β-aminoisobutyric acid accelerates the proliferation and differentiation of MC3T3-E1 cells via moderate activation of ROS signaling

Abstract

Backgroud: Osteoporosis is one of the bone-metabolic diseases associated with decreased bone renewal and bone mineral density. β-aminoisobutyric acid (BAIBA), a natural thymine catabolite, can reduce inflammation in skeletal muscle and alleviate hepatic endoplasmic reticulum stress. However, the roles of BAIBA in osteoblast proliferation and differentiation remain largely unknown.

Methods: The cultured MC3T3-E1 cells received various treatments in this study, including BAIBA alone, H₂O₂ alone, BAIBA plus N-acetyl-l-cysteine and BAIBA plus apocynin. Cell proliferation was determined by CCK-8 assay and ³H-Thymidine incorporation. Cell differentiation was evaluated by determining mRNA level of differentiation makers and ALP, and ALP activity. Reactive oxygen species (ROS) were determined by DHE staining while superoxide anion level and NAD(P)H oxidase activity were determined by the lucigenin-derived chemiluminescence method. The content of hydrogen peroxide (H₂O₂) was detected using a commercial kit. The level of NOX1, NOX2 and NOX4 was determined by Western-blot or qRT-PCR.

Results: We show that treatment of BAIBA stimulated the proliferation of MC3T3-E1 osteoprogenitor cells and enhanced the gene expression of osteoblast differentiation markers. Incubation of MC3T3-E1 cells with BAIBA evoked increases in NAD(P)H oxidase-derived reactive oxygen species (ROS). Scavenging of reactive oxygen species (N-acetyl-l-cysteine) or inhibition of NAD(P)H oxidase (apocynin) abolished the BAIBA-elicited proliferation and differentiation of MC3T3-E1 cells.

Conclusion: Our results provide the first evidence that BAIBA stimulates proliferation and differentiation of osteoprogenitor cells via activation of NAD(P)H oxidase/ROS signaling.

Keywords: Cell proliferation and differentiation; Osteoprogenitors; ROS signaling; β-aminoisobutyric acid.