5 - GROWTH AND DIFFERENTIATION OF THE EPIDERMIS Flashcards
EPIDERMAL STEM CELLS
Stem cells are progenitor cells that can both renew themselves and give rise to differentiated progeny over an extended time period. The ability of the epidermis to continually regenerate itself and to heal wounds indicates that it must contain stem cells.
Further, the pioneering work of Howard Green and colleagues allowed for the growth of these epidermal stem cells in culture, which was a great boon to understanding many aspects of epidermal stem cell biology. In addition, their work allowed for the clinical use of cultured epidermal stem cells in treating burn wounds, an early example of the utility of adult stem cells in regenerative therapy. 1 Here, we will describe some current aspects of the state of knowledge of stem cells within interfollicular epidermis. Of note, however, is that this has lagged behind our understanding of stem cells of the hair follicle (discussed in Chap. 7).
When cells are cultured from the human epidermis under standard conditions, they are heterogeneous in their self-renewing ability. Some undergo only a few rounds of division and may reflect transit-amplifying cells, a highly proliferative but short-lived cell population. The cells that self-renew robustly are putative stem cells. Consistent with this difference in activity, isolated basal cells express varying levels of a number of genes, including β1 integrin, LRIG1, and CD46.2-4 Cells with high expression of these genes have higher self-renewing capacity in vitro and tend to form clusters in intact skin. This data are consistent with the presence of a distinct stem cell pool within the basal layer. Single-cell sequencing approaches have further demonstrated that there is likely to be heterogeneity even in this enriched pool of stem cells. 4 Although it is difficult to determine the functional roles and consequences of this heterogeneity in human skin in situ, these types of studies are possible in the mouse. However, there has been continued debate about the hierarchical makeup of progenitors within the mouse epidermis. Although some studies suggest that the basal layer is composed of a single unipotent population, others have provided evidence for at least 2 types of progenitor cells that differ in proliferation dynamics and marker expression. 5-9 It is expected that in the next few years, a much clearer picture of the regulation and functions of these distinct cell types will emerge.
The tissue microenvironment in which stem cells normally exist is called their ______
niche
Niches are continually being more thoroughly defined and include factors such as direct cell-cell and cell-matrix communication, soluble secreted factors (including inflammatory, nervous, and other tissue resident cells), and local tissue mechanical properties. The niche for stem cells in the human interfollicular epidermis is not clear. Various studies have suggested that they are located dispersed throughout the basal layer, or at either the tops or bottoms of rete ridges. Part of this may reflect regional differences in skin architecture as well as the early stages of our ability to mark and track these specific cell types. Regardless of their position, the progenitor cells in the basal layer largely produce differentiated progeny that form in columns directly above them.10 Although progenitor cells can be lost or can expand laterally (to a limited degree) in a stochastic manner, they give rise to differentiated cells that move through the layers without significant lateral movement.
EPIDERMAL PROLIFERATION
The epidermis requires continuous proliferation for both homeostatic turnover and wound healing. However, inappropriate proliferation occurs in many pathologic conditions, including cancer and psoriasis. Uncovering the underlying mechanisms regulating proliferation is therefore expected to reveal pathologic mechanisms and treatment options. It is thus important to understand the hierarchy/types of progenitor cells
to determine whether there are specific kinds of cells that are amplified or hyperproliferative in response to different stimuli, whether cells are inhibited from differentiating and what molecular pathways are driving the overproliferation.
This is a complex area as many diverse external cues (chemical and mechanical) as well as genetic mutations can impact proliferation. These include endothelial signals, inflammatory signals, and extracellular matrix rigidity to name a few. Many of the major developmental signaling pathways including Wnt, Notch, Yap, and Hedgehog regulate epidermal proliferation in both physiologic and pathogenic states. Major downstream targets include the Ras-MAPK pathway and the PI3K/ AKT/PTEN, which regulate growth and entry into and passage through the cell cycle.
