Ethanol Inhibits Mesenchymal Stem Cell Osteochondral Lineage Differentiation Due in Part to an Activation of Forkhead Box Protein O‐Specific Signaling

Background During bone fracture repair, resident mesenchymal stem cells (MSCs) differentiate into chondrocytes, to form a cartilaginous fracture callus, and osteoblasts, to ossify the collagen matrix. Our laboratory previously reported that alcohol administration led to decreased cartilage formation within the fracture callus of rodents and this effect was mitigated by postfracture antioxidant treatment. Forkhead box protein O (FoxO) transcription factors are activated in response to intracellular reactive oxygen species (ROS), and alcohol has been shown to increase ROS. Activation of FoxOs has also been shown to inhibit canonical Wnt signaling, a necessary pathway for MSC differentiation. These findings have led to our hypothesis that alcohol exposure decreases osteochondrogenic differentiation of MSCs through the activation of FoxOs. Methods Primary rat MSCs were treated with ethanol (EtOH) and assayed for FoxO expression, FoxO activation, and downstream target expression. Next, MSCs were differentiated toward osteogenic or chondrogenic lineages in the presence of 50 mM EtOH and alterations in osteochondral lineage marker expression were determined. Lastly, osteochondral differentiation experiments were repeated with FoxO1/3 knockdown or with FoxO1/3 inhibitor AS1842856 and osteochondral lineage marker expression was determined. Results EtOH increased the expression of FoxO3a at mRNA and protein levels in primary cultured MSCs. This was accompanied by an increase in FoxO1 nuclear localization, FoxO1 activation, and downstream catalase expression. Moreover, EtOH exposure decreased expression of osteogenic and chondrogenic lineage markers. FoxO1/3 knockdown restored proosteogenic and prochondrogenic lineage marker expression in the presence of 50 mM EtOH. However, FoxO1/3 inhibitor only restored proosteogenic lineage marker expression. Conclusions These data show that EtOH has the ability to inhibit MSC differentiation, and this ability may rely, at least partially, on the activation of FoxO transcription factors. Mesenchymal stem cells (MSC) cultured in media supplemented with ethanol have increased Forkhead box protein O1/3 (FoxO1/3) expression and activity. MSC co‐cultured in osteogenic or chondrogenic differentiation media and ethanol show reduced expression of osteochondral lineage markers. FoxO1/3 attenuation rescues the expression of the pro‐osteogenic transcription factor osterix, but only partially rescues pro‐chondrogenic transcription factor sox