Betaine promotes cell differentiation of human osteoblasts in primary culture

Isabella Villa,Alessandro Rubinacci,Anna Montesano,Michela Bottani,Anita Ferraretto,Alice Spinello,Livio Luzi,Simona Bolamperti,Fernanda Vacante,Ileana Terruzzi,Pamela Senesi

doi:10.1186/s12967-017-1233-5

Isabella Villa, Alessandro Rubinacci + Show 9 more

Open Access

https://doi.org/10.1186/s12967-017-1233-5

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Abstract

BackgroundBetaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it also shows an antioxidant activity. Moreover, BET stimulates muscle differentiation via insulin like growth factor I (IGF-I). The processes of myogenesis and osteogenesis involve common mechanisms with skeletal muscle cells and osteoblasts sharing the same precursor. Therefore, we have hypothesized that BET might be effective on osteoblast cell differentiation.MethodsThe effect of BET was tested in human osteoblasts (hObs) derived from trabecular bone samples obtained from waste material of orthopedic surgery. Cells were treated with 10 mM BET at 5, 15, 60 min and 3, 6 and 24 h. The possible effects of BET on hObs differentiation were evaluated by real time PCR, western blot and immunofluorescence analysis. Calcium imaging was used to monitor intracellular calcium changes.ResultsReal time PCR results showed that BET stimulated significantly the expression of RUNX2, osterix, bone sialoprotein and osteopontin. Western blot and immunofluorescence confirmed BET stimulation of osteopontin protein synthesis. BET stimulated ERK signaling, key pathway involved in osteoblastogenesis and calcium signaling. BET induced a rise of intracellular calcium by means of the calcium ions influx from the extracellular milieu through the L-type calcium channels and CaMKII signaling activation. A significant rise in IGF-I mRNA at 3 and 6 h and a significant increase of IGF-I protein at 6 and 24 h after BET stimulus was detected. Furthermore, BET was able to increase significantly both SOD2 gene expression and protein content.ConclusionsOur study showed that three signaling pathways, i.e. cytosolic calcium influx, ERK activation and IGF-I production, are enhanced by BET in human osteoblasts. These pathways could have synergistic effects on osteogenic gene expression and protein synthesis, thus potentially leading to enhanced bone formation. Taken together, these results suggest that BET could be a promising nutraceutical therapeutic agent in the strategy to counteract the concomitant and interacting impact of sarcopenia and osteoporosis, i.e. the major determinants of senile frailty and related mortality.

Highlights

Betaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it shows an antioxidant activity
We have investigated if BET could induce osteoblast differentiation by evaluating the expression of osteogenic genes, i.e. runt-related transcription factor 2 (RUNX2), OSX, bone sialoprotein (BSP) and OPN, and if it could activate important intracellular pathway such as ERK pathway, protein synthesis and calcium signaling, which are fundamental for the recruitment and function of bone cells [22]
Since Superoxide Dismutase 2 (SOD2), a crucial mitochondrial enzyme playing a role in antioxidant activity [24], is associated to intracellular calcium perturbations [25], we have evaluated SOD2 gene expression in human osteoblasts after BET treatment

Summary

Introduction

Betaine (BET), a component of many foods, is an essential osmolyte and a source of methyl groups; it shows an antioxidant activity. BET stimulates muscle differentiation via insulin like growth factor I (IGF-I). As recently reviewed [4], BET favours lipolysis and inhibits lipogenesis via gene and protein expression; it promotes the autocrine/endocrine release of insulinlike growth factor type I (IGF-I), and modulates its relative receptor signaling pathway. It has been shown that BET enhances neo myotubes formation and differentiation while promoting IGF-I gene and protein expressions in C2C12 murine myoblasts [6], a model of skeletal muscle development [7]. IGF-I stimulates RUNX2 downstream gene expression, a critical step in osteogenesis, by up-regulating the protein levels of PI3K subunits, Akt, and increasing p70S6 kinase (p70 S6K) stimulating osteoblast differentiation, protein synthesis and cell growth [11]. The critical role of IGF-I in bone is further supported by the inhibition of osteoblast differentiation, protein synthesis and cell growth after treating the cells with antibodies against IGF-I [9,10,11]

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