O-GlcNAcylation drives calcium signaling towards osteoblast differentiation: a bioinformatics-oriented study
Objective: This study aimed to reveal the possible mechanisms by which O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulates osteoblast differentiation using a series of bioinformatics-oriented experiments. Methods: To examine the influence of O-GlcNAcylation levels on osteoblast differentiation, osteoblastic MC3T3-E1 cells were treated with O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) inhibitors. Correlations between the levels of O-GlcNAcylation and the expression of osteogenic markers as well as OGT were evaluated by qPCR and western blotting. The O-GlcNAcylated proteins assumed to correlate with Runx2 expression were retrieved from several public databases and used for further bioinformatics analysis. Following the findings of the bioinformatics analysis, intracellular calcium ([Ca2+]i) was monitored in the cells treated with OGT and OGA inhibitors using a confocal laser-scanning microscope (CLS). The interaction effect between O-GlcNAcylation and [Ca2+]i on osteogenic marker expression was determined using stable OGT knockdown MC3T3-E1 cells. Results: O-GlcNAcylation was positively and negatively associated with osteoblast differentiation. The time-course profile of global O-GlcNAcylated proteins showed a distinctive pattern with different molecular weights during osteoblast differentiation. The expression pattern of several O-GlcNAcylated proteins was significantly similar to that of Runx2 expression. Bioinformatic analysis of the retrieved Runx2-related-O-GlcNAcylated-proteins revealed the importance of [Ca2+]i. CLS showed that alteration of O-GlcNAcylation rapidly changed [Ca2+]i in MC3T3-E1 cells. O-GlcNAcylation and [Ca2+]i showed an interaction effect on the expression of osteogenic markers. OGT knockdown disrupted the [Ca2+]i-induced expression changes of osteogenic markers. Conclusions: O-GlcNAcylation interacts with [Ca2+]i and elicits osteoblast differentiation by regulating the expression of osteogenic markers. KEYWORDS: Osteoblast differentiation; O-linked-N-acetylglucosaminylation; Calcium; Bioinformatics; Literature-mining
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Mouse pre-osteoblastic MC3T3-E1 cells were cultured in a growth medium that is α-MEM containing 10% FBS, 1% Penicillin/Streptomycin at 37°C with 5% CO2. Differentiation medium, the growth medium with 50 µM ascorbic acid and 2 mM β-glycerophosphate, was used to induce osteoblast differentiation after the cell confluent. The medium was changed every two days. To inhibit OGT or OGA, cells were treated with 1, 5, 10 or 20 µM OSMI-1 or 50, 100 nM, 1,10 or 100 µM Thiamet-G.