Maranke I. Koster

p63 induces key target genes required for epidermal morphogenesis

Mice lacking p63, a single gene that encodes a group of transcription factors that either contain (TA) or lack (ΔN) a transactivation domain, fail to develop stratified epithelia as well as epithelial appendages and limbs. ΔNp63 isoforms are predominantly expressed during late embryonic and postnatal epidermal development, however, the function of these proteins remains elusive. Using an epidermal-specific inducible knockdown mouse model, we demonstrate that ΔNp63 proteins are essential for maintaining basement membrane integrity and terminal differentiation of keratinocytes. Furthermore, we have identified two ΔNp63α target genes that mediate these processes. We propose that ΔNp63α initially induces expression of the extracellular matrix component Fras1, which is required for maintaining the integrity of the epidermal–dermal interface at the basement membrane. Subsequently, induction of IκB kinase-α by ΔNp63α initiates epidermal terminal differentiation resulting in the formation of the spinous layer. Our data provide insights into the role of ΔNp63α in epidermal morphogenesis and homeostasis, and may contribute to our understanding of the pathogenic mechanisms underlying disorders caused by p63 mutations.

p63 in skin development and ectodermal dysplasias.

The transcription factor p63 is critically important for skin development and maintenance. Processes that require p63 include epidermal lineage commitment, epidermal differentiation, cell adhesion, and basement membrane formation. Not surprisingly, alterations in the p63 pathway underlie a subset of ectodermal dysplasias, developmental syndromes in which the skin and skin appendages do not develop normally. This review summarizes the current understanding of the role of p63 in normal development and ectodermal dysplasias.

Reactivation of Developmentally Expressed p63 Isoforms Predisposes to Tumor Development and Progression

Genes that are active during normal development are frequently reactivated during neoplastic transformation. We now report that developmentally expressed TAp63 isoforms are frequently reactivated in human squamous cell carcinomas. To determine the consequences of TAp63 reactivation, we induced TAp63alpha expression during chemically-induced skin carcinogenesis. Deregulated TAp63alpha expression dramatically accelerated tumor development and progression, frequently resulting in epithelial-mesenchymal transitions to spindle cell carcinomas and lung metastases. Consistent with this observation, we detected high levels of Twist and N-cadherin in tumors overexpressing TAp63alpha. Thus, as observed for other developmental pathways, aberrant reactivation of TAp63 predisposes to tumor development and progression.

Conflicting roles for p63 in skin development and carcinogenesis.

Epidermal morphogenesis is a complex process that culminates in the formation of a barrier that protects the organism from environmental substances and dehydration. p63, a transcription factor, is essential for normal epidermal morphogenesis as demonstrated by the failure of mice lacking p63 expression to develop an epidermis. However, since two independently generated p63-/- mouse models displayed different phenotypes, the role of p63 in epidermal morphogenesis has remained controversial. Furthermore, the tumor susceptibility phenotypes of both p63-/- mouse models were strikingly different. In this review, we discuss these conflicting findings and provide evidence for various roles of p63 in the epidermis under normal and pathological conditions.

Making an epidermis.

The skin functions as a barrier protecting the body from dehydration and environmental insults. This barrier function is mainly provided by the outermost layer of the skin, the epidermis. The epidermis is maintained by epidermal stem cells which reside in the basal layer and which generate daughter cells that move upward toward the surface of the skin. During this journey, keratinocytes undergo a series of biochemical and morphological changes that result in the formation of the various layers of the epidermis. Eventually, these cells turn into the outermost layer of dead cornified cells that are sloughed into the environment. This review summarizes our current understanding of the mechanisms that control proliferation and differentiation of epidermal stem cells, and thus addresses fundamental processes that control epidermal morphogenesis and function.

IKKα Is a p63 Transcriptional Target Involved in the Pathogenesis of Ectodermal Dysplasias

The transcription factor p63 plays a pivotal role in the development and differentiation of the epidermis and epithelial appendages. Indeed, mutations in p63 are associated with a group of ectodermal dysplasias characterized by skin, limb, and craniofacial defects. It was hypothesized that p63 exerts its functions by activating specific genes during epidermal development, which in turn regulate epidermal stratification and differentiation. We have identified I-kappaB kinase alpha (IKKalpha) as a direct transcriptional target of p63 that is induced at early phases of terminal differentiation of primary keratinocytes. We show that the DeltaNp63 isoform is required for IKKalpha expression in differentiating keratinocytes and that mutant p63 proteins expressed in ectodermal dysplasia patients exhibit defects in inducing IKKalpha. Furthermore, we observed reduced IKKalpha expression in the epidermis of an ankyloblepharon ectodermal dysplasia clefting patient. Our data demonstrate that a failure to properly express IKKalpha may play a role in the development of ectodermal dysplasias.

