The properties of embryonic stem (ES) cells are controlled in part by signalling pathways which translate inputs from external signals to changes in gene expression. In particular, the cytokine LIF (leukaemia inhibitory factor) binds to the gp130/LIFR heterodimeric complex and activates the JAK/STAT3 pathway to promote ES-cell self-renewal. LIF also activates the ERK1/2 pathway, which regulates early differentiation events. The balance between these two ‘competing’ pathways lies in favour of self-renewal. The addition of L-Proline to the culture medium results in the differentiation of ES cells to a population of cells that is equivalent to the primitive ectoderm of the pre-gastrulating embryo (Washington et al., 2010). The molecular mechanisms by which L-proline induces this conversion are largely unknown.
Using pathway inhibitors and Western blot/kinome array analysis, we show that L-proline works by i) changing the balance in activity of pathways already stimulated by LIF and ii) activating additional pathways. The output of the complete signalling circuitry, including pathway crosstalk, mediates ES-cell differentiation to primitive ectoderm but major players include L-Proline-mediated activation of the mTOR pathway, L-Proline and LIF-mediated activation of the ERK1/2 pathway, and L-proline-mediated inhibition of the JAK/STAT3 pathway. Pathway activity is also modulated as the ES cells transition to primitive ectoderm.
In addition to helping to elucidate the molecular mechanisms underlying normal and abnormal embryogenesis, this study can help in the development of protocols for generating specific cell types for use in animal models of human disease and injury.