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Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, USADepartment of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan
We present here a dynamic model of functional equilibrium between keratinocyte stem cells, transit amplifying populations and cells that are reversibly versus irreversibly committed to differentiation. According to this model, the size of keratinocyte stem cell populations can be controlled at multiple levels, including relative late steps in the sequence of events leading to terminal differentiation and by the influences of a heterogeneous extra-cellular environment. We discuss how work in our laboratory, on the interconnection between the cyclin/CDK inhibitor p21WAF1/Cip1 and the Notch1 signaling pathways, provides strong support to this dynamic model of stem cell versus committed and/or differentiated keratinocyte populations.
Emerging evidence indicates that the balance between epithelial cell growth and differentiation involves a complex interplay between classical cell regulatory pathways and development-related signals. Keratinocytes provide an ideal experimental system to dissect the extra and intracellular signaling pathways involved in growth/differentiation control of epithelial cells, under normal conditions and during tumor development (
). Within the proliferative compartment of the epidermis, at least two kinds of keratinocyte populations are thought to exist: multipotent “stem cells”, slow cycling but with an indefinite self renewal potential and capable of generating all other types of growing and differentiating keratinocytes; and “transient amplifying cells”, actively proliferating but capable of a limited number of cell divisions and already committed towards differentiation (
An attractive model, depicted in Figure 1, is that there is a dynamic equilibrium between keratinocyte stem cells, transit amplifying populations and cells that are reversibly versus irreversibly committed to differentiation. According to this model, the size of stem cell populations is likely to be influenced by the number of surrounding transit amplifying cells that are in turn in equilibrium with cells that have withdrawn reversibly versus irreversibly from the cell cycle. The rate of conversion from one cell population to the next is likely to be further controlled by the surrounding cellular environment, i.e. the stroma and associated blood supply. Such cellular environment is likely to be non-homogeneously distributed, but organized along discontinuous signaling centers that are more (niches) or less permissive for stem cell populations. It is therefore likely that the number of keratinocyte stem cells can be controlled through mechanisms acting at multiple levels, including (A) relative late steps in the sequence of events leading to terminal differentiation; (B) influences of the surrounding cellular and extracellular environment. Here we will discuss how work in our laboratory, on the interconnection between the cyclin/CDK inhibitor p21WAF1/Cip1 and the Notch1 signaling pathway, which provides strong support to the dynamic model illustrated above.
Results and Discussion
p21WAF1/Cip1 as a determinant of the balance among keratinocyte stem cells, transit amplifying and irreversibly differentiated populations
p21WAF1/Cip1 was originally identified as a mediator of p53-induced growth arrest (
). This protein belongs to the Cip/Kip family of CKI (p21WAF1/Cip1, p27Kip1, p57Kip2), which share significant sequence homology in their amino-terminal portions and recognize a broad, but not identical range of cyclin/CDK targets (
). The amino terminal domain of p21, like the corresponding domains of p27 or p57, is both necessary and sufficient to inhibit cyclin/CDK activity in vitro and in vivo. The unique carboxy-terminal domain of p21 associates with the proliferating nuclear antigen (PCNA), a subunit of DNA polymerase δ and can inhibit DNA replication directly, without affecting DNA repair (
), besides associating with cyclin/CDKs and PCNA, p21 participates in a number of other specific protein–protein interactions. Some of these interactions still bear on cell cycle control, whereas others point to separate functions of p21, as a modulator of apoptosis or transcription.
In keratinocytes, like in other cell types, increased p21 expression plays an important role in cell cycle withdrawal which is associated with differentiation (
). In contrast to the transition between growth and growth arrest of these “transit amplifying” populations, we have found that p21 is likely to play an essential role in restricting the number of keratinocytes with high growth/differentiation potential (putative stem cells). Several lines of evidence support this conclusion.
