Introduction

The epidermis, the outermost part of the skin, is a stratified and keratinized squamous epithelium mainly composed of keratinocytes, which forms a protective barrier. Epidermal differentiation, which starts in the mouse at embryonic day (E) 12, leads to formation of several distinct cell layers characterized by their ultrastructure, mitotic state and expression of specific molecular markers (Fuchs and Byrne, 1994; Koster and Roop, 2007). The basal layer develops from the surface ectoderm at approximately E9.5 in the mouse. The p63 gene, which specifies different isoforms of a transcription factor related to tumour suppressor p53, controls basal layer formation and maintenance as well as IB kinase (IKK) expression (Candi et al, 2007; Koster et al, 2007). Basal keratinocytes, including epidermal stem cells and transit-amplifying cells, are cuboidal, express cytokeratins (CKs) 5 and 14 and have a high proliferative potential (Koster and Roop, 2007). These cells form the embryonic periderm (M'Boneko and Merker, 1988), which is lost on establishment of the epidermal barrier. At E12 in the mouse, the basal cells give rise to the intermediate cell layer located between the embryonic basal layer and the periderm (Smart, 1970; Weiss and Zelickson, 1975). The intermediate cells divide several times before they withdraw from cell cycle and mature into postmitotic spinous cells (Smart, 1970; Koster and Roop, 2007). By contrast, adult basal keratinocytes directly become spinous cells when terminal differentiation is initiated, without involvement of an intermediate cell type (Koster and Roop, 2007). The spinous layer is characterized by a switch in keratin expression, from CK5 and CK14 to CK1 and CK10 (Fuchs and Green, 1980). Involucrin, a marker of early terminal differentiation is also synthesized in the upper part of this layer. The spinous cells continue their differentiation and maturation to form the granular layer, which is characterized by keratohyalin granules and expression of the late differentiation markers loricrin and filaggrin (Candi et al, 2005). Terminal differentiation gives rise to the cornified layer (stratum corneum), which consists of extremely flat, keratin-filled and anucleated keratinocytes, called corneocytes, which are mummified within a lipid matrix (Candi et al, 2005; Segre, 2006). The stratum corneum is primarily responsible for the barrier function of the skin (Elias, 2004; Segre, 2006), which is established around E17.5 in mouse (Hardman et al, 1998). Although major progress has been made in understanding the molecular changes that characterize epidermal differentiation, less is known about the signalling pathways that control these events. Here, we provide an overview of the current understanding of the central function of IKK in the control of epidermal differentiation, homoeostasis and tumorigenesis.

Defective epidermal morphogenesis in Ikk-deficient mice

The oligomeric IKK complex is composed of two catalytic subunits, IKK and IKK (DiDonato et al, 1997; Mercurio et al, 1997; Woronicz et al, 1997; Zandi et al, 1997), and a regulatory subunit named IKK or NEMO (nuclear factor B (NF-B) essential modulator) (Rothwarf et al, 1998; Yamaoka et al, 1998). This complex is the key mediator of NF-B activation in response to proinflammatory and innate immune challenges (Rothwarf et al, 1998). Although IKK and IKK share considerable sequence identity, it is IKK that usually serves the more critical function in the activation of classical NF-B signalling (Tanaka et al, 1999; Li et al, 1999b, 1999c). To determine the unique functions of IKK, several groups have disrupted the Ikk locus in mice and were surprised to find that it has an essential function in epidermal differentiation and morphogenesis (Hu et al, 1999; Li et al, 1999a; Takeda et al, 1999). Newborn Ikk^–/– mice present with multiple morphological defects, including shiny and translucent skin, absence of erupted whiskers, shortened limbs and truncated snout and tail (Figure 1) (Hu et al, 1999; Takeda et al, 1999; Yoshida et al, 2000). These mice develop to term but die shortly after birth, probably as a consequence of a major skin barrier defect that results in severe dehydration.

