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Title: Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin}
Article:
Abstract:
Extracellular signal-regulated kinase( ERK) is a key effector of many growth signalling pathways. In this study, we visualise epidermal ERK activity in living mice using an ERK FRET biosensor. Under steady-state conditions, the epidermis occasionally revealed bursts of ERK activation patterns where ERK activity radially propagated from cell to cell. The frequency of this spatial propagation of radial ERK activity distribution( SPREAD) correlated with the rate of epidermal cell division. SPREADs and proliferation were stimulated by 12-O-tetradecanoylphorbol 13-acetate( TPA) in a manner dependent on EGF receptors and their cognate ligands. At the wounded skin, ERK activation propagated as trigger wave in parallel to the wound edge, suggesting that ERK activation propagation can be superimposed. Furthermore, by visualising the cell cycle, we found that SPREADs were associated with G2/M cell cycle progression. Our results provide new insights into how cell proliferation and transient ERK activity are synchronised in a living tissue.
Introduction:
Exquisite control of extracellular signal-regulated kinase( ERK) MAPK signalling is required for homeostasis in the epidermis and other tissues( Pouysségur et al., 2002; Kholodenko, 2006; Khavari and Rinn, 2007; Roberts and Der, 2007). ERK activation is triggered by the activation of epidermal growth factor receptors( EGFRs) through binding to their cognate ligands( Yuspa, 1994; Sibilia et al., 2007; Schneider et al., 2008). Precursors of EGFR ligands including EGF, transforming growth factor α( TGFα), and heparin binding EGF-like growth factor( HB-EGF), are generated as membrane-bound proteins and are released through cleavage by matrix metalloproteinases( MMPs) including ADAM17( Massagué and Pandiella, 1993; Sahin et al., 2004). The critical role of MMP-mediated release of EGFR ligands is corroborated by the close resemblance of the phenotypes among knockout mice of TGFα, ADAM17, and EGFR( Luetteke et al., 1993; Peschon et al., 1998; Franzke et al., 2012). However, little is known about how such cell-to-cell communication leading to activation of the Ras/ERK pathway is dynamically regulated in vivo. To address this issue, we live imaged transgenic( Eisuke) mice expressing a Förster resonance energy transfer( FRET) biosensor for ERK in order to visualise ERK activity in living epidermis under two photon excitation microscopy( Kamioka et al., 2012). During long-time time lapse imaging of the steady-state mouse skin, we noticed the epidermis occasionally appear with bursts of ERK activation patterns, where ERK activation is propagated from cell to cell in a radial wave. We named this new phenomenon as Spatial Propagation of Radial ERK Activity Distribution( SPREAD) and investigated characteristics, mechanisms, and roles of SPREAD. Single-cell analysis of SPREAD demonstrated that ERK activation in SPREAD originated from a cluster of a few cells and propagated up to about 50 μm with an average velocity of 1. 5 μm/min. The amplitude of ERK activation and efficiency in propagation gradually decreased until SPREAD disappeared. Interestingly, the frequency of SPREAD was spatio-temporally associated with that of cell division. SPREADs were significantly stimulated by topical treatment of mitotic stimulation, 12-O-tetradecanoylphorbol 13-acetate( TPA). The induction was dependent on EGF receptors and the production of their cognate ligands by MMPs. The role of SPREAD in cell cycle progression was studied with Fucci mice, in which G0/G1 cells and S/G2/M cells are visualised by the expression of mKO2-Cdt1 and mAG-Geminin, respectively( Sakaue-Sawano et al., 2008). Inhibition of SPREAD in Fucci mice led to significant delay of entry to G0/G1 phase from S/G2/M phase under TPA treatment, suggesting the role of SPREAD in G2/M progression of cell cycle. We also imaged ERK activity of ear skin subjected to epithelial wounding. Interestingly, wounded skin revealed another type of ERK activation propagation pattern, where waves of ERK activation propagated in parallel with the wound edge. Single cell analysis revealed similarity and difference between the wound-induced waves and SPREAD. While the velocity of ERK activity propagation in wound-induced waves was similar to that of SPREAD, the ERK activation waves from the wound edge was relatively maintained during propagation during propagation. In this study, we propose how dynamically growth signal is regulated in steady-state and wounded skin in a living tissue by showing two types of novel ERK activation patterns.
