Annatto-Derived Tocotrienol Promotes Mineralization of MC3T3-E1 Cells by Enhancing BMP-2 Protein Expression via Inhibiting RhoA Activation and HMG-CoA Reductase Gene Expression

Abstract

Purpose

Annatto-derived tocotrienol (AnTT) has been shown to improve bone formation in animal models of osteoporosis and promote differentiation of pre-osteoblastic cells. However, the mechanism of action of AnTT in achieving these effects is unclear. This study aims to investigate the mechanism of action of AnTT on MC3T3-E1 pre-osteoblasts via the mevalonate pathway.

Methods

Murine pre-osteoblastic cells, MC3T3-E1, were cultured with the density of 1 × 10⁴ cells/mL and treated with 4 concentrations of AnTT (0.001–1 µg/mL). Expression of HMG-CoA reductase (HMGR) gene was carried out using qPCR after treatment with AnTT for 21 days. RhoA activation and bone morphogenetic protein-2 (BMP-2) were measured using immunoassay after 9 and 15 days of AnTT treatment. Lovastatin was used as the positive control. Mineralized nodules were detected using Von Kossa staining after 21 days of AnTT treatment.

Results

The results showed that HMGR was up-regulated in the lovastatin group on day 9 and 21 compared to the control. Lovastatin also inhibited RhoA activation (day 9 and 15) and increased BMP-2 protein (day 15). On the other hand, AnTT at 0.001 μg/mL (day 3) and 0.1 μg/mL (day 21) significantly down-regulated HMGR gene expression compared to the control. On day 21, HMGR gene expression was significantly reduced in all groups compared to day 15. AnTT at 0.1 μg/mL significantly decreased RhoA activation on day 9 compared to the control. AnTT at 1 μg/mL significantly increased BMP-2 protein on day 15 compared to the control (P<0.05). Mineralized calcium nodules were more abundant in AnTT treated groups compared to the control on day 21.

Conclusion

AnTT suppresses the mevalonate pathway by downregulating HMGR gene expression and inhibiting RhoA activation, leading to increased BMP-2 protein in MC3T3-E1 cells. This explains the stimulating effects of AnTT on osteoblast mineralization.

Keywords:

• bone
• osteogenic
• osteoporosis
• tocotrienol
• vitamin E

Acknowledgments

We express our gratitude to American River Nutrition (USA) for providing the annatto-derived tocotrienol, DeltaGold^®. This research was funded by ERGS/1/2013/SKK03/UKM/01/1 grant provided by the Ministry of Education, Malaysia, and GUP-2017-012 grant by Universiti Kebangsaan Malaysia. We would like to thank Puan Nur Farhana Mohd Fozi, Puan Nurul Hafizah Abas and James Jam Jolly from Pharmacology Department, UKM for technical assistance.

Disclosure

The authors report no conflicts of interest in this work.