CCAAT/enhancer binding protein-β is a mediator of keratinocyte survival and skin tumorigenesis involving oncogenic Ras signaling

Abstract

The basic leucine zipper transcription factor CCAAT͞enhancer binding protein-␤ (C͞EBP␤) is expressed in many cell types, including keratinocytes. C͞EBP␤ activity can be increased by phosphorylation through pathways stimulated by oncogenic Ras, although the biological implications of Ras-C͞EBP␤ signaling are not currently understood. We report here that C͞EBP␤-nullizygous mice are completely refractory to skin tumor development induced by a variety of carcinogens and carcinogenesis protocols, including 7,12-dimethylbenz-[a]anthracene-initiation͞12-O-tetradecanoylphorbol 13-acetate promotion, that produce tumors containing oncogenic Ras mutations. No significant differences in TPA-induced epidermal keratinocyte proliferation were observed in C͞EBP␤-null versus wild-type mice. However, apoptosis was significantly elevated (17-fold) in the epidermal keratinocytes of 7,12-dimethylbenz[a]anthracene-treated C͞EBP␤-null mice compared with wild-type mice. In v-Ha-ras transgenic mice, C͞EBP␤ deficiency also led to greatly reduced skin tumor multiplicity and size, providing additional evidence for a tumorigenesis pathway linking Ras and C͞EBP␤. Oncogenic Ras potently stimulated C͞EBP␤ to activate a C͞EBP-responsive promoter-reporter in keratinocytes and mutating an ERK1͞2 phosphorylation site (T188) in C͞EBP␤ abolished this Ras effect. Finally, we observed that C͞EBP␤ participates in oncogenic Ras-induced transformation of NIH 3T3 cells. These findings indicate that C͞EBP␤ has a critical role in Ras-mediated tumorigenesis and cell survival and implicate C͞EBP␤ as a target for tumor inhibition.T he Ras family of GTP binding proteins function as intracellular mediators of extracellular signals to regulate cell proliferation, apoptosis, survival, senescence, and differentiation (1-5). Ras protooncogenes are frequently mutated in tumors, and Ϸ25% of human cancers contain transforming mutations in ras. Therefore, understanding oncogenic Ras-signaling pathways is critical for elucidating the mechanisms that underlie cellular transformation and for designing effective therapeutic strategies to prevent the development or block the growth of many classes of tumors. Ras has numerous effectors, and its pathways are multifaceted (3, 6, 7). Ras activation by growth factors or oncogenic mutations elicits activation of several transcription factors, which in turn regulate the expression of genes that control the cellular responses to Ras signaling, including oncogenesis. The transcription factors Ets, c-jun, c-myc, and NF-B are known to have roles in oncogenic ras-induced cellular transformation (8-11).The basic leucine zipper (bZIP) transcription factor CCAAT͞ enhancer binding protein-␤ (C͞EBP␤, also known as NF-IL6, IL-6DBP, LAP, CRP2, and NF-M) is expressed in a variety of cell types (12, 13) including keratinocytes (14,15), where it plays a role in squamous differentiation (16). C͞EBP␤ is also involved in regulating differentiation of specific mesenchymal, epithelial, and hematopoietic cell types (17-21). C͞EBP␤ activity can be activ...

Conditional ablation of C/EBPβ demonstrates its keratinocyte-specific requirement for cell survival and mouse skin tumorigenesis

“…C/EBPb À/À mice also display elevated levels of DMBA-induced apoptosis suggesting that C/EBPb has a role in keratinocyte survival in response to oncogenic Ras signaling and/or DNA damage (Zhu et al, 2002;Johnson, 2005). These data demonstrate an important role for C/EBPb in keratinocytes survival and skin tumorigenesis.…”

“…For example, C/EBPb can cooperate with Ras to transform NIH 3T3 cells in a phosphorylation-dependent manner (Zhu et al, 2002;Shuman et al, 2004). The C/EBPb gene expresses three different isoforms by virtue of alternative translation initiation or regulated proteolysis (Ramji and Foka, 2002).…”

“…C/EBPb is abundantly expressed in keratinocytes (Oh and Smart, 1998) and we previously reported that C/EBPb À/À mice are completely refractory to skin tumorigenesis induced by carcinogens, such as 7,12-dimethylbenz[a]anthracene (DMBA), that produce oncogenic Ras mutations in epidermal keratinocytes (Zhu et al, 2002). C/EBPb À/À mice also display elevated levels of DMBA-induced apoptosis suggesting that C/EBPb has a role in keratinocyte survival in response to oncogenic Ras signaling and/or DNA damage (Zhu et al, 2002;Johnson, 2005).…”

“…HF --hair follicle, E -epidermis, and SG -sebaceous gland. The number of apoptotic keratinocytes/cm length of epidermis was determined as described (Zhu et al, 2002). Interfollicular keratinocytes were scored from three H&E stained sections (B1.5 cm length)/mouse using the following criteria; presence of dark pyknotic nuclei, cytoplasmic eosinophilia and the absence of cellular contacts.…”

Decreased survival of C/EBPβ-deficient keratinocytes is due to aberrant regulation of p53 levels and function

“…C/EBPb is expressed in many tissues, including epidermis (Maytin and Habener, 1998;Oh and Smart, 1998) where it plays a role in the early stages of stratified squamous differentiation (Zhu et al, 1999). Recent studies have identified a role for C/EBPb in tumor development (Zhu et al, 2002), senescence (Sebastian et al, 2005), cell survival (Buck et al, 2001;Zhu et al, 2002;Wessells et al, 2004;Li et al, 2005) and cellular transformation (Zhu et al, 2002). In terms of cell transformation, C/EBPb can cooperate with Ras12V to transform NIH3T3 cells (Zhu et al, 1999) and more recently, it has been shown that cell-cycledependent phosphorylation of C/EBPb on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH3T3 cells (Shuman et al, 2004).…”