p63/p51-induced onset of keratinocyte differentiation via the c-Jun N-terminal kinase pathway is counteracted by keratinocyte growth factor

p63/p51, a homolog of the tumor suppressor protein p53, is chiefly expressed in epithelial tissues, including the epidermis. p63 affects cell death similar to p53, and also plays important roles in the development of epithelial tissues and the maintenance of epithelial stem cells. Because it remains unclear how p63 regulates epithelial cell differentiation, we examined the function(s) of p63 in keratinocyte differentiation through the use of a keratinocyte culture system. DeltaNp63alpha (DeltaNp51B), a p63 isoform specifically expressed in basal keratinocytes, suppressed the differentiation of specific late-stage proteins, such as filaggrin and loricrin. In contrast, DeltaNp63alpha induced keratin 1 (K1), which is expressed at the start of differentiation, via c-Jun N-terminal kinase (JNK)/AP-1 activation. However, p63 did not induce K1 expression in the basal layer in vivo, although basal keratinocytes had high levels of p63. This discrepancy was explained by the suppression of K1 expression by dermis-secreted keratinocyte growth factor. This suppression occurred via extracellular signal-related kinase (ERK) signaling, and counteracted the p63-mediated induction of K1. Thus, a precise balance between p63 and keratinocyte growth factor mediates the onset of epithelial cell differentiation, through JNK and ERK signaling. These data may provide mechanistic explanations for the pathological features of skin diseases, including psoriasis.     

Repetin (Rptn), a New Member of the “Fused Gene” Subgroup within the S100 Gene Family Encoding a Murine Epidermal Differentiation Protein

We report the cloning and characterization of a murine epidermal differentiation gene, repetin (Rptn), exhibiting striking similarity to the genes of the intermediate filament-associated proteins profilaggrin and trichohyalin. The repetin gene consists of three exons and two introns. The first exon is short and untranslated. The deduced amino acid sequence distributed between exons II and III contains 1130 amino acids with a calculated molecular mass of 130 kDa and pIof 7.7. The amino terminus exhibits significant homology to the S100 proteins containing two calcium-binding motifs of the EF-hand type. The remainder coding sequence contains a central segment consisting of 49 tandem repeats of a 12-amino-acid sequence rich in glutamines. By fluorescencein situhybridization the repetin gene was localized to chromosome band 3 F1-2. Expression of repetin mRNA is detectable in the stratified internal epithelia of forestomach and tongue and to a lesser degree in normal skin epidermis, where it is restricted to the differentiated suprabasal cell layers. Based on its chromosomal localization, its genomic organization, and its stage-specific expression during late epidermal differentiation, as well as on the structural features of the encoded protein, we conclude that the repetin gene represents a novel member of the “fused gene” subgroup of the S100 gene family encoding multifunctional epidermal matrix proteins.     

Identification of markers for nipple epidermis: changes in expression during pregnancy and lactation

In vertebrates, specific regions of skin crucial for interaction with and manipulation of elements in the environment are characterized by specialized epidermis. Regions of specialized epidermis show distinct patterns of cellular differentiation and express specific keratins that provide an increased ability to withstand mechanical strain. The nipple, which must endure the mechanical strain of nursing, is a type of specialized epidermis. The entire ventral skin of the keratin 14 promoter driven PTHrP mouse provides a model for nipple development. To identify novel markers for this specialized epidermis, we have used two-dimensional (2-D) gels, mass spectrometric protein identification, Western blotting and immunohistochemistry to compare intermediate filament preparations from the nipple-like K14-PTHrP ventral skin to that of wild-type littermates. We identified 64 spots on 2-D gels that were increased in expression in the nipple-like skin of the female K14-PTHrP mouse and 11 spots that were elevated in the wild type. Microsequencing suggested that K17 and epiplakin were among the proteins with the greatest increase in expression in the K14-PTHrP ventral skin. Using Western blots and immunohistochemistry, we evaluated the expression of these proteins as well as K6 in the wild-type nipple, K14-PTHrP ventral skin and wild-type ventral skin. In addition, we found that the expression of K6 was minimally changed in the pregnant and lactating nipple, but the expression of a previously identified marker, K2e, was reduced during lactation. Using a model of the mechanical strain induced by nursing, we found that K2e but not K6 expression was responsive to this condition. The identification of epidermal markers and their expression patterns will provide insight into the cellular differentiation patterns of the nipple and the underlying epidermal-mesenchymal interactions that direct this differentiation.     

Dominant-negative retinoic acid receptors elicit epidermal defects through a non-canonical pathway

Previous work has shown that a dominant-negative retinoic acid receptor alpha (dnRARalpha), expressed under the K14 promoter, causes severe epidermal defects. Similar defects are, however, not seen in RARalphagamma double null mutant mice, which lack the entire complement of RARs expressed in the epidermis. To investigate the mechanism of action of these dominant-negative receptors, dnRARalpha or a DNA binding-deficient variant, dnRARalpha(DBD), were targeted to the basal epidermis. Expression of either receptor type led to similar epidermal phenotypes suggesting that both RAR mutants acted through a common mechanism. The epidermal phenotype was reminiscent of defects seen in p63(-/-) mice. Consistent with this, reduced p63 expression was observed in transgenic offspring expressing either RAR mutant, suggesting that down-regulation of p63 might underlie the effects of these receptors on epidermal development. By contrast, expression of p63 in the epidermis of RARalphagamma(-/-) offspring was unaffected, indicating that RARs were not essential for p63 expression. These findings suggest that dnRARs may impact on epidermal development through one or more non-canonical pathways, which are independent of receptor-DNA interaction.