characterized by their mitotic activity, which is necessary for normal epidermal turnover and wound healing
Basal progenitor cells
Basal progenitor cells are most often marked by the expression of ________
keratins 5 and 14
Figure 5-1 Structural markers of epidermal differentiation. Basal cells are defined by basement membrane attachment, the presence of hemidesmosomes, and the expression of keratins 5 and 14. Keratin 1 and 10, in contrast, mark all differentiated cell layers, whereas loricrin and filaggrin are expressed in upper spinous and granular cells. All cells contain desmosomes, though the makeup of desmosomes changes, with desmoglein 3 (Dsg3) and plakophilin 2 (Pkp2) being highly expressed in basal cells whereas Dsg1 and Pkp1 are highly expressed in differentiated epidermis. Tight junctions and keratohyalin granules mark the granular cells.
Keratins are members of the intermediate filament family of proteins and are obligate heterodimers that assemble to form polymers in the cell. 13 This large family of proteins shows tissue and cell type–specific expression (Table 5-1). In the epidermis, the keratin filaments stabilize both cell-cell adhesions called desmosomes and cellsubstratum adhesions called hemidesmosomes. Both of these provide mechanical anchoring roles, and mutations in hemidesmosomal, desmosomal, and keratin genes result in various forms of epidermolysis
bullosa (discussed in detail in Chap. 60; see Table 5-2). In addition to hemidesmosomes, basal keratinocytes are attached to the underlying substratum by focal adhesions, which bind to filamentous actin in the cell and play many roles, including promoting survival and proliferation of keratinocytes. 14 All keratinocytes, including basal keratinocytes, have cell-cell adhesions called adherens junctions, which are actin-based structures. Adherens junctions’ components have diverse functions from regulating cellular architecture to growth control and inflammation.15
Basal keratinocytes give rise to spinous cells during epidermal homeostasis. The production of cells in the suprabasal cell layers is driven by mitotic spindle reorientation and asymmetric cell divisions during embryonic development. 16,17 However, in the adult mouse skin, delamination of basal cells from the underlying basement membrane and their subsequent migration upward appears to predominate. 7 The relative contributions of cell division orientation and delamination is not yet clear in the human epidermis. However, the pathways that control the commitment to differentiation are established in some detail. Here we will first describe characteristics of differentiated cell types followed by the intricate pathways that control differentiation.
SPINOUS CELLS
Basal to spinous differentiation is a highly regulated transition. Cells switch from a mitotic, keratin 5/14expressing type to a postmitotic state characterized by the expression of keratins 1 and 10. There is an upregulation of desmosomes in these cells that gives them a spiny appearance in histologic sections. The composition of desmosomes also changes on differentiation. Although basal cells are high in the desmosomal cadherin, Dsg3, the levels of this protein decrease through differentiation whereas Dsg1 is upregulated. 18 Consistent with this, disruption of Dsg3-based junctions (as occurs in pemphigus vulgaris) results in disruption of cell-cell adhesion between basal cells and between the basal cells and the first layer of spinous cells. Perturbation of Dsg1 (which occurs in pemphigus foliaceus) results in blistering in more superficial layers of the epidermis.
What is the identifying feature of granular cells?
Keratohyalin granules
These granules are composed of keratins, profilaggrin and loricrin, and other proteins that make up the bulk of the cornified envelopes. Filaggrin and loricrin are also commonly used markers for these cell layers. Upon secretion, keratins and loricrin as well as other proteins, including small proline-rich proteins, become highly crosslinked by transglutaminase to the plasma membrane to generate cornified envelopes.
Profilaggrin is eventually metabolized into amino acids and pyrrolidone carboxylic acid and urocanic acid (sometimes referred to as natural moisturizing factor [NMF]) to provide hydration and UV protective functions.
Many of the genes required for formation of the terminal differentiated epidermis are contained in the epidermal differentiation complex, a region on chromosome 1q21. These include loricrin, involucrin, small proline-rich proteins, and late cornified envelope proteins to name a few. The transcriptional regulation of this complex is under tight control and is one of the major targets of the differentiation cascade.
