Epithelial Stem Cell Maintenance Research Articles

Tcf proteins are deeply rooted in skin

Adult mammalian tissues are maintained by multipotent stem cells, many of which are highly responsive to soluble Wnt proteins. A new study reports the requirement of two Tcf family members, Tcf3 and Tcf4, in the development and maintenance of epithelial stem cells in skin through Wnt-dependent and -independent processes.

Conformational stability and activity of p73 require a second helix in the tetramerization domain

p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional alpha-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family.

SnapShot: Ca 2+-Calcineurin-NFATSignaling

Ca2+-calcineurin-NFAT signaling transmits signals to the nucleus from a wide variety of receptors and is required for developmental events as diverse as axon outgrowth, cardiac morphogenesis, lung morphogenesis, neural crest diversification, epithelial stem cell maintenance, and immune responses. The pathway appears to be particularly dedicated to vertebrate-specific morphogenic events, perhaps reflecting its first evolutionary appearance in vertebrates. Calcineurin-NFAT signaling is the target of the drugs cyclosporin A and FK506 and several viral immune modulators. In addition, its malfunction is implicated in Down’s syndrome, diabetes, and cardiac hypertrophy. Receptors that induce the entry of Ca2+ into the cell resulting in the activation of calcineurin phosphatase are shown in the lower left. Ca2+ binds to the regulatory subunit of calcineurin as well as to calmodulin to activate the phosphatase activity of calcineurin (Stemmer and Klee, 1994). The immunosuppressive drugs FK506 and cyclosporin A inhibit calcineurin by binding first to the abundant intracellular proteins, FKBP and cyclophilin, respectively. These protein complexes then bind to calcineurin, preventing substrate access (Liu et al., 1991). The specificity of these calcineurin inhibitors is due to the large composite surface arising from FKBP bound to FK506 (see inset structure) (Griffith et al., 1995) or cyclosporin A bound to cyclophilin. FK506 and cyclosporin A are highly specific probes of the NFAT signaling pathway in tissues where the concentration of calcineurin is less than the concentration of FKBP or cyclophilin (Graef et al., 2001). Calcineurin removes several phosphate residues from the N terminus of NFATc proteins, the Ca2+-calcineurin-sensitive subunits of NFAT transcription complexes. Removal of the phosphates exposes nuclear localization sequences in NFATc proteins leading to their rapid entry into the nucleus (Beals et al., 1997a). Once in the nucleus, the NFATc proteins assemble on DNA with partner proteins generically termed NFATn (lower right) that are often the endpoints of other signaling pathways (Crabtree and Olson, 2002). In most cases, NFAT-dependent transcription requires that the Ca2+ signaling be coincident with MAP kinase signaling, providing a mechanism for signal integration and coincidence detection. The targets of NFAT signaling (panel, upper right) are largely cytokines, growth factors and their receptors, proteins involved in cell-cell interactions, as well as many microRNAs. Of particular importance are positive feedback loops initiated by direct binding of NFATc1 and NFATc4 to their promoters as well as the regulation of Ca2+ channels, such as the IP3 receptor, responsible for the release of intracellular Ca2+ stores (Genazzani et al., 1999). This positive feedback loop appears to be important in committing cells to specific fates. A negative feedback loop mediated by the NFAT-dependent activation of RCAN (DSCR1) gene expression appears to constrain the pathway (Arron et al., 2006). NFATc proteins are actively removed from the nucleus by first priming with Dyrk1a or protein kinase A (PKA) followed by phosphorylation by GSK3 (Arron et al., 2006; Beals et al., 1997b; Gwack et al., 2006). The inhibition of GSK3 by AKT and PI3 kinase (PI3K) signaling increases the level of NFATc proteins in the nucleus and hence provides a second means of coincidence detection and signal integration. Many of the phenotypes of Down’s syndrome are thought to be due to the duplication of the DSCR and Dyrk1a genes on chromosome 21. Increased gene dosage both reduces NFATc import into the nucleus and also facilitates export leading to a reduction in NFATc function and compromised positive feedback (Arron et al., 2006).