The Ikk^–/– epidermis is characterized by the presence of basal and suprabasal layers, both in a highly proliferative state, and complete absence of the granular and the cornified layers (Figure 2A) (Hu et al, 1999; Takeda et al, 1999). At the molecular level, these anomalies are characterized by the expression of CK5 and CK14 as well as proliferating cell markers, such as CK6, PCNA (proliferating cell nuclear antigen) and Ki67 in basal and suprabasal layers (Hu et al, 1999; Takeda et al, 1999). In contrast, CK1 and CK10 are appropriately expressed in the first suprabasal layers of the Ikk^–/– epidermis (Hu et al, 1999; Takeda et al, 1999). Involucrin is also synthesized in the upper part of the suprabasal layer of these mice (Takeda et al, 1999), indicating that an early step in the differentiation process still takes place, although late terminal differentiation markers, including loricrin and filaggrin, are not expressed (Hu et al, 1999; Takeda et al, 1999) (Figure 2B). It was suggested that the highly proliferative suprabasal cells of the Ikk^–/– epidermis, which express CK1 and CK10, are reminiscent of intermediate cells rather than spinous cells (Koster and Roop, 2007). Hence, the most critical function of IKK may be induction of cell cycle exit needed for converting basal and intermediate keratinocytes to spinous cells. When this step fails, all subsequent differentiation states are aborted (Figure 3).

Nuclear IKK controls terminal differentiation of keratinocytes

Control of epidermal proliferation and differentiation by IKK does not involve its protein kinase function and is completely independent from NF-B activation (Hu et al, 2001). Ikk^–/– keratinocytes do not exhibit a primary defect in NF-B activation (Hu et al, 1999; Takeda et al, 1999). In contrast, Ikk^–/– keratinocytes display higher IKK and NF-B activities than wild-type (WT) cells after incubation with either tumour necrosis factor- or interleukin-1 (Hu et al, 2001). The observations that transgenic mice overexpressing a dominant inhibitor of NF-B function (IBM) in the epidermis or mice lacking both RelA and c-Rel display epidermal hyperplasia that does not disrupt terminal differentiation and stratum corneum formation (Seitz et al, 1998; Gugasyan et al, 2004; Zhang et al, 2004), are consistent with the NF-B-independent action of IKK in the epidermis.

As observed in vivo, isolated Ikk^–/– keratinocytes are hyperproliferative and do not respond to differentiation-inducing signals such as confluence or high Ca²⁺ (Hu et al, 1999, 2001). Ikk^–/– keratinocytes, however, do differentiate in vitro when transduced by an adenovirus expressing a 'kinase-dead' form of IKK, indicating that the kinase function is dispensable for keratinocyte differentiation (Hu et al, 2001). Instead, IKK needs to enter the nucleus to induce keratinocyte cell cycle arrest and terminal differentiation (Sil et al, 2004). Nuclear entry depends on a nuclear localization sequence (NLS) within the IKK kinase domain, the disruption of which prevents the induction of keratinocyte differentiation (Sil et al, 2004). An NLS is absent from IKK, which cannot substitute for IKK in keratinocyte differentiation and growth arrest. IKK is also nuclear in basal and suprabasal cells of the epidermis (Descargues et al, 2008) and the oral epithelium (Maeda et al, 2007), findings that are consistent with the cell culture results.

The function of IKK in the keratinocyte nucleus is linked to the production of a yet-to-be identified soluble factor or group of factors termed keratinocyte differentiation-inducing factor (kDIF) that can induce the expression of terminal differentiation markers, even in Ikk^–/– cells (Hu et al, 2001). Consistent with the existence of kDIF, transplantation of Ikk^–/– skin onto the back of immuodeficient WT mice allows the Ikk-deficient epidermis to undergo normal differentiation, suggesting that the requirement for IKK can be bypassed by factors produced by normal skin (Hu et al, 2001). These experiments do not exclude the possibility that another important source of kDIF or similarly acting factors are dermal fibroblasts, which are well known to produce factors that control epidermal morphogenesis (Wessells, 1977). In support of this hypothesis, a keratinocyte-specific Ikk disruption results in a less severe epidermal differentiation defect with altered skin barrier function than the total Ikk knockout (Gareus et al, 2007). These results were interpreted to suggest that Ikk functions non-autonomously in the dermis to control epidermal differentiation. However, Ikk-deficient keratinocytes from the epidermal-specific knockout mouse still fail to differentiate in vitro (Gareus et al, 2007), similar to keratinocytes from total Ikk knockout mice (Hu et al, 2001). Collectively, these results indicate that IKK functions within epidermal keratinocytes and probably in dermal fibroblasts to induce keratinocyte differentiation. As keratinocyte-restricted expression of IKK, unlike the keratinocyte-specific knockout, does not result in a differentiation defect (Sil et al, 2004), it appears that the keratinocyte is the major site of IKK action with respect to keratinocyte differentiation and epidermal-directed morphogenetic events.

