p21WAF1/Cip1 Suppresses Keratinocyte Differentiation Independently of the Cell Cycle through Transcriptional Up-regulation of the IGF-I Gene

Abstract

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...

Various peroxisome proliferator‐activated receptor (PPAR)‐γ agonists differently induce differentiation of cultured human keratinocytes

“…This discrepancy is probably due to more potent structure-specific PPAR-independent activity ('off-target' effects) of ciglitazone and troglitazone than rosiglitazone and pioglitazone (15). Previous studies suggested that such strong off-target effects can reduce cornification marker expression in keratinocytes during later stages of differentiation via p63-mediated sustained p21 expression (16)(17)(18)(19)(20)(21).…”

Crosstalk between androgen receptor and epidermal growth factor receptor-signalling pathways: a molecular switch for epithelial cell differentiation

“…Cip1 was originally known as a mediator of p53-induced growth arrest; however, there is increasing evidence that, depending on the tissue, this protein is rather involved in cell proliferation, as has been shown in keratinocytes (Devgan et al 2006) and in intestinal epithelial cells using p27 kip1 and p21 Cip1 conditional knockout mice (Stehr et al 2005). The association of b-catenin within cell-cell junctions in differentiated cells ongoing conformational changes (Manin et al 2000) may explain the reduction of functionally competent p53 through an ubiquitin-mediated proteolysis, since b-catenin can no longer interact with p53 and interfere with the normal proteasomal degradation (Damalas et al 1999).…”

Fos cooperation with PTEN loss elicits keratoacanthoma not carcinoma, owing to p53/p21WAF-induced differentiation triggered by GSK3β inactivation and reduced AKT activity

“…Analysis of p21 WAF was doubly attractive, as p21 WAF possesses roles in keratinocyte differentiation (Topley et al, 1999) separate to that of cell cycle regulation (Devgan et al, 2006) and can be an early response to PTEN loss (Yoo et al, 2006). All HK1.fos/Δ5Pten flx KAs exhibiting P-GSK3β hyper-inactivation and high p53 expression also exhibited novel high p21 WAF expression levels (Fig.…”

“…Given the roles for p21 WAF in epidermal differentiation (Topley et al, 1999), this basal layer expression of p21 WAF would be consistent with the premature commitment of HK1.fos/ΔPten flx keratinocytes to terminal differentiation, as indicated by novel basal layer K1 expression (above). The confused atypical nature of epidermal differentiation, however, may be due to continued, p21 WAF expression in the suprabasal/granular layers when normally p21 expression shuts down (Devgan et al, 2006).…”

“…Importantly, this study found that in the context of HK1.fos/Δ5Pten flx benign tumours, significant reexpression of p53 and p21 WAF , previously lost in control phenotypes, now inhibited further malignant progression. This compensatory p53/p21 WAF expression profile was triggered by increasing levels of GSK3β inactivation (Ghosh and Altieri, 2005), which inhibited P-AKT activity in basal layer keratinocytes (Lei et al, 2006) to reduce proliferation, as indicated by BrdU labelling, and initiate p21 WAF -mediated differentiation (Devgan et al, 2006;Topley et al, 1999;Zhou et al, 2001) that is associated with novel basal layer expression of keratin K1, and with premature loricrin and filaggrin expression. This potential sentinel mechanism, which is deployed at the benign tumour stage and crucially dependent on normal p53 and p21 WAF functions, was able to block malignant progression by continually switching keratinocyte hyperproliferation into differentiation, resulting in the hallmark keratosis of KA.…”