Deletion of p66Shc dysregulates ERK and STAT3 activity in embryonic stem cells, enhancing their naïve-like self-renewal in the presence of LIF.

Abstract

The ShcA adapter protein is necessary for early embryonic development. The role of ShcA in development is primarily attributed to its 52kDa and 46kDa isoforms, that transduce receptor tyrosine kinase (RTK) signaling via the extracellular signal regulated kinase (ERK). During embryogenesis, ERK acts as the primary signalling effector, driving fate acquisition and germ layer specification. P66Shc, the largest of the ShcA isoforms, has been observed to antagonize ERK in several contexts, however its role during embryonic development remains poorly understood. We hypothesized that p66Shc could act as a negative regulator of ERK activity during embryonic development, antagonizing early lineage commitment. To explore the role of p66Shc in stem cell self-renewal and differentiation, we created a p66Shc knockout (KO) murine embryonic stem cell (mESC) line. Deletion of p66Shc enhanced basal ERK activity, but surprisingly, instead of inducing mESC differentiation, loss of p66Shc enhanced the expression of core and naïve pluripotency markers. Using pharmacologic inhibitors to interrogate potential signalling mechanisms, we discovered that p66Shc deletion permits the self-renewal of naïve mESCs in the absence of conventional growth factors, by increasing their responsiveness to leukemia inhibitory factor (LIF). We discovered that loss of p66Shc enhanced not only increased ERK phosphorylation but also increased phosphorylation of Signal transducer and activator of transcription (STAT3(S727)) in mESCs, which may be acting to stabilize their naïve-like identity, desensitizing them to ERK-mediated differentiation cues. These findings identify p66Shc as a regulator of both LIF-mediated ESC pluripotency and of signaling cascades that initiate post-implantation embryonic development and ESC commitment.