of MC3T3-E1 subclone 14 cells after treatment with different concentrations of ICT. The effects of ICT and noggin on the levels of alkaline phosphatase(ALP), type I collagen (Col I) and bone Gla protein (BGP) in MC3T3-E1 subclone 14 cells were observed after ER was blocked by ICI182780. The relative mRNA levels of BMPs (2, 4, 7) were detected by real-time PCR. The protein phosphorylation of Smad1/5/8 was determined by Western blotting after ER signaling pathway was blocked by ICI182780. RESULTS: ICT at concentrations of 0.1 μmol/L and 1 μmol/L increased the levels of ALP, Col I and BGP, and the numbers of mineralized nodules in MC3T3-E1 subclone 14 cells, indicating that ICT-promoted the differentiation, but did not affect the cell viability and proliferation. After the ER receptor signaling was blocked, ICT-promoted differentiation was significantly decreased. ICT improved the mRNA expression of BMP-2, 4, but did not affect the mRNA expression of BMP-7. After the ER receptor signaling was blocked, ICT-promoted phosphorylation of Smad1/5/8 was significantly decreased. Blockage of BMP/Smad signaling inhibited the effect of ICT on the differentiation. CONCLUSION: Icaritin induces the differentiation of MC3T3-E1 subclone 14 cells by activating BMP/Smad signaling pathway through ER.