Transgenic mouse models provide new insights into the role of p63 in epidermal development.

The epidermis is a stratified epithelium which provides a barrier between the organism and the environment protecting it from dehydration and pathogenic insult. A gene essential for development of the epidermis and other stratified epithelia is the transcription factor p63. The p63 gene is transcribed into isoforms that contain (TA) or lack (DN) a transactivation domain. Of these isoforms, only TAp63 isoforms are expressed in the uncommitted surface ectoderm, while DNp63 isoforms are expressed after the surface ectoderm has committed to a stratification program. Consistent with these embryonic expression profiles, we found that TAp63a functions as the master switch for initiation of epithelial stratification. Furthermore, TAp63a induces proliferation and inhibits terminal differentiation. This inhibition is overcome by the subsequent expression of DNp63a which, in this context, acts as a dominant-negative molecule and allows basal keratinocytes to withdraw from the cell cycle and commit to terminal differentiation. These data demonstrate that TA- and DNp63 isoforms have fundamentally different roles during epidermal development and provide new insight into the molecular events required for normal epidermal morphogenesis.

Genetic pathways required for epidermal morphogenesis

The epidermis is composed of keratinocytes which undergo a highly reproducible terminal differentiation program resulting in the formation of a protective barrier, which is established during embryogenesis. Significant progress has recently been made in understanding the genetic pathways associated with the earliest event characteristic of epidermal morphogenesis, commitment to stratification. This process depends on the expression of p63, a transcription factor which is transcribed into isoforms that contain (TA) or lack (AN) a transactivation domain. In the absence of p63 expression, epithelia remain single-layered, while ectopic TAp63alpha expression in single-layered epithelia initiates stratification. Later events during epidermal morphogenesis require withdrawal from the cell cycle and commitment to terminal differentiation. Some of the genetic pathways underlying these events are beginning to be elucidated, however, the exact molecular events remain to be determined. In this review, we summarize the involvement of several signaling pathways in different stages of epidermal morphogenesis.

ΔNp63 Knockdown Mice: A Mouse Model for AEC Syndrome

Dominant mutations in TP63 cause ankyloblepharon ectodermal dysplasia and clefting (AEC), an ectodermal dysplasia characterized by skin fragility. Since ΔNp63α is the predominantly expressed TP63 isoform in postnatal skin, we hypothesized that mutant ΔNp63α proteins are primarily responsible for skin fragility in AEC patients. We found that mutant ΔNp63α proteins expressed in AEC patients function as dominant‐negative molecules, suggesting that the human AEC skin phenotype could be mimicked in mouse skin by downregulating ΔNp63α. Indeed, downregulating ΔNp63 expression in mouse epidermis caused severe skin erosions, which resembled lesions that develop in AEC patients. In both cases, lesions were characterized by suprabasal epidermal proliferation, delayed terminal differentiation, and basement membrane abnormalities. By failing to provide structural stability to the epidermis, these defects likely contribute to the observed skin fragility. The development of a mouse model for AEC will allow us to further unravel the genetic pathways that are normally regulated by ΔNp63 and that may be perturbed in AEC patients. Ultimately, these studies will not only contribute to our understanding of the molecular mechanisms that cause skin fragility in AEC patients, but may also result in the identification of targets for novel therapeutic approaches aimed at treating skin erosions.

Roles of TGFβ signaling in epidermal/appendage development

The transforming growth factor β (TGFβ) superfamily encompasses a number of structurally related proteins that can be divided into several subfamilies including TGFβs, activins/inhibins and bone morphogenetic proteins (BMPs). The Smads are major intracellular mediators in transducing the signals of TGFβ superfamily members, and are abundantly expressed in the developing epidermis and epidermal appendages. Moreover, the phenotypes of transgenic/knockout mice with altered components of the TGFβ superfamily signaling pathway suggest that TGFβ superfamily signaling is required for epidermal/appendage development. TGFβ superfamily members are involved in most events during epidermal/appendage development through the TGFβ signal transduction pathway and through cross talk with other signaling pathways. Future studies will be instrumental in defining the precise roles for TGFβ superfamily signaling in epidermal/appendage development.