The skin is the largest organ in the human body, and is comprised of two main layers: the epidermis, a stratified layer of epithelial cells (predominantly keratinocytes) that provides a protective barrier from the surrounding environment, and the thicker dermis, made up predominantly of collagen and other connective tissues that provides a protective cushion surrounding the delicate muscles beneath. Basal epidermal cells are anchored to a basement membrane that segregates the two layers.
The outermost epidermal layer maintains dynamic homeostasis throughout adult life through continuous proliferation of pluripotent stem cells in the basal epidermal cell compartment (Watt, 1998; Taylor et al., 2000; Watt and Hogan, 2000; Oshima et al., 2001) (Figure 1.2.2). The predominant model for epidermal cell replenishment is the idea that basal stem cells divide infrequently to form daughter cells with limited proliferative capacity, known as transit-amplifying (TA) cells (Mackenzie, 1970; Potten, 1974). TA cells remain within the basal layer and undergo a small number of divisions before withdrawing from the cell cycle, breaking away from the basement membrane, migrating into the suprabasal epidermis and committing to a program of terminal differentiation (Lajtha, 1979; Alonso and Fuchs, 2003). However, the role of TA cells in skin homeostasis has recently been called into question, with recent studies suggesting that proliferation of epidermal keratinocytes is attributable to a single progenitor cell (Clayton et al., 2007; Jones et al., 2007).
Terminal differentiation involves migration towards the skin surface in conjunction with accumulation of tough keratin filaments, which provide resilience to mechanical stress. Keratinocytes within the top layer of the suprabasal layer produce small basophilic granules, such as membrane-coated lamellar granules and keratohyalin granules, in their cytoplasm. These assist in strengthening the cell. Throughout migration, keratinocytes gradually begin to die due to a lack of nutrients and oxygen, and by the time they reach the outer level of the epidermis, they become dead, flattened, denucleated, hyperkeratinised
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squames, which provide a waterproof barrier from the environment. Squamous cells are continuously sloughed off from the skin surface to be replaced by new differentiating suprabasal keratinocytes.
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Figure 1.2.2: Homeostasis in skin epidermis is maintained by continuous proliferation of pluripotent stem cells in the basal layer
Homeostasis in the skin epidermis is maintained by dynamic equilibrium between keratinocyte proliferation and cell loss. In this way, the skin is kept in a constant state of renewal, which is particularly important for rapid wound healing response. The epidermis is a stratified epithelial tissue, made up of several distinct layers. Cells in the basal layer (red) are attached to the basement membrane, which separates the epidermis from the protective dermis beneath. Keratinocyte stem cells (or TA cells) in the basal layer replicate to form daughter cells (green), which detach from the basement membrane and migrate into the suprabasal layer, (blue) where they become committed to a program of terminal differentiation. Cells accumulate tough structural keratin proteins, which protect the cell from mechanical stress. Further accumulation of keratin and formation of membrane-coated lamellar granules and keratohyalin granules accompanies further migration, until cells become denucleated hyperkeratinised squames. These provide a protective waterproof barrier, and are continuously shed from the epidermis surface to be replenished by further differentiating keratinocytes.
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In vivo models that target Myc activation to epidermal cell compartments allow more accurate studies of the effects of Myc deregulation, whilst taking the all- important context of the specific tissue into account. In particular, the MycERTAM switchable transgenic model allows controlled activation (and deactivation) of deregulated Myc in suprabasal keratinocytes using the suprabasal keratinocyte specific Involucrin promoter (inv-mycERTAM). Activation of MycERTAM through daily topical administration of 4OHT results in loss of differentiation and induction of proliferation in suprabasal keratinocytes that are committed to a program of terminal differentiation. Continued activation of MycERTAM leads to the formation of benign tumours resembling papillomas, or pre-malignant lesions that resemble actinic keratosis (Pelengaris et al., 1999) (Figure 1.2.3).
This also leads to a loss in cell-cell contact and increased angiogenic growth (Pelengaris et al., 1999; Knies-Bamforth et al., 2004). Interestingly, the resulting phenotype is entirely dependent on continued administration of 4OHT, and is fully reversed upon cessation of 4OHT administration (Pelengaris et al., 1999). However, despite showing many of the hallmarks of cancerous growth, over- representation of the Myc protein alone is insufficient to result in malignancy. This may be due to the fact that the keratinocyte population is self-limiting, since completion of terminal differentiation results in the ultimate loss of affected cells (Flores et al., 2004).
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Figure 1.2.3: Activation of MycERTAM in suprabasal keratinocytes results in increased proliferation and formation of benign papilloma-like tumours
The MycERTAM transgenic construct can be targeted to suprabasal keratinocytes via the Involucrin promoter. In wild type (WT) and vehicle-treated (VT) transgenic animals, proliferation is confined to the basal compartment. Post-mitotic cells enter a program of terminal differentiation and migrate towards the tissue surface. Activation of the MycERTAM protein in terminally differentiating suprabasal cells results in disruption of differentiation and induction of proliferation, leading to formation of benign papilloma-like growths. a) Cartoon representation of induction of proliferation in suprabasal keratinocytes. b) H&E staining for skin tissue from a transgenic untreated mouse, and a mouse treated with 4OHT for 7 days (taken with permission from Pelengaris, Littlewood et al., 1999). hf, hair follicle; gr, granular cell; pa, parakeratosis.
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