SummaryTransient activation of β-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours

Using K14deltaNbeta-cateninER transgenic mice, we show that short-term, low-level beta-catenin activation stimulates de novo hair follicle formation from sebaceous glands and interfollicular epidermis, while only sustained, high-level activation induces new follicles from preexisting follicles. The Hedgehog pathway is upregulated by beta-catenin activation, and inhibition of Hedgehog signaling converts the low beta-catenin phenotype to wild-type epidermis and the high phenotype to low. beta-catenin-induced follicles contain clonogenic keratinocytes that express bulge markers; the follicles induce dermal papillae and provide a niche for melanocytes, and they undergo 4OHT-dependent cycles of growth and regression. New follicles induced in interfollicular epidermis are derived from that cellular compartment and not through bulge stem cell migration or division. These results demonstrate the remarkable capacity of adult epidermis to be reprogrammed by titrating beta-catenin and Hedgehog signal strength and establish that cells from interfollicular epidermis can acquire certain characteristics of bulge stem cells.

Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear β-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector β-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the selfrenewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve the therapeutic outcome.

Loss-of-function mutations in Whn (Hfh 11), a winged-helix/forkhead transcription factor, result in the nude mouse phenotype. To determine the whn expression pattern during development, we utilized mice in which a beta-galactosidase reporter gene was placed under the control of the wild-type whn promoter by homologous recombination (M. Nehls et al., 1996, Science 272, 886-889). Sites of reporter expression were confirmed by immunohistochemical staining for Whn protein or by in situ hybridization for whn mRNA. At all developmental stages, whn expression is restricted to epithelial cells. In addition to the skin and thymus, whn is expressed in the developing nails, nasal passages, tongue, palate, and teeth. In embryonic epidermis, suprabasal cells induce whn expression at the same time that terminal differentiation markers first appear. As the epidermis matures, whn promoter activity is found primarily in the first suprabasal layer, which contains keratinocytes in the early stages of terminal differentiation. In developing and mature anagen hair follicles, whn is expressed at high levels in the postmitotic precursor cells of the hair shaft and inner root sheath. Though principally associated with terminal differentiation, whn expression is also detected in progenitor cell compartments; in the hair bulb matrix and basal epidermal layer, a small subclass of cells expresses whn, while in the outer root sheath, whn promoter activity is induced as the follicle completes its elongation. Within these compartments, rare cells exhibit both whn expression and the nuclear proliferation marker Ki-67. The results suggest that whn expression encompasses the transition from a proliferative to a postmitotic state and that whn regulates the initiation of terminal differentiation.

The recent discovery of the human coun-terpart of the hairlessmousephenotype1has helped our understandingof the molecular genetics of hair growth.But there are no reports of a defect in thehuman homologue of the best known of the'bald' mouse phenotypes, the nudemouse2.This may be because affected individualsare so gravely ill from the accompanyingimmunodeficiency that their baldness goesunnoticed. We have carried out a geneticanalysis that reveals a human homologue ofthe nudemouse.The nudemouse is characterized by acongenital absence of hair and a severeimmunodeficiency2, resulting from muta-tions in the whn(winged-helix-nude;Hfh11nu) gene, which encodes a member ofthe forkhead/winged-helix transcriptionfactor family with restricted expression inthymus and skin3. The simultaneous occur-rence of severe functional T-cell immunodeficiency, congenital alopecia and nail dys-trophy (MIM database no. 601705) in twoaffected sisters led to the recognition thatthe clinical phenotype was reminiscent ofthe nudemouse4. We therefore investigatedwhether this syndrome represents thehuman counterpart of the nudemousephenotype.We obtained DNA samples from mem-bers of the sisters' family in a small villagein southern Italy. The affected sisters wereborn with a complete absence of scalp hair (Fig. 1a), eyebrows and eyelashes and haddystrophic nails, and no thymic shadow wasevident upon X-ray examination. The firstaffected child revealed a striking impair-ment of T-cell function shortly after birth,and died at the age of 12 months. Her sisterhad similar immunological abnormalities,but bonemarrow transplantation at fivemonths of age led to full immunologicalreconstitution, although the alopecia andnail dystrophy are still present4.We performed linkage analysis usingmicrosatellite markers near the humanWHNlocus on chromosome 17, and founda lod score of 1.32, suggestive of linkage. Wethen sequenced the human WHNgene5andfound a homozygous C-to-T transition atnucleotide position 792 of the WHNcDNA(GenBank accession no. Y11739) (Fig. 1b).This leads to a nonsense mutation atresidue 255 (R255X) in exon 5, and predictsthe complete absence of functional proteinas a result of nonsense-mediated decay ofmessenger RNA.Because the proband's bonemarrowtransplant was from her brother, we exam-ined her leukocyte DNA both before andafter the graft for the presence of chi-maerism. Genotyping the proband beforethe transplant showed that her leukocyteDNA was homozygous only for the mutantallele (Fig. 1c). Four years after the transplant, we detected the haplotype specific forthe wild-type paternal WHNallele receivedfrom the brother, as well as the mutantallele, indicative of chimaerism. Genderdetermination revealed that the proband'sleukocyte DNA was genotypically XXbefore the transplant, and the brother'sDNA was XY. Afterwards, the proband'sleukocyte DNA was found to be XY (Fig.1c), providing evidence of longtermengraftment and expansion of the bone-marrow graft.The WHNgene encodes a transcriptionfactor, which is developmentally regulatedand directs cel...

