Notch signaling promotes commitment of keratinocytes to differentiation and suppresses tumorigenesis. p63, a p53 family member, has been implicated in establishment of the keratinocyte cell fate and/or maintenance of epithelial self-renewal. Here we show that p63 expression is suppressed by Notch1 activation in both mouse and human keratinocytes through a mechanism independent of cell cycle withdrawal and requiring down-modulation of selected interferon-responsive genes, including IRF7 and/or IRF3. In turn, elevated p63 expression counteracts the ability of Notch1 to restrict growth and promote differentiation. p63 functions as a selective modulator of Notch1-dependent transcription and function, with the Hes-1 gene as one of its direct negative targets. Thus, a complex cross-talk between Notch and p63 is involved in the balance between keratinocyte self-renewal and differentiation. Normal tissue homeostasis is determined by a complex interplay between developmental signals and other cell regulatory pathways. Notch cell surface receptors and their ligands belonging to the Delta and Serrate/Jagged families play a crucial role in cell fate determination and differentiation, functioning in a cell-and context-specific manner (Artavanis-Tsakonas et al. 1999). In mammalian cells, Notch activation is generally thought to maintain stem cell potential and inhibit differentiation, thereby promoting carcinogenesis (Artavanis-Tsakonas et al. 1999). However, in specific cell types such as keratinocytes, increased Notch activity causes exit from the cell cycle and commitment to differentiation (Lowell et al. 2000;Rangarajan et al. 2001;Nickoloff et al. 2002), whereas down-modulation or loss of Notch1 function promotes carcinogenesis (Talora et al. 2002;Nicolas et al. 2003).In the human epidermis, localized expression of the Notch-ligand Delta in putative "stem cells" has been proposed to induce commitment of neighboring Notch1-expressing keratinocytes to a "transit-amplifying" phenotype, through a negative feedback mechanism of lateral inhibition (Lowell et al. 2000). On the other hand, in both mouse and human epidermis, Jagged 1/2, Notch1, and Notch2 are coexpressed in differentiating keratinocytes of the supra-basal layers, consistent with a positive feedback loop between these molecules that serves to reinforce and synchronize Notch activation with differentiation (Luo et al. 1997;Rangarajan et al. 2001;Nickoloff et al. 2002).The best characterized "canonical" pathway of Notch activation involves proteolytic cleavage and translocation of the cytoplasmic domain of the receptor to the nucleus, where it associates with the DNA-binding protein RBP-J (CBF-1, CSL), converting it from a repressor

Little is known about the regulation and function of the Notch1 gene in negative control of human tumors.Here we show that Notch1 gene expression and activity are substantially down-modulated in keratinocyte cancer cell lines and tumors, with expression of this gene being under p53 control in these cells. Genetic suppression of Notch signaling in primary human keratinocytes is sufficient, together with activated ras, to cause aggressive squamous cell carcinoma formation. Similar tumor-promoting effects are also caused by in vivo treatment of mice, grafted with keratinocytes expressing oncogenic ras alone, with a pharmacological inhibitor of endogenous Notch signaling. These effects are linked with a lesser commitment of keratinocytes to differentiation, an expansion of stem cell populations, and a mechanism involving up-regulation of ROCK1/2 and MRCK␣ kinases, two key effectors of small Rho GTPases previously implicated in neoplastic progression. Thus, the Notch1 gene is a p53 target with a role in human tumor suppression through negative regulation of Rho effectors.[Keywords: Notch; p53; ROCK/MRCK; stem cells; squamous cell carcinoma; in vivo siRNA delivery] Supplemental material is available at http://www.genesdev.org.

In keratinocytes, the cyclin/CDK inhibitor p21 WAF1/Cip1 is a direct transcriptional target of Notch1 activation; loss of either the p21 or Notch1 genes expands stem cell populations and facilitates tumor development. The Notch1 tumor-suppressor function was associated with down-regulation of Wnt signaling. Here, we show that suppression of Wnt signaling by Notch1 activation is mediated, at least in part, by down-modulation of Wnts gene expression. p21 is a negative regulator of Wnts transcription downstream of Notch1 activation, independently of effects on the cell cycle. More specifically, expression of the Wnt4 gene is under negative control of endogenous p21 both in vitro and in vivo. p21 associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone hypoacetylation at this promoter. Thus, p21 acts as a selective negative regulator of transcription and links the Notch and Wnt signaling pathways in keratinocyte growth control.[Keywords: Differentiation; stem cell potential; transcription; chromatin; E2F-1; c-Myc; p300] Supplemental material is available at http://www.genesdev.org.

p21 plays a dual role in keratinocyte growth and differentiation control. It restricts the number of keratinocyte stem cell populations while inhibiting the later stages of differentiation independently of the cell cycle. The molecular/biochemical mechanism for the differentiation suppressive function of p21 is unknown. Here we show that elevated p21 expression leads to activation of MAPK family members in a keratinocyte-specific and cell cycle-independent manner, and up-regulation of MAPK activity can explain the inhibitory effects of p21 on differentiation. p21 induces transcription of several genes with MAPK activation potential. Although several of these genes are induced by p21 in a MAPK-dependent manner, expression of IGF-I is induced upstream of MAPK activation. IGF-I stimulation is by itself sufficient to cause MAPK activation and inhibit differentiation and suppression of IGF-I signaling by knock down of the cognate receptor (IGF-R1), diminishing the ability of p21 to activate MAPK and suppress differentiation. Thus, in keratinocytes, the ability of p21 to suppress differentiation can be explained by cell type-specific activation of the MAPK cascade by transcriptional up-regulation of the IGF-I gene. p21WAF1/Cip1 was originally identified as a mediator of p53-induced growth arrest (1), a direct inhibitor of CDK 2 activity (CKI) (2), and a gene whose expression is induced with cellular senescence (3). This protein belongs to the Cip/Kip family of, p27Kip1 , and p57 Kip2), which share significant sequence homology in their amino-terminal portions and recognize a broad but not identical range of cyclin/CDK targets (4). 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 carboxyl-terminal domain of p21 associates with the proliferating nuclear antigen, a subunit of DNA polymerase ␦, and can inhibit DNA replication directly without affecting DNA repair (4). p21 has also been variously implicated as either a negative or positive regulator of apoptosis with cell type-specific mechanisms that may depend on its effects on the cell cycle or more direct association and modulation of proapoptotic molecules (5-7). Additionally p21 has been implicated in control of transcription through a mechanism that may be coupled to its CKI activity but also direct association and modulation of transcription factors and/or transcription coactivators (8 -11).This complexity of p21 function is well illustrated by its role in keratinocyte self-renewal and differentiation: p21 expression promotes the initial commitment of keratinocyte stem cell populations to differentiation (12) and contributes to differentiation-associated growth arrest (13). However, after an initial increase, at later stages of differentiation the p21 protein is down-modulated both in vitro and in the intact skin in vivo; sustained p21 expression under these conditions suppresses differentiation, suggesting that p21 is part of a negative r...