Inhibition of CCAAT/Enhancer Binding Protein Family DNA Binding in Mouse Epidermis Prevents and Regresses Papillomas

Abstract

The CCAAT/enhancer binding proteins (C/EBP) are a family of B-ZIP DNA binding proteins that act as transcription factors to regulate growth and differentiation of many cell types, including keratinocytes. To examine the consequences of inhibiting the C/EBP family of transcription factors in skin, we generated transgenic mice that use the tetracycline system to conditionally express A-C/EBP, a dominant negative that inhibits the DNA binding of C/EBP family members. We expressed A-C/EBP in the basal layer of the skin epidermis during a two-step skin carcinogenesis protocol. A-C/EBP expression caused hyperplasia of the basal epidermis and increased apoptosis in the suprabasal epidermis. The mice developed fewer papillomas and had systemic hair loss. A-C/ EBP expression caused C/EBPB protein to disappear whereas C/EBPA, p53, Bax, and caspase-3 protein levels were dramatically up-regulated in the suprabasal layer. Primary keratinocytes recapitulate the A-C/EBP induction of cell growth and increase in p53 protein. A-C/EBP expression after papilloma development caused the papillomas to regress with an associated increase in apoptosis and up-regulation of p53 protein. Furthermore, A-C/EBP-expressing mice heterozygous for p53 were more susceptible to papilloma formation, suggesting that the suppression of papilloma formation has a p53-dependent mechanism. These results implicate DNA binding of C/EBP family members as a potential molecular therapeutic target.

Epigenetic Regulation of Epidermal Differentiation

“…Expression of C/EBPa increases on differentiation and seems to be specific to the suprabasal layers, which suggests it might play a role in epidermal differentiation (Rishi et al 2010). Further, the overexpression of C/EBPa in the skin leads to hyperplasia of the basal layer of the epidermis whereas its down-regulation in vitro results in inhibition of differentiation (Oh et al 2007;Rishi et al 2010). These phenotypes are compatible with the role of C/EBPa in inducing expression of methylated genes involved in skin differentiation.…”

“…In mammalian genomes, up to 80% of the CpG sequences are methylated, and most of the unmethylated CpG are found in regions of high CpG content, known as CpG islands (Reik 2007), close to the promoters and first exons of actively transcribed genes. Although DNA methylation is generally associated with transcriptional repression, there are some examples in which 5mC is thought to mediate gene expression by recruitment of the mCpG-binding protein C/EBPa (CRE enhancer-binding protein alpha-isoform), which functions as a transcriptional activator (Oh et al 2007;Rishi et al 2010). …”

IL-17 promotes keratinocyte proliferation via the downregulation of C/EBPα

“…C/EBPα is expressed in various highly differentiated cells and tissues, including peripheral blood mononuclear cells, and adipose, liver and lung tissues (7). Previous studies have demonstrated that C/EBPα is expressed in every layer of the epidermis; therefore, C/EBPα may regulate the proliferation and differentiation of keratinocytes (8,9).…”

CpG methylation of half-CRE sequences creates C/EBPα binding sites that activate some tissue-specific genes

“…1C). We chose to study C/EBPα in more detail because it is involved in the activation of differentiation in several cell types (15), including keratinocytes (16)(17)(18) and adipocytes (19)(20)(21), potentially linking C/EBPα binding to methylated promoters with activation of methylated promoters upon differentiation (see below).…”

“…First, we cultured primary keratinocytes from transgenic mice expressing a dominant-negative protein (A-C/EBP) that inhibits the DNA binding of C/EBP family members (18). Second, we used siRNA to deplete C/EBPα and C/EBPβ (SI Appendix, Fig.…”