ERK basal and pulsatile activity are differentially regulated in mammalian epidermis to control proliferation and exit from the stem cell compartment

Abstract

Fluctuation in signal transduction pathways is frequently observed during mammalian development. However, its role in regulating stem cells has not been explored. Here we tracked spatiotemporal ERK MAPK dynamics in human epidermal stem cells. While stem cells and differentiated cells were distinguished by high and low stable basal ERK activity, respectively, we also found cells with pulsatile ERK activity. Transitions from Basalhi-Pulselo (stem) to Basalhi-Pulsehi, Basalmid-Pulsehi, and Basallo-Pulselo (differentiated) cells occurred in expanding keratinocyte colonies and in response to a range of differentiation stimuli. Pharmacological inhibition of ERK induced differentiation only when cells were in the Basalmid-Pulsehi state. Basal ERK activity and pulses were differentially regulated by DUSP10 and DUSP6, leading us to speculate that DUSP6-mediated ERK pulse downregulation promotes initiation of differentiation whereas DUSP10-mediated downregulation of mean ERK activity promotes and stabilizes post-commitment differentiation. Quantification of MAPK1/3, DUSP6 and DUSP10 transcripts in individual cells demonstrated that ERK activity is controlled both transcriptionally and post-transcriptionally. When cells were cultured on a topography that mimics the epidermal-dermal interface, spatial segregation of mean ERK activity and pulses was observed. In vivo imaging of mouse epidermis revealed a patterned distribution of basal cells with pulsatile ERK activity and downregulation was linked to the onset of differentiation. Our findings demonstrate that ERK MAPK signal fluctuations link kinase activity to stem cell dynamics.SignificanceUnderstanding how intracellular signaling cascades control cell fate is a key issue in stem cell biology. Here we show that exit from the stem cell compartment in mammalian epidermis is characterised by pulsatile ERK MAPK activity. Basal activity and pulses are differentially regulated by DUSP10 and DUSP6, two phosphatases that have been shown previously to regulate differentiation commitment in the epidermis. ERK activity is controlled both transcriptionally and post-transcriptionally. Spatial segregation of mean ERK activity and pulses is observed both in reconstituted human epidermis and in mouse epidermis. Our findings demonstrate the tight spatial and temporal regulation of ERK MAPK expression and activity in mammalian epidermis.

Dynamics and heterogeneity of Erk-induced immediate-early gene expression

“…We used prior data from the Erk signaling pathway and immediate-early gene expression to infer reasonable parameter values where possible. Erk typically remains active for 10 min to 2 h after stimulation, providing a typical signaling timescale [6][7][8] ; immediate-early gene expression is rapid 13 , leading us to assume that ktxn is fast (~1 min -1 ) compared to experimentally measured signaling dynamics; and the mRNA degradation rate for the fos IEG has been experimentally measured (kdeg ~20 min -1 ) 19 . We varied other parameters, in particular the inactive-to-listening transition rates, as we examined different response regimes.…”

“…Erk has been found to be highly dynamic in vivo 31 and associated with exit from the basal stem cell compartment 8 ; furthermore, Fos protein accumulation has been associated with the process of differentiation in keratinocytes in vivo 33 . We stained mouse epidermis for either doubly phosphorylated Erk or Fos protein and imaged the basal and suprabasal layers of the inter-follicular epidermis (Figure 5A).…”

“…Many cell signaling pathways do not simply switch from off to on when stimulated, but instead exhibit complex dynamics such as pulses whose frequency, amplitude, or width depend on the stimulus or cellular context [1][2][3][4][5][6][7][8] . Although they vary between pathways and contexts, timescales of signaling dynamics typically range from minutes to a few hours (e.g.…”

“…Although they vary between pathways and contexts, timescales of signaling dynamics typically range from minutes to a few hours (e.g. ~10 min signaling pulses for the Erk kinase 7,8 ; ~1 h pulses for the p53 [1][2][3] and NF-kB transcription factors 5 ). Signaling dynamics have been proposed to activate distinct sets of target genes compared to constant-on and constant-off signaling states [9][10][11] .…”

“…Here, we set out to address these questions using the activation of the Ras/Erk pathway and induction of IEGs as a testbed. The Ras/Erk pathway has been shown to demonstrate rich, context-dependent dynamics, with pulses and traveling waves of activity that can be observed in both cultured cells and in vivo tissues 7,8 . Erk activity also triggers a potent transcriptional response, particularly by activating a set of ~100 immediate-early genes (IEGs) within tens of minutes 13 .…”