Background and Aims: While there is evidence of cross-communication between the skeletal and neural systems, knowledge of the underlying molecular mechanisms is limited. The Eph receptors, EphA and EphB, are the largest family of receptor tyrosine kinases that interact with their corresponding ephrinA and ephrinB ligands. Eph/ephrin signalling mediates bone homeostasis and neuronal patterning. We hypothesise Eph/ephrin interactions mediate communication within the neuro-osteogenic network. We assessed the Eph /ephrin expression profile of neural cells and investigated the effect of manipulating these Eph/ephrin interactions on neural differentiation, adhesion and migration.
Methodology: A human neuroblastoma line was cultured under basal and neural-inductive conditions, to determine changes in Eph and ephrin gene expression levels during neural differentiation by qPCR. Cells adhesion and neurite extension was assessed in the presence of bound Eph-Fc fusion proteins over time, following staining with DAPI and Phalloidin. Chick embryo dorsal root ganglia (DRG) were cultured with bound Eph-Fc then stained with anti-Neurofilament Medium Chain (NF-M) to determine total neurite growth and percentage of invading sensory neurites.
Results: Gene expression levels of ephrinA2, ephrinA5, ephrinB1 and ephrinB2 were significantly upregulated in neural differentiated cells, which bind to EphA3, EphA4, EphB2 and EphB4 expressed by bone marrow stromal cells, respectively. During neural differentiation, NESTIN and PERIPHERIN expression were decreased in response to EphA4-Fc and EphB2-Fc, whereas NESTIN and NF-M expression were upregulated in response to EphB4-Fc. In addition, β-III TUBULIN expression was reduced in response to EphB2-Fc. In the neural differentiated cells, neurite extension was enhanced in response to EphA4-Fc, while EphB4-Fc inhibited neural adhesion. There was a trend of EphA3-Fc inhibiting DRG neurite extension that did not reach statistical significance.
Conclusion: Human neuroblastoma expressed ephrinA2, ephrinA5, ephrinB1 and ephrinB2 with increasing neural differentiation. EphA4-Fc, EphB2-Fc and EphB4-Fc fusion proteins differentially affected neural differentiation, while EphB4-Fc inhibited neural adhesion suggesting Eph/ephrin interactions mediate crosstalk between neural and stromal populations.