Stem Cell Reports Short Article Sustained ERK Activation Underlies Reprogramming in Regeneration- Competent Salamander Cells and Distinguishes Them from Their Mammalian Counterparts Maximina H. Yun, 1, * Phillip B. Gates, 1 and Jeremy P. Brockes 1 1 Division of Biosciences, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK *Correspondence: maximina.yun@ucl.ac.uk http://dx.doi.org/10.1016/j.stemcr.2014.05.009 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). SUMMARY In regeneration-competent vertebrates, such as salamanders, regeneration depends on the ability of various differentiated adult cell types to undergo natural reprogramming. This ability is rarely observed in regeneration-incompetent species such as mammals, providing an explanation for their poor regenerative potential. To date, little is known about the molecular mechanisms mediating natural reprogram- ming during regeneration. Here, we have identified the extent of extracellular signal-regulated kinase (ERK) activation as a key compo- nent of such mechanisms. We show that sustained ERK activation following serum induction is required for re-entry into the cell cycle of postmitotic salamander muscle cells, partially by promoting the downregulation of p53 activity. Moreover, ERK activation induces epige- netic modifications and downregulation of muscle-specific genes such as Sox6. Remarkably, while long-term ERK activation is found in salamander myotubes, only transient activation is seen in their mammalian counterparts, suggesting that the extent of ERK activation could underlie differences in regenerative competence between species. INTRODUCTION In most vertebrates, the process of myogenic differentia- tion entails the withdrawal of precursors from the cell cycle, followed by their fusion into myotubes. The multi- nucleate state is characterized by a permanent postmitotic arrest, which renders the myotubes unable to respond to proliferative cues (Pajalunga et al., 2008; Walsh and Perl- man, 1997). In contrast, salamander myotubes remain responsive to such cues, being able to re-enter the cell cycle upon serum stimulation in culture (Tanaka et al., 1997) or after implantation within regenerating structures (Kumar et al., 2000). In salamander (Notophthalmus viridescens) A1 myotubes (Ferretti and Brockes, 1988), serum stimulation induces a reprogramming process that includes partial dedifferentia- tion, as suggested by the downregulation of the myogenic gene Myf5 (Imokawa et al., 2004), and re-entry into the cell cycle, which is also considered an aspect of dedifferentia- tion. The latter depends on the phosphorylation of Rb (Tanaka et al., 1997) and the downregulation of p53 activ- ity (Yun et al., 2013). The serum component that triggers these responses is not a conventional growth factor but an as-yet-unidentified thrombin-activated serum compo- nent that acts as a mitogen for myotubes, but not for mononucleate precursors (Lo ¨o ¨f et al., 2007; Straube et al., 2004; Tanaka et al., 1999). Even though mammalian myo- tube nuclei cannot be reprogrammed upon exposure to this factor (Lo ¨o ¨f et al., 2007), they are able to re-enter the cell cycle after forming heterokaryons with salamander myo- tubes (Velloso et al., 2001). This suggests that even when the initial response may be different, part of the pathway leading to serum-mediated reprogramming is conserved. Both the identity of the serum factor and the signaling pathways driving the reversal of the differentiated state in regeneration-competent salamander cells remain un- known, although extensive efforts to identify the serum factor are ongoing (Straube et al., 2004). In proliferating cells, the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases (MAPKs) plays a critical role in driving cell-cycle progres- sion in a variety of cell types (Albeck et al., 2013; Cook and McCormick, 1996; Murphy et al., 2002; Weber et al., 1997; Yamamoto et al., 2006). In fibroblasts, sustained ERK activation is required for successful S phase progres- sion by promoting the downregulation of antiproliferative genes during G1 phase and controlling the state of Rb phosphorylation (Yamamoto et al., 2006). Hence, it is possible that ERK activation plays a role during the reprog- ramming of differentiated salamander cells. Herein, we have tested this hypothesis using the salamander A1 cell line as a model for serum-induced reprogramming. RESULTS AND DISCUSSION Sustained ERK Activation in Cell-Cycle Re-entry of Salamander Myotubes Serum stimulation of A1 myotubes triggers an early activa- tion of the ERK pathway, which is sustained for up to 48 hr post stimulation (Figures 1A and 1B). This is accompanied by a long-term increase in the protein levels of c-FOS Stem Cell Reports j Vol. 3 j 1–9 j July 8, 2014 j ª2014 The Authors 1 Please cite this article in press as: Yun et al., Sustained ERK Activation Underlies Reprogramming in Regeneration-Competent Salamander Cells and Distinguishes Them from..., Stem Cell Reports (2014), http://dx.doi.org/10.1016/j.stemcr.2014.05.009