High glucose impairs osteogenic differentiation of embryonic stem cells via early diversion of beta‐catenin from Forkhead box O to T cell factor interaction

Background Diabetes, which is characterized by an increase in blood glucose concentration, is accompanied by low bone turnover, increased fracture risk, and the formation of embryonic skeletal malformations. Yet, there are few studies elucidating the underlying alterations in signaling pathways leading to these osteogenic defects. We hypothesized here that bone formation deficiencies in a high glucose environment result from altered activity of beta‐catenin (CTNNB1), a key contributor to osteogenic differentiation, dysregulation of which has also been implicated in the development of diabetes. Methods To test this hypothesis, we used a previously established embryonic stem cell (ESC) model of differentiation that mimics the diabetic environment of the developing embryo. We differentiated murine ESCs within osteogenic‐inducing media containing either high (diabetic) or low (physiological) levels of D‐glucose and performed time course analyses to study the influence of high glucose on early and late bone cell differentiation. Results Endpoint measures for osteogenic differentiation were reduced in a glucose‐dependent manner and expression of precursor‐specific markers altered at multiple time points. Furthermore, transcriptional activity of the lymphoid enhancer factor (LEF)/T cell factor (TCF) transcription factors during precursor formation stages was significantly elevated while levels of CTNNB1 complexed with Forkhead box O 3a (FOXO3a) declined. Modulation of AKT, a known upstream regulator of both LEF/TCF and FOXO3a, as well as CTNNB1 rescued some of the reductions in osteogenic output seen in the high glucose condition. Conclusions Within our in vitro model, we found a clear involvement of LEF/TCF and FOXO3a signaling pathways in the regulation of osteogenic differentiation, which may account for the skeletal deficiencies found in newborns of diabetic mothers. Murine embryonic stem cells osteogenically differentiated in 4.5 g/L glucose fail to mineralize to the same extent as cells grown in 1.0 g/L glucose. At the end of the differentiation period, mRNAs associated with extracellular matrix assembly and mineralization were downregulated. During earlier differentiation, before cells enter the pre‐osteoblast stage, cell fate seems partially diverted into tangential lineages and altered transcription factor usage by beta‐catenin (CTNNB1) was observed.