Rescue of key features of the p63-null epithelial phenotype by inactivation of Ink4a and Arf

Mice lacking p63 cannot form skin, exhibit craniofacial and skeletal defects, and die soon after birth. The p63 gene regulates a complex network of target genes, and disruption of p63 has been shown to affect the maintenance of epithelial stem cells, the differentiation of keratinocytes, and the preservation of the adhesive properties of stratified epithelium. Here, we show that inactivation of p63 in mice is accompanied by aberrantly increased expression of the Ink4a and Arf tumour suppressor genes. In turn, anomalies of the p63-null mouse affecting the skin and skeleton are partially ameliorated in mice lacking either Ink4a or Arf. Rescue of epithelialization is accompanied by restoration of keratinocyte proliferative capacity both in vivo and in vitro and by expression of markers of squamous differentiation. Thus, in the absence of p63, abnormal upregulation of Ink4a and Arf is incompatible with skin development.

Tissue Engineering for Damaged Surface and Lining Epithelia: Stem Cells, Current Clinical Applications, and Available Engineered Tissues

Tissue engineering is an important tool for the treatment of damaged surface and lining epithelia. A source of cells and biocompatible substrates upon which cells can grow and differentiate are key components of this technology. Cultured normal human epithelial cells reconstitute sheets of stratified epithelia that retain biochemical and histological characteristics as well as specific differentiation features of the original donor site. Maintenance of epithelial stem cells in culture and a well-prepared receiving wound bed allow to permanently regenerate full-thickness wounds by means of in vitro reconstituted epithelia. Further, cultured cells produce growth factors and extracellular matrix (ECM) components that help resident cells to contribute to the wound-healing process. Biological matrices enhance the performance of the in vitro reconstituted epithelia. Owing to their similarity to the ECM, natural polymers offer the advantage of being similar to macromolecules that the human environment is prepared to recognize. They also maintain biological information and physical and chemical features that are instructive for cells used to populate them. This article discusses the developments of tissues engineered for cutaneous and mucosal regeneration. Native tissues and their stem cells are also considered, to enhance understanding of the extensive field of tissue reconstruction.

P63/p51-induced Onset of Keratinocyte Differentiation via the c-Jun N-terminal Kinase Pathway Is Counteracted by Keratinocyte Growth Factor

p63/p51, a homolog of the tumor suppressor protein p53, is chiefly expressed in epithelial tissues, including the epidermis. p63 affects cell death similar to p53, and also plays important roles in the development of epithelial tissues and the maintenance of epithelial stem cells. Because it remains unclear how p63 regulates epithelial cell differentiation, we examined the function(s) of p63 in keratinocyte differentiation through the use of a keratinocyte culture system. DeltaNp63alpha (DeltaNp51B), a p63 isoform specifically expressed in basal keratinocytes, suppressed the differentiation of specific late-stage proteins, such as filaggrin and loricrin. In contrast, DeltaNp63alpha induced keratin 1 (K1), which is expressed at the start of differentiation, via c-Jun N-terminal kinase (JNK)/AP-1 activation. However, p63 did not induce K1 expression in the basal layer in vivo, although basal keratinocytes had high levels of p63. This discrepancy was explained by the suppression of K1 expression by dermis-secreted keratinocyte growth factor. This suppression occurred via extracellular signal-related kinase (ERK) signaling, and counteracted the p63-mediated induction of K1. Thus, a precise balance between p63 and keratinocyte growth factor mediates the onset of epithelial cell differentiation, through JNK and ERK signaling. These data may provide mechanistic explanations for the pathological features of skin diseases, including psoriasis.

BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties

Hair follicle (HF) formation is initiated when epithelial stem cells receive cues from specialized mesenchymal dermal papilla (DP) cells. In culture, DP cells lose their HF-inducing properties, but during hair growth in vivo, they reside within the HF bulb and instruct surrounding epithelial progenitors to orchestrate the complex hair differentiation program. To gain insights into the molecular program that maintains DP cell fate, we previously purified DP cells and four neighboring populations and defined their cell-type-specific molecular signatures. Here, we exploit this information to show that the bulb microenvironment is rich in bone morphogenetic proteins (BMPs) that act on DP cells to maintain key signature features in vitro and hair-inducing activity in vivo. By employing a novel in vitro/in vivo hybrid knockout assay, we ablate BMP receptor 1a in purified DP cells. When DPs cannot receive BMP signals, they lose signature characteristics in vitro and fail to generate HFs when engrafted with epithelial stem cells in vivo. These results reveal that BMP signaling, in addition to its key role in epithelial stem cell maintenance and progenitor cell differentiation, is essential for DP cell function, and suggest that it is a critical feature of the complex epithelial-mesenchymal cross-talk necessary to make hair.

