Epidermal growth factor can signal via β-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling

Bone marrow mesenchymal stem/stromal cells (MSCs) maintain bone homeostasis and repair through the ability to expand in response to mitotic stimuli and differentiate into skeletal lineages. Signalling mechanisms that enable precise control of MSC function remain unclear. Here we report that by initially examining differences in signalling pathway expression profiles of individual MSC clones, we identified a previously unrecognised signalling mechanism regulated by epidermal growth factor (EGF) in primary human MSCs. We demonstrate that EGF is able to activate β-catenin, a key component of the canonical Wnt signalling pathway. EGF is able to induce nuclear translocation of β-catenin in human MSCs but does not drive expression of Wnt target genes or T cell factor (TCF) activity in MSC reporter cell lines. Using an efficient Design of Experiments (DoE) statistical analysis, with different combinations and concentrations of EGF and Wnt ligands, we were able to confirm that EGF does not influence the Wnt/β-catenin pathway in MSCs. We show that the effects of EGF on MSCs are temporally regulated to initiate early “classical” EGF signalling mechanisms (e.g via mitogen activated protein kinase) with delayed activation of β-catenin. By RNA-sequencing, we identified gene sets that were exclusively regulated by the EGF/β-catenin pathway, which were distinct from classical EGF-regulated genes. However, subsets of classical EGF gene targets were significantly influenced by EGF/β-catenin activation. These signalling pathways cooperate to enable EGF-mediated proliferation of MSCs by alleviating the suppression of cell cycle pathways induced by classical EGF signalling.