Abstract
Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here, we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA - promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.
Highlights
Commitment is a transient state during which a cell becomes restricted to a particular differentiated fate
By determining when cells recovered from suspension could no longer resume clonal growth on replating (Fig.1b; Fig.1-Figure Supplement 1a), we confirmed that there is a marked drop in colony forming ability between 4 and 8 hours. This correlates with an increase in the proportion of cells expressing the terminal differentiation markers involucrin (IVL) and transglutaminase 1 (TGM1) (Fig.1c, d; Fig.1-Figure Supplement 1b) and downregulation of genes that are expressed in the basal layer of the epidermis, including integrin α6 (ITGα6)
Given that DUSP10 knockdown differed from the other protein phosphatases in decreasing the clonogenicity (Fig.2b; Fig.2-Figure Supplement 3a) and growth rate of keratinocytes (Fig.2-Figure Supplement 3b, e), we examined the effect of DUSP10 knockdown on ERK1/2 activity and Activator Protein 1 (AP1) factor expression
Summary
Commitment is a transient state during which a cell becomes restricted to a particular differentiated fate. While commitment is a well-defined concept in developmental biology, it is still poorly understood in the context of adult tissues (Simons & Clevers 2011; Semrau & van Oudenaarden 2015; Nimmo et al 2015). This is because end-point analysis fail to capture dynamic changes in cell state, and rapid cell state transitions can depend on post-translational events, such as protein phosphorylation and dephosphorylation (Avraham & Yarden 2011). Cells that leave the basal layer undergo a process of terminal differentiation as they move through the suprabasal layers. In the final stage of terminal differentiation the cell nucleus and cytoplasmic organelles are lost and cells assemble an insoluble barrier, called the cornified envelope, which is formed of transglutaminase cross
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