Like in other organ systems, keratinocyte stem cells are only a small fraction of cells in the epidermis, with no unequivocal biochemical markers, so that their analysis is ultimately dependent on functional rather than biochemical assays. Stem cells should have two fundamental properties: (1) an indefinite life span, and (2) the capability to give rise to all other types of partially committed and differentiated cells. The existence of keratinocyte subpopulations with increased growth potential, capable of generating rapidly expanding colonies in culture has been well established (
). When tested in a growth clonogenicity assays, however, p21-/- keratinocyte cultures contain a much higher fraction of keratinocytes with clonogenic properties than their wild type counterpart (0.1% of total proliferating cells, vs <0.005%) (
Keratinocytes in the interfollicular epidermis undergo a vertical differentiation program along a single lineage. By contrast, keratinocyte differentiation in hair follicles proceeds along a minimum of six distinct lineages (
). To take into account this complex program of differentiation, we developed an in vivo skin/hair reconstitution assay with genetically labeled cells. The interfollicular epidermis reconstituted in these assays was composed of well-demarcated columnar units likely to originate from single progenitor cells. By contrast, reconstituted hair follicles were composed of cells of multiple origin, consistent with distinct progenitor cells for the shaft, inner (IRS) and outer root sheath (ORS), respectively (
Exposure of primary keratinocytes to increased extracellular calcium concentrations induces a differentiation program similar to that occurring in vivo, including establishment of adherens junctions and desmosomes, induction of several enzymatic and structural markers of the upper epidermal layers and exit from the cell cycle (
). Importantly, establishment of this differentiation program can be reversed at early times (within 24 h) by simple removal of the calcium pro-differentiation stimulus, and only at later times (2–3 d and beyond) differentiation becomes irreversible. p21 appears also to be a critical determinant of the irreversibility of differentiation, as a significant fraction of primary keratinocytes from p21-/- mice (15%–30%), but not wild-type cells, can resume proliferation even after 3–6 d of exposure to elevated extracellular calcium (
Thus, although not essential for exit of the vast majority of “transit amplifying” keratinocytes from the cell cycle, p21 plays a key role in restricting the number of keratinocyte stem cell populations (as determined by clonogenicity and skin/hair reconstitution assays) as well as, further downstream, determining the irreversibility of differentiation. Thus, p21 may exert these functions by acting at two distinct levels (increasing k1/k2 as well as k5 in Figure 1). On the basis of our dynamic model, however, the same balance of keratinocyte stem cells versus committed populations could be achieved if p21 were to act at a single level (i.e. by simply increasing k5 in Figure 1).
Notch1, a direct upstream regulator of p21 expression, plays a key role in the coordinate control of keratinocyte self renewal and differentiation
The Notch gene family encodes evolutionarily conserved transmembrane receptors with a key role in cell-fate determination and differentiation (
). Notch activation is triggered by interactions with ligands of the Delta and Serrate/Jagged families, and results in release of the intracellular region of the receptor by proteolytic cleavage and nuclear translocation (
). The activated Notch intracellular region binds to a ubiquitous DNA-binding protein of the CSL family (Drosophila Suppressor of hairless (Su(H)) or its mammalian homolog RBP-Jκ/CBF1), converting it from a repressor into an activator of transcription (
Most work has focused on the ability of Notch to direct divergent pathways of differentiation among initially equipotent cells (Figure 3a). One-way Notch and ligands such as Delta act to promote divergent differentiation is termed lateral inhibition, a mechanism first described and characterized in the developing neuroectoderm of Drosophila (
). Notch and its ligands, however, can also be co-expressed in the same cells undergoing coordinate differentiation to a single fate (Figure 3b). Examples of such cells include those in developing mammalian muscle and skin, in which the Serrate-like ligands Jagged 1 and 2 are co-expressed with Notch (
). The relative contribution of negative and positive feedback mechanisms for modulation of Notch signaling appears to be further controlled by ancillary proteins, such as members of the Fringe family that selectively inhibit Notch responsiveness to Serrate-like ligands (
A variety of data suggest that all these mechanisms for regulation of Notch activity operate in mammalian skin. A complex pattern of expression of Notch and its ligands is seen in hair follicles and feathers, consistent with their multiple keratinocyte cell lineages (
). A role for Notch in keratinocyte skin appendages formation and/or differentiation is supported by ectopic expression of Notch or its ligands, with Notch activation in one layer of the hair follicle modulating differentiation and/or lineage commitment in neighboring layers (