The cyclin-dependent kinase inhibitor p21 is induced in several in vitro terminal differentiation systems as well as in differentiating tissues in vivo. To determine the mechanism responsible for p21 induction during differentiation of mouse primary keratinocytes, we performed a deletion analysis of the p21 promoter. The minimal region of the p21 promoter required for its induction in keratinocyte differentiation consists of a contiguous stretch of 78 base pairs, which contains a GC-rich region as well as the TATA box. We determined that transcription factors Sp1 and Sp3, present in primary keratinocyte nuclear extracts, bind the GC region concomitantly. Expression studies established that both Sp1 and Sp3 activate the p21 promoter, but showed that only Sp3 overexpression enhances promoter inducibility during differentiation. Furthermore, disruption of the GC-rich region dramatically decreases transcription factor binding as well as promoter activity and inducibility upon differentiation. The overexpression of either Sp1 or Sp3 restores the basal activity of the disrupted promoter, but only Sp3 can restore its inducibility. These findings show that both Sp1 and Sp3 can contribute to the basal activity of the p21 promoter, and establish Sp3 as a specific transcription factor involved in the induction of p21 promoter during keratinocyte differentiation.Induction of terminal differentiation is a complex process involving the regulation of many genes, including those directly responsible for cell cycle withdrawal and growth arrest. The activity of the cyclin-dependent kinases (CDKs) 1 is tightly regulated during passage through the cell cycle by cyclin association, subunit phosphorylation, and interaction with CDK inhibitors (1, 2). Induction of the CDK inhibitor p21 occurs in several in vitro terminal differentiation systems (3), as well as in differentiating tissues in vivo (4, 5), suggesting that this molecule plays an important role in regulating terminal differentiation-associated growth arrest.Control of p21 induction upon differentiation is of great interest, as it may involve mechanisms common to most cell types and others that are cell type-specific. In all the differentiation systems analyzed so far the induction of p21 occurs by p53-independent mechanisms (5-9). During differentiation of myoblasts to myotubes, p21 up-regulation has been reported to be under the control of MyoD (6), although other myogenic regulatory factors may also be involved, at least in MyoD knockout mice (5). In differentiating primary keratinocytes, the induction of p21 depends on the function of the nuclear phosphoprotein p300 (9), a novel transcriptional coactivator, which is thought to act as a bridge between specific transcription factors and the basal transcription complex. Little else is known about the regulation of p21 expression during normal differentiation.Recent data indicate that the GC-rich region of the p21 promoter, located next to the TATA box, binds the Sp1 transcription factor and can exert important regulatory ...

Nude mice are characterized by the absence of visible hair, epidermal defects, and the failure to develop a thymus. This phenotype results from loss-of-function mutations in Whn (Hfh11), a winged-helix transcription factor. In murine epidermis and hair follicles, endogenous whn expression is induced as epithelial cells initiate terminal differentiation. Using the promoter for the differentiation marker involucrin, transgenic mice that ectopically express whn in stratified squamous epithelia, hair follicles, and the transitional epithelium of the urinary tract were generated. Transgenic epidermis and hair follicles displayed impaired terminal differentiation and a subset of hair defects, such as delayed growth, a waved coat, and curly whiskers, correlated with decreased transforming growth factor (TGF)-alpha expression. The exogenous Whn protein also stimulated epithelial cell multiplication. In the epidermis, basal keratinocytes exhibited hyperproliferation, though transgene expression was restricted to suprabasal, postmitotic cells. Hair follicles failed to enter telogen (a resting period) and remained continuously in an abnormal anagen (the growth phase of the hair cycle). Ureter epithelium developed severe hyperplasia, leading to the obstruction of urine outflow and death from hydronephrosis. Though an immune infiltrate was present occasionally in transgenic skin, the infiltrate was not the primary cause of the epithelial hyperproliferation, as the immune reaction was not observed in all affected transgenics, and the transgene induced identical skin and urinary tract abnormalities in immunodeficient Rag1-null mice. Given the effects of the transgene on cell proliferation and TGFalpha expression, the results suggest that Whn modulates growth factor production by differentiating epithelial cells, thereby regulating the balance between proliferative and postmitotic populations in self-renewing epithelia.

The forkhead transcription factor FOXN1 is required for normal cutaneous and thymic epithelial development. Mutations in FOXN1 give rise to the nude phenotype in mice, rats and man. However, the genes that are regulated by FOXN1 are unknown. To investigate FOXN1 function we expressed an inducible form of the protein, FOXN1ER, that is activated by 4-hydroxytamoxifen in primary human epidermal keratinocytes. Transient activation of FOXN1 decreased the proportion of keratinocytes that formed actively growing clones attributable to stem cell founders and increased the number of abortive clones, without inducing apoptosis. Within 24 hours the majority of cells had initiated terminal differentiation, as assessed by involucrin expression. We performed a cDNA microarray experiment to analyse changes in the transcription of approximately 6000 genes. Following FOXN1 activation we detected increases of two fold or greater in the RNA levels of over 30 genes. Genes promoting growth arrest, survival and differentiation featured prominently and markers of early events in keratinocyte differentiation were also detected. Since one of the induced genes was Akt we investigated whether Akt played a role in terminal differentiation. Activation of PI 3-kinase but not Akt was necessary for FOXN1-induced differentiation. In reconstituted epidermis FOXN1 promoted early stages of terminal differentiation whereas Akt activation was sufficient to induce late stages, including formation of the cornified layers. These results establish a role for FOXN1 in initiation of terminal differentiation and implicate Akt in subsequent events.