IKK is a critical component of a Smad4-independent TGF–Smad2/3 signalling pathway

The most immediate effect of IKK re-expression in Ikk^–/– keratinocytes is cell cycle withdrawal, which precedes the expression of differentiation markers (Hu et al, 2001). Thus, to understand the molecular function of IKK, we searched for cell cycle-related target genes, the expression of which is IKK dependent. This search netted several genes encoding negative cell cycle regulators, the expression of which is downregulated in Ikk^–/– keratinocytes and epidermis, including Mad1 and Ovol1 (Descargues et al, 2008), which encode negative regulators of Myc. The c-Myc oncogene is thought to influence the balance between keratinocyte proliferation and differentiation, depending on the intensity and timing of its activity (Watt et al, 2008). Mad1 is a basic region/helix-loop-helix/leucine zipper transcriptional regulator that dimerizes with Max to form Mad:Max heterodimers that antagonize the transcriptional function of Myc:Max dimers (Ayer et al, 1993; Grandori et al, 2000). Mad1^–/– mice, however, are viable, phenotypically normal (Foley et al, 1998; Grandori et al, 2000) and do not show any epidermal defect. Most likely, other Mad genes, including Mad2, Mad3 and Mad4, are functionally redundant with Mad1 and compensate for its loss. Indeed Mad2 and Mad3 are also induced in keratinocytes in an IKK-dependent manner (unpublished data). Ovol1 is a zinc-finger-containing transcription factor, which, similar to Mad1, is also expressed in differentiating suprabasal keratinocytes (Dai et al, 1998; Descargues et al, 2008). Ovol1^–/– adult mice present with aberrant hair formation but normal epidermal differentiation (Dai et al, 1998). However, the suprabasal epidermis of Ovol1^–/– embryos shows increased proliferation and in vitro, Ovol1^–/– keratinocytes fail to exit the cell cycle in response to growth-inhibitory signals, such as high Ca²⁺ or TGF (Nair et al, 2006). This defect may be explained in part by abnormal upregulation of c-Myc, which is a direct target of Ovol1 in keratinocytes (Nair et al, 2006). Thus, IKK controls keratinocyte proliferation and cycling through the regulation of several Myc antagonists to allow keratinocytes to embark on their differentiation pathway (Figure 4). Interestingly, another TGF-related mechanism involving Smad3/4 and E2F4/5 transcription factors has been shown earlier to directly inhibit c-Myc expression in keratinocytes (Chen et al, 2002) (Figure 4). Although this signalling pathway triggers antiproliferative effects of TGF in keratinocytes, its impact on keratinocyte differentiation is unknown.

Mad1 and Ovol1 are involved in the inhibition of keratinocyte proliferation induced by TGF family members (Vastrik et al, 1995; Gomis et al, 2006), suggesting a link between IKK and the TGF signalling pathway. TGF family members, including TGFs, activins and BMPs, are cytokines that control cell growth, differentiation and deposition of extracellular matrix through binding to heterodimeric cell surface receptor complexes composed of type I and II subunits, and intracellular Smad transcription factors (Shi and Massague, 2003; Feng and Derynck, 2005; Schmierer and Hill, 2007). The eight mammalian Smad proteins are divided into three distinct groups: receptor-activated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5 and Smad8), a unique common Smad mediator (Co-Smads: Smad4) and inhibitory Smads (i-Smads: Smad6 and Smad7). On ligand binding, the type II receptor activates the type I receptor through its kinase domain, and the type I receptor in turn phosphorylates R-Smads. The activated R-Smads form heterodimeric complexes with Smad4, which accumulate in the nucleus and directly repress or activate specific target genes (Shi and Massague, 2003; Feng and Derynck, 2005; Schmierer and Hill, 2007).

