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      Regulation of ERK basal and pulsatile activity control proliferation and exit from the stem cell compartment in mammalian epidermis

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          Significance

          Understanding 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 characterized 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 posttranscriptionally. 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.

          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 Basal hi-Pulse lo (stem) to Basal hi-Pulse hi, Basal mid-Pulse hi, and Basal lo-Pulse lo (differentiated) cells occurred in expanding keratinocyte colonies and in response to differentiation stimuli. Pharmacological inhibition of ERK induced differentiation only when cells were in the Basal mid-Pulse hi state. Basal ERK activity and pulses were differentially regulated by DUSP10 and DUSP6, leading us to speculate that DUSP6-mediated ERK pulse down-regulation promotes initiation of differentiation, whereas DUSP10-mediated down-regulation of mean ERK activity promotes and stabilizes postcommitment differentiation. Levels of MAPK1/MAPK3 transcripts correlated with DUSP6 and DUSP10 transcripts in individual cells, suggesting that ERK activity is negatively regulated by transcriptional and posttranslational mechanisms. 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 down-regulation was linked to the onset of differentiation. Our findings demonstrate that ERK MAPK signal fluctuations link kinase activity to stem cell dynamics.

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          Most cited references55

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          Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.

          C Marshall (1995)
          A number of different intracellular signaling pathways have been shown to be activated by receptor tyrosine kinases. These activation events include the phosphoinositide 3-kinase, 70 kDa S6 kinase, mitogen-activated protein kinase (MAPK), phospholipase C-gamma, and the Jak/STAT pathways. The precise role of each of these pathways in cell signaling remains to be resolved, but studies on the differentiation of mammalian PC12 cells in tissue culture and the genetics of cell fate determination in Drosophila and Caenorhabditis suggest that the extracellular signal-regulated kinase (ERK-regulated) MAPK pathway may be sufficient for these cellular responses. Experiments with PC12 cells also suggest that the duration of ERK activation is critical for cell signaling decisions.
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            Stem cells and niches: mechanisms that promote stem cell maintenance throughout life.

            Niches are local tissue microenvironments that maintain and regulate stem cells. Long-predicted from mammalian studies, these structures have recently been characterized within several invertebrate tissues using methods that reliably identify individual stem cells and their functional requirements. Although similar single-cell resolution has usually not been achieved in mammalian tissues, principles likely to govern the behavior of niches in diverse organisms are emerging. Considerable progress has been made in elucidating how the microenvironment promotes stem cell maintenance. Mechanisms of stem cell maintenance are key to the regulation of homeostasis and likely contribute to aging and tumorigenesis when altered during adulthood.
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              Oscillations in NF-kappaB signaling control the dynamics of gene expression.

              Signaling by the transcription factor nuclear factor kappa B (NF-kappaB) involves its release from inhibitor kappa B (IkappaB) in the cytosol, followed by translocation into the nucleus. NF-kappaB regulation of IkappaBalpha transcription represents a delayed negative feedback loop that drives oscillations in NF-kappaB translocation. Single-cell time-lapse imaging and computational modeling of NF-kappaB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IkappaBalpha transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-kappaB signaling may thus depend on number, period, and amplitude of oscillations.
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                Author and article information

                Journal
                Proc Natl Acad Sci U S A
                Proc. Natl. Acad. Sci. U.S.A
                pnas
                pnas
                PNAS
                Proceedings of the National Academy of Sciences of the United States of America
                National Academy of Sciences
                0027-8424
                1091-6490
                28 July 2020
                10 July 2020
                10 July 2020
                : 117
                : 30
                : 17796-17807
                Affiliations
                [1] aCentre for Stem Cells and Regenerative Medicine, King’s College London , SE1 9RT London, United Kingdom;
                [2] bDepartment of Mathematics, Imperial College London , SW7 2BZ London, United Kingdom;
                [3] cDepartment of Applied Mathematics and Theoretical Physics, Centre for Mathematical Sciences, University of Cambridge , CB3 0WA Cambridge, United Kingdom;
                [4] dThe Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge , CB2 1QN Cambridge, United Kingdom
                Author notes
                1To whom correspondence may be addressed. Email: fiona.watt@ 123456kcl.ac.uk .

                Contributed by Fiona M. Watt, June 2, 2020 (sent for review April 14, 2020; reviewed by Joshua M. Brickman and Valerie Horsley)

                Author contributions: T.H., I.B., and F.M.W. designed research; T.H. performed research; T.H. and I.B. contributed new reagents/analytic tools; T.H., I.B., G.P., and F.M.W. analyzed data; and T.H., I.B., and F.M.W. wrote the paper.

                Reviewers: J.M.B., University of Copenhagen; and V.H., Yale University.

                Author information
                http://orcid.org/0000-0002-5359-2690
                http://orcid.org/0000-0002-1017-777X
                http://orcid.org/0000-0002-4704-9632
                http://orcid.org/0000-0001-9151-5154
                Article
                202006965
                10.1073/pnas.2006965117
                7395546
                32651268
                1be015e3-ec01-4183-b922-ec8f101ee7ad
                Copyright © 2020 the Author(s). Published by PNAS.

                This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

                History
                Page count
                Pages: 12
                Funding
                Funded by: RCUK | Medical Research Council (MRC) 501100000265
                Award ID: MR/PO18823/1
                Award Recipient : Fiona M. Watt
                Funded by: Wellcome Trust
                Award ID: 206439/Z/17/Z
                Award Recipient : Fiona M. Watt
                Funded by: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) 501100000268
                Award ID: BB/M007219/1
                Award Recipient : Fiona M. Watt
                Categories
                Biological Sciences
                Cell Biology

                erk,stem cells,keratinocytes,live imaging,cell signaling
                erk, stem cells, keratinocytes, live imaging, cell signaling

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