Discussion:
Cell proliferation at proper time and place is essential for tissue maintenance in multicellular organisms. In steady-state skin, epidermal cells undergo continuous turnover through autonomous activation of growth signal pathway triggered by the interaction of EGFRs and their cognate receptors( Kholodenko, 2006; Khavari and Rinn, 2007). ERK plays a central role as a downstream target of a variety of growth signal pathways and its tight regulation in time, place, and degree of activation determine cell behaviours such as cell proliferation, migration, and differentiation( Pouysségur et al., 2002). Aberrant activation of ERK has been reported in many types of cancers especially by the oncogenic mutation of Ras and Raf( Roberts and Der, 2007). Despite the significance of ERK activity regulation, technical limitations have obscured how growth signal activation is generated and propagated throughout the tissue. In this study, we exploited transgenic mice expressing a FRET biosensor of ERK and monitored ERK activity in epidermal cells at single cell resolution. Our long time lapse imaging of intact epidermis revealed radial propagation of ERK activity, where ERK activation originates from a few cells and propagate radially up to a radius of about 50 μm over 30 min until disappearance. We named this novel phenomenon as SPREAD and investigated its mechanism of ERK propagation and its role in cell cycle progression. Although we previously reported stochastic propagation of ERK activity pulse in a single cell to its neighbouring cells( Aoki et al., 2013), the propagation was restricted within an array of a few cells rather than firework-like round propagation covering more than 100 cells. Our single cell analysis of SPREAD and inhibitor treatment revealed similarities and differences between the two patterns of ERK activation in vitro and in vivo. While they share in common the dependence of EGFRs and the production of their cognate ligands for ERK activation propagation, they differ in the efficiency and amplitude of ERK activation during propagation. Together with the difference in ERK propagation manner between SPREADs in steady-state skin and ERK activation waves, this suggests that ERK propagation pattern might be altered depending on the surrounding environment of the cells, including the involvement of stem cells, cellular density, and pathological conditions. The induction of SPREADs by double TPA treatment raises the possibility that the alterations in SPREADs could be involved in tumorigenesis. Thus, it will be of special interest to observe SPREADs in various pathological conditions including cancer. The dependency on EGFRs and MMPs strongly suggests that a common molecular mechanism operates between SPREADs and ERK activation propagation in tissue culture cells( Aoki et al., 2013). Unlike the ERK activation propagation in tissue culture cells, however, we could not conclude whether the inhibitors against EGFRs and MMPs suppress ERK activation in the initiator cell or abrogate intercellular propagation of the ERK activation. This is because we cannot clearly identify the ERK activation pulse at single cell resolution due partly to the long interval of image acquisition in vivo and low signal-to-noise ratio of in vivo FRET imaging. As well as the established role of ERK activation in G1/S progression of cell cycle( Torii et al., 2006), the necessity of ERK to G2/M progression has also been reported in mammalian cells( Tamemoto et al., 1992; Wright et al., 1999). On the other hand, excessive ERK activation in G2 phase arrests cells in G2-phase. High ERK activity due to BRCA1 mutation, lack of Vaccinia H1-related( VHR) dual-specific protein tyrosine phosphatase, or stimulation by TPA induces G2-phase arrest( Yan et al., 2005; Dangi et al., 2006; Rahmouni et al., 2006; Chambard et al., 2007). Intriguingly, ERK activity is high around the G2/M phase in synchronized cells, but low when cells are arrested in M phase( Torii et al., 2006). These previous reports together with our findings show that exquisite regulation of ERK activation in terms of time, place, and degree is required for cell cycle progression and the localised transient activation of ERK in SPREAD can contribute to it. To validate the causal relationship between SPREADs and cell cycle progression, we need to develop methods to control cell cycle and/or ERK activity with the single cell resolution in the tissues. We used the C57BL/6 strain and the FVB/N strain for the expression of the Fucci biosensor and the FRET biosensor, respectively. This is because bright fluorescence of melanin granules in C57BL/6 mice hampered quantification of FRET signal and because Fucci mice with FVB/N background were not available. However, we speculate that the difference of the mouse strain will not affect our interpretation significantly by the following reasons. First, we observed significant increase of cell division in TPA-treated C57BL/6 mice, indicating that TPA promotes cell cycle in C57BL/6 as in FVB/N( Figure 5). Second, we observed SPREADs in C57BL/6 mice as in FVB/N, although quantitative data analysis was hampered by the fluorescence from the melanin granule. In summary, here we revealed localized propagations of ERK activation regulate cell cycle progression in mouse epidermis. The propagation of ERK activity was achieved by cellular interaction via EGFRs and the production of their cognate ligands. Our finding of SPREAD achieved by in vivo imaging of dynamic changes in kinase activity pave a new way to understanding intrinsic relationship between cell cycle and the underlying growth signal in vivo regulated by an intercellular communications via EGFR ligands.
Our skin is our largest organ; it provides a barrier that protects the underlying tissues and internal organs from the external environment and acts as one of our first lines of defense against infection . Both of these roles subject the skin to wear and tear and so it must constantly create new skin cells to replace those lost or damaged . However , if this renewal process goes awry it can lead to excessive cell growth or skin cancer . To avoid this , cells tightly regulate the pathways that stimulate skin renewal . Skin renewal involves growth signals activating an enzyme called ERK . When and where the ERK enzyme is activated is normally tightly regulated , and many kinds of cancer have been linked to ERK becoming active at the wrong time or in the wrong place . Despite the importance of ERK in skin cells , a number of technical challenges have made it difficult to study how these signals are passed from cell to cell . Hiratsuka et al . have now examined genetically altered mice that produce a fluorescent sensor molecule that makes it possible to see ERK activity in living skin cells . The skin of anesthetized mice was observed under a microscope , and time-lapse videos revealed occasional ‘firework-like’ bursts of ERK activity . At first the ERK enzyme was active in a small cluster of skin cells , then ERK activity was seen in the surrounding cells—appearing to spread outwards over the course of several minutes—before the activity stopped . Hiratsuka et al . named this pattern of activity a ‘Spatial Propagation of Radial ERK Activity Distribution’ , or SPREAD for short . By studying SPREADs in the skin on the ears and the back of these mice , Hiratsuka et al . learned that these bursts of ERK activity coincided with skin cell growth; the bursts happened more frequently in the areas where the skin cells were dividing . Applying a chemical that stimulates cell division to the skin of the mice triggered more bursts of ERK activity; whereas fewer bursts were observed if Hiratsuka et al . used other chemicals to block the activity of some of the signaling proteins that work upstream of ERK . Further experiments suggested that SPREADs encourage cells to progress through the cycle of events that leads a cell to divide; blocking these bursts caused the cell to pause at the stage just before it would normally divide . Hiratsuka et al . also observed similar patterns of ERK activity moving out like waves from the edges of skin wounds . Further research using similar methods will reveal how growth signals are triggered and propagated in healthy and diseased tissues , not only in the skin but also other organs such as the liver , intestine , and muscles .