TGF family members, their receptors and Smad transcription factors are abundantly expressed in epidermal keratinocytes, suggesting homoeostatic and regulatory functions (He et al, 2001; Li et al, 2003). Phosphorylated Smad2 and Smad3 proteins are concentrated in the nuclei of basal and suprabasal keratinocytes, whereas nuclear Smad4 staining is more preeminent in basal cells (Descargues et al, 2008). Nonetheless, alterations of TGF signalling in transgenic/knockout mice have often resulted only in minor epidermal defects, thereby obscuring its exact functions in this tissue (Li et al, 2003). We recently found that IKK interacts strongly with Smad3 and weakly with Smad2, but does not bind Smad4 (Descargues et al, 2008). IKK associates with the C-terminal MH2 domain of Smad3 through its kinase domain (unpublished observations). The R-Smad MH2 domain is known to be essential for trans-activation, phosphorylation by type I receptors and Smad4 binding (Massague, 2000; Feng and Derynck, 2005). On stimulation by TGF1, IKK and Smad3 form a transcriptional complex that accumulates in the keratinocyte nucleus to directly control the transcription of Mad1 (Descargues et al, 2008). Similar findings were made for Ovol1 (unpublished data). In Ikk^–/– keratinocytes stimulated with TGF1, Smad3 is no longer recruited to the Mad1 regulatory region despite its normal association with Smad4. Furthermore, nuclear staining for activated Smad2 and Smad3 is dramatically diminished in the Ikk^–/– epidermis (Descargues et al, 2008). Taken together, these results indicate that IKK is required for nuclear accumulation and chromatin recruitment of phosphorylated Smad2 and Smad3 to IKK-regulated genes in response to TGF1. Interestingly, activin A, an important regulator of epidermal differentiation (Owens et al, 2008), is involved in the induction of Mad1 expression in keratinocytes (Werner et al, 2001). As a result, one may speculate that the IKK–Smad2/3 complex forms and activates anti-Myc genes, including Mad1, on activin A signals. As activin A also downregulates the Id1, Id2 and Id3 genes in keratinocytes (Rotzer et al, 2006), it could also be interesting to analyse whether this signalling pathway depends on IKK. Finally, we found that kDIF functions downstream of the IKK–Smad2/3 signalling pathway as it can induce differentiation of Ikk^–/– keratinocytes without inducing Mad1 expression (Figure 5). Exactly how kDIF functions and what it is composed of remain to be determined.

The results mentioned above provided the first clear evidence for a critical function for Smad transcription factors in epidermal differentiation. Although Smad3 deficiency alone does not result in any cutaneous defect (Zhu et al, 1998; Datto et al, 1999; Yang et al, 1999) and the loss of Smad2 leads to early embryonic lethality (Nomura and Li, 1998; Waldrip et al, 1998; Weinstein et al, 1998; Heyer et al, 1999), loricrin and filaggrin expression is barely detectable in E15.5 embryos lacking both Smad3 alleles and one Smad2 allele in their epidermis (Descargues et al, 2008). Similarly, siRNA-mediated Smad2 knockdown in Smad3-deficient keratinocytes inhibited the expression of loricrin and filaggrin, as well as Mad1, in response to high Ca²⁺ (Descargues et al, 2008). These results suggest that Smad2 and Smad3 are functionally redundant. The importance of the TGF–Smad2/3–IKK axis for proper epidermal differentiation is also underscored by the analysis of transgenic mice in which the i-Smad Smad7 is inducibly expressed in the epidermis, resulting in defective expression of loricrin and filaggrin and failed stratum corneum formation (Descargues et al, 2008). Consequently, these mutant mice display epidermal hyperplasia due to abnormal proliferation of suprabasal keratinocytes and loss of nuclear IKK and Mad1 (Descargues et al, 2008).

