Keratin 10 Gene Expression during Differentiation of Mouse Epidermis Requires Transcription Factors C/EBP and AP-2

Abstract

The epidermis forms a vital barrier composed of stratified keratinocytes and their differentiated products. One of these products, keratin K10, is critical to epidermal integrity, because mutations in k10 lead to abnormal blistering. For the normal expression of k10, differentiation-associated transcription factors C/EBPalpha, C/EBPbeta, and AP-2 are well positioned to play an important role. Here, regulation of the k10 gene is examined in keratinocytes in the skin of normal mice and in transgenic mice carrying targeted deletions of c/ebpbeta and ap-2alpha. In cultured cells, C/EBPalpha and C/EBPbeta are each capable of activating the k10 promoter via three binding sites, identified by site-directed mutagenesis. In a given epidermal cell in vivo, however, the selection of C/EBPalpha versus C/EBPbeta for k10 regulation is determined via a third transcription factor, AP-2. This novel regulatory scheme involves: (1) unique gradients of expression for each transcription factor, i.e., C/EBPbeta and AP-2 most abundant in the lower epidermis, C/EBPalpha in the upper; (2) C/EBP-binding sites in the ap-2alpha gene promoter, through which C/EBPbeta stimulates ap-2alpha; and (3) AP-2 binding sites in the c/ebpalpha promoter, through which AP-2 represses c/ebpalpha. Promoter-analysis and gene-expression data presented herein support a regulatory model in which C/EBPbeta activates and maintains AP-2 expression in basal keratinocytes, whereas AP-2 represses C/EBPalpha in those cells. In response to differentiation signals, loss of AP-2 expression leads to derepression of the c/ebpalpha promoter and activation of k10 as cells migrate upward.

Proliferation and cornification during development of the mammalian epidermis

“…4D). The K10 target gene is upregulated in these ectopic locations as well, driven by C/EBP␣ (Maytin et al, 1999). These data represent just one example of the many complex interactions (cross-talk) that certainly must occur between various transcription factors as they work together in complex ways to regulate keratinocyte differentiation during epidermal development.…”

“…Despite these strong connections, AP-2␣ Ϫ/Ϫ skin appears normal (Zhang et al, 1996) and the expression of K5/K14 is unaffected (Talbot et al, 1999), perhaps due to functional redundancy in the basal cell layer. However, K10 is upregulated in AP-2␣ Ϫ/Ϫ suprabasal cells (Maytin et al, 1999) together with the aberrant regulation of another gene that is dynamically expressed in differentiating epidermis: C/EBP␣ (Maytin et and Habener, 1998). The interplay between AP-2␣ and C/EBP␣ is described in the next section.…”

“…Transfection of differentiated keratinocytes with a C/EBP-responsive promoter construct results in dramatic increases in luciferase activity as compared to undifferentiated cells (Zhu et al, 1999). Both C/EBP␣ and C/EBP␤ can bind to and activate the K10 promoter (Maytin et al, 1999). In addition, forced expression of C/EBP␤ in primary mouse keratinocytes inhibits cell growth, while C/EBP␤ deficient keratinocytes are resistant to Ca 2ϩ -induced growth arrest (Zhu et al, 1999).…”

“…An interesting example of how transcriptional regulators of growth and differentiation can work together in fetal epidermis comes from a study that looked at AP-2 and C/EBP factors in the skin (Maytin et al, 1999). With the use of a combination of cell culture and in vivo mouse studies, it was shown that C/EBP␣, C/EBP␤, and AP-2␣ are linked together in an interactive regulatory system that operates at the level of the respective gene promoters (Fig.…”

“…For example, the loss of individual members of the C/EBP family of genes does not appear to provoke any obvious skin phenotype in newborn mice, although subclini-cal abnormalities do occur at the microscopic level (Maytin et al, 1999). Yet it seems quite likely that under stress or disease conditions, the loss of a single family member may manifest itself in the skin.…”