O-GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics-oriented study

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. 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. O-GlcNAcylation was positively 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. O-GlcNAcylation interacts with [Ca²⁺]_i and elicits osteoblast differentiation by regulating the expression of osteogenic markers.