WNT signaling in the normal intestine and colorectal cancer

The intestinal epithelium is a self-renewing tissue that represents a unique model for studying interconnected cellular processes such as proliferation, differentiation, cell migration and carcinogenesis. This review covers work from the past decade and highlights the importance of the canonical Wnt pathway in regulating multiple aspects of intestinal homeostasis. Numerous in vivo studies combined with gene profiling experiments have shown that Wnt signaling promotes maintenance of epithelial stem cells and early progenitors by driving transcription of genes associated with proliferation. These studies also revealed strong similarities between the genetic program initiated by Wnt signals in normal crypt progenitors and in colorectal cancer cells. More recently it has become apparent that Wnts do not act alone but rather cooperate with Notch signals in maintaining progenitor cell populations. Processes associated with differentiated epithelial cells also appear to be regulated by Wnt signals. For instance, Paneth cells employ active Wnt signals for terminal differentiation. Moreover, through transcriptional regulation of members of the Eph and Ephrin families, Wnt signaling promotes compartmentalization of epithelial cells along the crypt-villus axis. The Eph/Ephrin system also operates to limit progression of colorectal cancer beyond the early stages.

Lhx2—decisive role in epithelial stem cell maintenance, or just the “tip of the iceberg”?

Stem cell self renewal, maintenance and differentiation are influenced by the convergence of intrinsic cellular signals and extrinsic microenvironmental cues from the surrounding stem cell niche. However, the specific signals involved are often still poorly understood. This is also true for skin epithelial stem cells. Recently, by transcriptionally profiling of embryonic hair progenitors in mice, Rhee et al. have managed to define how murine hair follicle epithelial stem cells are specified and maintained in an undifferentiated state. These authors have identified Lhx2 as a transcription factor functionally positioned downstream of signals necessary to specify hair follicle stem cells such as p63 or NFkappaB, but upstream of signals like Wnt/beta-catenin, Bmp or Shh that are required to drive activated stem cells via the production of transient amplifying cells into terminal differentiation.

Deregulated expression of c-Myc depletes epidermal stem cells.

The beta-catenin/TCF signaling pathway is essential for the maintenance of epithelial stem cells in the small intestine. c-Myc a downstream target of beta-catenin/TCF (ref. 2), can induce differentiation of epidermal stem cells in vitro. To determine the role of c-Myc in epidermal stem cells in vivo, we have targeted expression of human MYC2 to the hair follicles and the basal layer of mouse epidermis using a keratin 14 vector (K14.MYC2). Adult K14.MYC2 mice gradually lose their hair and develop spontaneous ulcerated lesions due to a severe impairment in wound healing; their keratinocytes show impaired migration in response to wounding. The expression of beta1 integrin, which is preferentially expressed in epidermal stem cells is unusually low in the epidermis of K14.MYC2 mice. Label-retaining analysis to identify epidermal stem cells reveals a 75% reduction in the number of stem cells in 3-month-old K14.MYC2 mice, compared with wildtype mice. We conclude that deregulated expression of c-Myc in stem cells reduces beta1 integrin expression, which is essential to both keratinocyte migration and stem cell maintenance.

P73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours.

p73 (ref. 1) has high homology with the tumour suppressor p53 (refs 2-4), as well as with p63, a gene implicated in the maintenance of epithelial stem cells. Despite the localization of the p73 gene to chromosome 1p36.3, a region of frequent aberration in a wide range of human cancers, and the ability of p73 to transactivate p53 target genes, it is unclear whether p73 functions as a tumour suppressor. Here we show that mice functionally deficient for all p73 isoforms exhibit profound defects, including hippocampal dysgenesis, hydrocephalus, chronic infections and inflammation, as well as abnormalities in pheromone sensory pathways. In contrast to p53-deficient mice, however, those lacking p73 show no increased susceptibility to spontaneous tumorigenesis. We report the mechanistic basis of the hippocampal dysgenesis and the loss of pheromone responses, and show that new, potentially dominant-negative, p73 variants are the predominant expression products of this gene in developing and adult tissues. Our data suggest that there is a marked divergence in the physiological functions of the p53 family members, and reveal unique roles for p73 in neurogenesis, sensory pathways and homeostatic control.