It is also in the granular cells that one barrier to loss of fluids occurs. Tight junctions are cell-cell adhesions that restrict the flow of fluids and ions and act as a barrier to membrane diffusion. These structures form specifically in the granular layer, thus allowing diffusion in the intercellular spaces of live cells within the lower layers of the epidermis. At the core of tight junctions are claudins and genetic disruption of claudin 1 results in lethal barrier defects in mice and neonatal ichthyosis in humans. 19,20 The mechanisms underlying the specific formation of tight junctions in granular cells are not currently known.
The last function of granular cells is to die. The resulting cornified envelopes are not living cells, and thus nuclear and cytoplasmic contents must be eliminated. This is thought to be a modified form of programmed cell death.
Keratin-Based, Inherited Skin Bullous Disease Affecting Primarily the Epidermisa
STRATUM CORNEUM
Cornified envelopes are the products of terminal differentiation in the epidermis. They are acellular and anuclear structures. The core consists of keratins surrounded by highly crosslinked networks of proteins, especially loricrin. Specialized lipids surround these, rich is ceramides that are also crosslinked. The cross-linking is largely contributed by transglutaminases. Transglutaminase expression begins in the spinous layer, but it is inactive there. Both increased calcium and cofactors are thought to specifically activate transglutaminase in the upper granular layer. The crosslinking of proteins and the presence of specialized lipids result in mechanical stability and relative impermeability of the epidermis. While the stratum corneum forms an outside-in barrier, the tight junctions form an inside-out barrier and thus collaborate to form an effective barrier to the external environment.
Give some criteria used to describe differentiation of epidermis
These include, but are not limited to, proliferation rate, expression of transcription factors, keratins and other structural proteins, and ultrastructural analyses.
The structural changes that accompany differentiation are highly regulated by a cascade of pathways, including signaling pathways, transcriptional control, epigenetic factors, and posttranscriptional regulation. Though complex, we present some of the major players below that highlight the roles of these various pathways in generating the differentiated structures that are necessary for epidermal barrier formation.
controls epidermal specification, but it also plays an important role in maintaining basal cell fate/ proliferation and induction of differentiation.
p63
Direct targets of p63 include both structural proteins and other transcription factors that regulate differentiation. One of these transcription factors, ZNF750, is in turn required for expression of another transcription factor Klf4, which plays a major role in expression of granular genes. 22 Klf4 upregulates the expression of a number of lipid-modifying enzymes and structural proteins important for cornified envelope production. 23,24 p63 also acts by controlling the epigenetic status of the genome, discussed below.
commitment factor for transition of basal cells to spinous cells
Notch signaling
It is absent in basal cells and activated in spinous cells. Inhibiting Notch signaling prevents many aspects of spinous cell fate, whereas activating it in basal cells is sufficient for some aspects of spinous differentiation. Although some direct targets of Notch are known, such as Hes1, a direct pathway to spinous gene expression is unclear. In some contexts, Notch collaborates with transcription factors of the AP-2 gene family to function. For example, Notch induces the expression of a transcription factor, C/EBP, in spinous cells that binds along with AP-2 factors to the promoter of keratin 10, regulating its expression.
Transcription factors of the Grainyhead-like (GRHL) are also key promoters of epidermal differentiation. Most prominently, GRHL3 is required for efficient barrier formation, in part through regulating the expression of transglutaminase-1.30
Some major regulators of epidermal differentiation are highlighted in Fig. 5-2, which shows their general expression profiles and sites of action.
control the organization and/ or accessibility of chromatin for transcriptional regulation
Epigenetic regulators
In the epidermis, differentiation is driven by coincident expression of transcription factors and chromatin states.
DNA METHYLATION
Methylated DNA is usually associated with a repressive transcriptional environment. In the epidermis, this is important for suppressing expression of differentiation genes in the basal layer. Loss of the methyltransferase DNMT1 results in loss of progenitor function and premature expression of differentiation-associated genes.
Regulators of epidermal differentiation
Figure 5-2 Regulators of epidermal differentiation. Transcription factors and epigenetic regulators of epidermal proliferation and differentiation are indicated. The position of the labels corresponds to the area of the epidermis where the factor is first expressed. Although direct interactions and regulations are known between some of these factors, this is both complex and incompletely understood. The text provides additional insight into cross-regulation and structural target genes.