Surprisingly, this new TGF response pathway centred around Smad2/3–IKK complex formation is independent of Smad4. Smad4-deficient keratinocytes display normal Mad1 expression (Descargues et al, 2008) and undergo terminal differentiation in response to high Ca²⁺ (unpublished data). These results are consistent with the phenotype of mice with epidermal-specific deletion of Smad4 (Smad4^/ mice). These mice present with degeneration of hair follicles and dermal cysts that progress to skin tumours in old animals, but do not show any perturbated epidermal differentiation and stratum corneum formation (Yang et al, 2005; Qiao et al, 2006). Furthermore, activated Smad2 and Smad3, as well as IKK, are normally localized in the nuclei of Smad4^/ keratinocytes, which display normal Mad1 expression (Descargues et al, 2008). Taken together, these results strongly indicate that Smad4 is not required for epidermal differentiation. The Smad4 independence of the TGF–Smad2/3–IKK signalling pathway is reminiscent of another TGF signalling operative during erythroid development in which TIF1 (also called TRIM33 or ectodermin) replaces Smad4 (He et al, 2006). However, TIF1 is a RING-type ubiquitin ligase that can target Smad4 to degradation and can therefore function as a negative regulator of Smad4-dependent TGF signalling (Dupont et al, 2005). Hence the exact function of TIF1 in TGF signalling is not fully understood, and it is not known whether it affects the IKK-dependent pathway.

IKK and squamous cell carcinoma

IKK was recently identified as a tumour suppressor in squamous cell carcinoma (SCC) (Liu et al, 2006; Maeda et al, 2007). SCC is a cancer derived from squamous epithelia of the skin (epidermis), head and neck tissues (mouth, throat, oral and nasal cavities, esophagus) as well as other sites. SCC is the second most common skin cancer in Caucasians with an estimated incidence of 100 000–150 000 new cases per year in the United States (Johnson et al, 1992). Sun exposure and immune suppression increase the risk of SCC development and so does tobacco use (Rudolph and Zelac, 2004; Hampton, 2005). SCC of the oral cavity is one of the most prevalent cancers of the head and neck region with a worldwide incidence of 300 000 new cases per year, the occurrence of which is linked to tobacco use and betel nut chewing (Silverman, 2001). SCCs of the oral cavity are more aggressive than those developing from the skin, and are associated with a 5-year survival rate of about 50–55% (Silverman, 2001).

Molecular changes in SCCs are characterized by a marked heterogeneity and include activation of oncogenes, such as RAS, MYC, EGFR and Cyclin D1, as well inactivation of tumour suppressors, including p53 and p16 (Hardisson, 2003). These genetic alterations are thought to influence malignant keratinocyte behaviour and tumour progression, but the precise molecular pathogenesis of SCC is poorly understood. Interestingly, mutations in exon 15 of the IKK locus were described in a few high-grade and poorly differentiated human SCCs of the skin and were shown to be associated with reduced IKK expression (Liu et al, 2006). However, it seems that downregulation of IKK due to epigenetic silencing of the IKK locus is a more common occurrence seen in close to 30% of invasive oral SCCs (Maeda et al, 2007). It was also reported that overexpression of IKK in the suprabasal compartment, which results in increased epidermal differentiation and reduced keratinocyte proliferation, inhibits chemically induced SCC formation and progression in mice (Liu et al, 2006). These results, together with enhanced SCC incidence in Ikk^+/– mice subjected to two-stage skin carcinogenesis and loss of Ikk heterozygosity in the tumours, provide evidence that IKK is a tumour suppressor in the epidermis (Liu et al, 2006; Park et al, 2007). This function of IKK is probably mediated through the control of keratinocyte proliferation. The loss of nuclear IKK contributes to malignant conversion of keratinocytes into less differentiated and proliferative carcinoma cells. However, a recent study has suggested that increased IKK may be found in acantholytic SCC (ASCC) (Moreno-Maldonado et al, 2008), an histologic variant of SCC showing positive staining for CKs (Rinker et al, 2001). Unfortunately, the authors of that study have not carefully analysed whether ASCCs present with loss of nuclear IKK, which would make their results more consistent with other studies mentioned above.