). In human interfollicular epidermis, localized expression of Delta in putative “stem cells” has been proposed to induce commitment of neighboring Notch1-expressing cells to a “transit amplifying” phenotype, a scenario reminiscent of lateral inhibition (
), we have found that in newborn mouse epidermis high expression of Jagged 1 and 2 coincide with Notch 1 and 2 in differentiating keratinocytes of the supra-basal layers, with both Jagged 1/2 and Notch1 expression being turned off in the terminally differentiated outermost layers (Rangarajan et al, 2001b). In mouse epidermis around birth, all three forms of Fringe are expressed (
). Of note, the spinous layer is the only region of the epidermis free of Fringe expression, thereby potentially creating a Notch signaling boundary between this layer and cells of the immediately underlying (basal) and overlying (granular) layers (
An essential role of Notch1 in epidermal border formation was demonstrated by the analysis of mice with a conditional keratinocyte-specific deletion of this gene. Loss of Notch1 results in an hyperplastic phenotype of the interfollicular epidermis, with inappropriate co-expression of keratins of the basal and upper epidermal layers, and a striking upregulation of integrin β1 and β4 expression in the supra-basal epidermal layers, where expression of these integrins is usually low or undetectable (
). Additionally, endogenous Notch1 activity is required for induction of p21 expression in differentiation. In fact, in mouse keratinocytes, the p21 gene is a direct transcriptional target of Notch1 activation. p21 expression is induced in these cells by activated Notch1 through RBP-Jκ-dependent transcription, and the RBP-Jκ protein binds directly to the endogenous p21 promoter (
). Importantly, Notch1 activity itself is induced in parallel with p21 expression in the transition between transit amplifying and differentiating cells (Figure 4a). In turn, increased Notch1 activity can also induce differentiation, through a mechanism likely to involve down-regulation of integrin α6/β4 and α3/β1 expression (
) (Figure 4b). Thus, Notch1 can function both upstream and in parallel with p21 in regulating the balance between keratinocyte stem cells and more committed populations. Like p21, Notch1 may exert this function acting at distinct multiple levels (increasing k1/k2 as well as k5 in Figure 1), but the same end result could be achieved if Notch1, like p21, were to act at a single level (i.e. only increasing k5).
Future directions: p21 and Notch1 as keratinocyte tumor suppressors
Keratinocyte stem cells are the putative targets for mouse skin carcinogenesis (
). Consistent with an increased size of stem cell populations, mice with a disruption of the p21 gene exhibit a substantially enhanced susceptibility to chemically induced skin carcinogenesis, with an increase numbers of tumors that are either malignant or benign, depending on protocol and genetic background (
). Strikingly, as for mice lacking the p21WAF1/Cip1 gene, mice with induced deletion of Notch1 exhibit a substantially increased susceptibility to chemically-induced skin carcinogenesis and, as with p21-/- cells, Notch1-/- keratinocytes infected with a retrovirus transducing the ras oncogene and injected subcutaneously into nude mice form aggressive squamous cell carcinomas, whereas Notch1+/+ cells do not (
Although p21 is one of the important downstream targets of Notch function in keratinocytes, Notch signaling is likely to affect other signaling pathways with a key regulatory function in this cell type. In fact, mice with an induced deletion of the Notch1 gene differ in other aspects of their phenotype from p21-/- animals. In particular, mice with the Notch1 deletion develop after 3 mo of age extensive hyperplasia and keratinization of the corneal epithelium that results in opaque plaque formation and blindness. Concurrently, they develop also spontaneous skin tumors in various parts of the body that are highly vascularized and consist of small proliferating basophilic cells similar to those in basal cell carcinomas (BCC) (
). In both hyperplastic epidermis and BCC-like tumors, Notch1 deficiency is associated with increased and sustained expression of Gli2, a downstream target of the sonic hedgehog (SHH) signaling pathway that has been causally linked to BCC tumor formation (
). Additionally, the Wnt/β-catenin pathway appears to be elevated in keratinocytes and tumors as a consequence of loss of Notch1 function, whereas β-catenin signaling can be suppressed by activated Notch1 expression (
) in keratinocytes with elevated Notch1 activity. Importantly, inhibition of the latter pathway has been recently connected with a growth inhibitory function of Notch1 in an important type of human keratinocyte-derived tumors: HPV-induced cervical carcinomas (
Thus, an exciting area of study for the future is the close interconnection among classical cell cycle and development-related pathways in control of keratinocyte stem cell potential, differentiation and tumorigenesis.
Notch signaling: Cell fate control and signal integration in development.