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. 2002 Jan 8;99(1):207-12.
doi: 10.1073/pnas.012437299. Epub 2001 Dec 26.

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

Songyun Zhu  1 , Kyungsil Yoon, Esta Sterneck, Peter F Johnson, Robert C Smart
Affiliations

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

Songyun Zhu et al. Proc Natl Acad Sci U S A. .

Abstract

The basic leucine zipper transcription factor CCAAT/enhancer binding protein-beta (C/EBPbeta) is expressed in many cell types, including keratinocytes. C/EBPbeta activity can be increased by phosphorylation through pathways stimulated by oncogenic Ras, although the biological implications of Ras-C/EBPbeta signaling are not currently understood. We report here that C/EBPbeta-nullizygous mice are completely refractory to skin tumor development induced by a variety of carcinogens and carcinogenesis protocols, including 7,12-dimethylbenz[a]anthracene-initiation/12-O-tetradecanoylphorbol 13-acetate promotion, that produce tumors containing oncogenic Ras mutations. No significant differences in TPA-induced epidermal keratinocyte proliferation were observed in C/EBPbeta-null versus wild-type mice. However, apoptosis was significantly elevated (17-fold) in the epidermal keratinocytes of 7,12-dimethylbenz[a]anthracene-treated C/EBPbeta-null mice compared with wild-type mice. In v-Ha-ras transgenic mice, C/EBPbeta deficiency also led to greatly reduced skin tumor multiplicity and size, providing additional evidence for a tumorigenesis pathway linking Ras and C/EBPbeta. Oncogenic Ras potently stimulated C/EBPbeta to activate a C/EBP-responsive promoter-reporter in keratinocytes and mutating an ERK1/2 phosphorylation site (T188) in C/EBPbeta abolished this Ras effect. Finally, we observed that C/EBPbeta participates in oncogenic Ras-induced transformation of NIH 3T3 cells. These findings indicate that C/EBPbeta has a critical role in Ras-mediated tumorigenesis and cell survival and implicate C/EBPbeta as a target for tumor inhibition.

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Figures

Figure 1
Figure 1
C/EBPβ-null mice are completely refractory to carcinogen-induced skin tumorigenesis. C/EBPβ-null (○), wild-type (□), or heterozygous (◊) mice littermates (7–8 weeks old) were treated with a single application of 200 nmol DMBA followed 1 week later with thrice weekly treatment with 5 nmol of TPA (n = 20 C/EBPβ+/+, 21 C/EBPβ−/−, 12 C/EBPβ+/−) (a and b); a single application of 2.5 μmol MNNG followed 1 week later with thrice weekly application of 5 nmol (n = 22 C/EBPβ+/+, 19 C/EBPβ−/−) (c and d); or 100 nmol of DMBA once a week for 25 weeks (n = 16 C/EBPβ+/+, 18 C/EBPβ−/−) (e and f). All agents were applied in 200 μl of acetone, and all experiments were repeated twice with similar results.
Figure 2
Figure 2
C/EBPβ-deficient v-Ha-ras transgenic mice display decreased tumor multiplicity and tumor size. v-Ha-ras+/− C/EBPβ+/+ mice (n = 16) and v-Ha-ras+/− C/EBPβ−/− mice (n = 14) were treated twice weekly with 5 nmol of TPA in 200 μl of acetone. (a) Tumor multiplicity in v-Ha-ras+/− C/EBPβ−/− mice (○) is decreased compared v-Ha-ras+/− C/EBPβ+/+ mice (□; P < 0.05, F test). (b) Tumor size distribution in v-Ha-ras+/− C/EBPβ+/+ mice (closed bars) and v-Ha-ras+/− C/EBPβ−/− mice (open bars; P < 0.05, Fisher's Exact test).
Figure 3
Figure 3
Oncogenic Ha-ras stimulates C/EBPβ transactivation function. (a and b) BALB/MK2 keratinocytes were transfected with pcDNA3-C/EBPβ (0.5 μg) and/or pcDNA3-Ha-ras(12V) (0.5 μg) and 1.0 μg of the specified C/EBP-dependent promoter/reporter as described in the text. Luciferase activity is expressed as fluorescent units per μg of protein, and each value represents the mean ± SD of triplicate dishes per treatment. Similar results were obtained from two repeat experiments. Inclusion of pSV-β-galactosidase and subsequent normalization of luciferase to β-galactosidase activity produced similar results to those normalized to protein levels. (c) BALB/MK2 keratinocytes were transfected with Ha-ras(12V) and/or C/EBPβ, and 48 h later, lysates were prepared and Western analysis was conducted.
Figure 4
Figure 4
Activation of C/EBPβ by oncogenic Ha-ras involves a T188 and requires the presence of the C/EBPβ transactivation domain. BALB/MK2 keratinocytes were transfected with 1.0 μg of the promoter/reporter MGF82-luc and 0.5 μg of one or more of the specified vectors. The experimental procedures were carried out as described in the legend to Fig. 3. Each value represents the mean ± SD of triplicate dishes per treatment. Similar results were obtained from two repeat experiments. Inclusion of pSV-β-galactosidase and subsequent normalization of luciferase to β-galactosidase activity produced similar results to those normalized to protein levels.
Figure 5
Figure 5
Endogenous C/EBPβ is a downstream mediator of oncogenic Ha-ras signaling in keratinocytes. Primary keratinocytes were isolated from C/EBPβ−/− or C/EBPβ+/+ newborn littermates (2–3 days old) and cultured as described (14, 16). Primary keratinocytes were transfected with the specified vector (0.5 μg each) and 1.0 μg of the C/EBP-dependent promoter/reporter vector and were processed as described in the legend to Fig. 3. Each value represents the mean ± SD of triplicate dishes per treatment. Similar results were obtained from two repeat experiments.
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