SCC and other carcinoma cells are known to overproduce TGF1 to modify their microenvironment through local immunosupression, extracellular matrix remodelling and neoangiogenesis, and these changes are required for tumour progression and invasiveness (Oft et al, 1996, 1998; Siegel and Massague, 2003; Li et al, 2005a, 2006). At the same time, carcinoma cells become resistant to TGF-induced growth arrest (Siegel and Massague, 2003; Li et al, 2005a, 2006). Altered IKK function may contribute, at least in part, to acquired resistance to TGF1-induced growth arrest.

Conclusions and future directions

Although IKK was first identified as a catalytic subunit of the IKK complex, which mediates NF-B activation (DiDonato et al, 1997; Mercurio et al, 1997; Regnier et al, 1997; Zandi et al, 1997), it has quickly emerged as a multifunctional protein with several unexpected and surprising activities. In the epidermis, IKK has turned out to be a critical regulator of keratinocyte proliferation, differentiation and oncogenic transformation, and this function is completely unrelated to its protein kinase activity or NF-B signalling. Instead, IKK functions as a cofactor for Smad2/3 in a Smad4-independent pathway that inhibits keratinocytes proliferation (Descargues et al, 2008) (Figure 6). In this capacity, IKK is required for the induction of a specific subset of TGF-responsive genes that include the Myc antagonists Mad1 and Ovol1, but is not needed for other well-known TGF target genes, such as p21, p15 and p27, encoding inhibitors of cyclin-dependent kinases (CDKs) (Descargues et al, 2008). This is reminiscent of the two different classes of antiproliferative gene responses: Myc repression and inhibition of CDKs, respectively, that are induced during TGF-mediated cell cycle arrest (Massague et al, 2000). It is attractive to speculate that the tumour suppressive function of IKK is exerted through this pathway as well.

Other proteins may be part of the TGF–Smad2/3–IKK signalling pathway, as revealed by two mouse models with functional alterations of 14-3-3 (repeated epilation mutant mice) and IRF6, the disruption of which faithfully mimics the phenotype of Ikk^–/– mice (Herron et al, 2005; Li et al, 2005b; Ingraham et al, 2006; Richardson et al, 2006). 14-3-3 belongs to a family of adaptors that can interact with target proteins in a sequence-specific manner, although its exact function is poorly understood (Mhawech, 2005). It was reported that IKK may protect the 14-3-3 locus from hypermethylation in keratinocytes by interacting with histone H3 (Zhu et al, 2007). In that study, the authors showed that 14-3-3 is downregulated in Ikk^–/– keratinocytes, suggesting that this gene is a downstream target of IKK (Zhu et al, 2007). The human IRF6 locus is defective in Van der Woude (VWS, OMIM: 119300) and popliteal pterygium (PPS, OMIM: 11500) syndromes, which are characterized by orofacial defects such as cleft lip and palate (Kondo et al, 2002). IRF6 belongs to a family of transcription factors that share a highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain. Interestingly, this protein-binding domain is related to the C-terminal MH2 domain of Smad proteins and has been referred to SMIR (Smad and IRF) domain (Eroshkin and Mushegian, 1999). As DNA binding by Smad transcription factors depends on their association with other DNA-bound transcription factors (Derynck and Zhang, 2003; ten Dijke and Hill, 2004), one can speculate that IRF6 may be a component of the Smad2/3–IKK transcriptional complex that accumulates in the keratinocyte nucleus to induce the obligatory cell cycle exit that precedes terminal differentiation (Figure 6). In addition, IKK may also interact with other transcription factors, such as RARs to control epidermal barrier formation (Gareus et al, 2007). The identification of other IKK-interacting proteins and additional IKK target genes will provide an ever better understanding of how this critical regulator of epidermal proliferation and differentiation carries out its daily work.

Acknowledgements

This study was supported by The International Human Frontier Science Program Organization (to PD), National Institutes of Health grants (to MK) and an American Cancer Society Research Professorship (to MK).

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