AP1 factor inactivation in the suprabasal epidermis causes increased epidermal hyperproliferation and hyperkeratosis but reduced carcinogen-dependent tumor formation

Abstract

Activator protein one (AP1) (jun/fos) factors comprise a family of transcriptional regulators (c-jun, junB, junD, c-fos, FosB, Fra-1 and Fra-2) that are key controllers of epidermal keratinocyte survival and differentiation, and are important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each member is expressed in defined cell layers during epidermal differentiation, and because AP1 factors regulate competing processes (that is, proliferation, apoptosis and differentiation). We have proposed that AP1 factors function differently in basal versus suprabasal epidermis. To test this, we inactivated suprabasal AP1 factor function in mouse epidermis by targeted expression of dominant-negative c-jun (TAM67), which inactivates function of all AP1 factors. This produces increased basal keratinocyte proliferation, delayed differentiation and extensive hyperkeratosis. These findings contrast with previous studies showing that basal layer AP1 factor inactivation does not perturb resting epidermis. It is interesting that in spite of extensive keratinocyte hyperproliferation, susceptibility to carcinogen-dependent tumor induction is markedly attenuated. These novel observations strongly suggest that AP1 factors have distinct roles in the basal versus suprabasal epidermis, confirm that AP1 factor function is required for normal terminal differentiation, and suggest that AP1 factors have a different role in normal epidermis versus cancer progression.

Fig. 1

Suprabasal TAM67 expression produces epidermal hyperproliferation and hyperkeratosis. A TAM67 expression alters epidermal phenotype. TAM67 and TAM67-rTA (bi-transgenic) mice were treated with drinking water (-Dox) or drinking water supplemented with 2 mg/ml doxycycline (+Dox) for 12 d. The dorsal epidermis was harvested on day twelve and sections were prepared and stained with H&E. The epidermis is indicated (Epi). B TAM67-FLAG is expressed in Dox-treated TAM67-rTA mice. Total cell extracts, prepared from dorsal epidermis, were electrophoresed and immunoblotted with HRP-conjugated mouse monoclonal anti-FLAG M2 (Sigma, A8592) and signal was detected using chemiluminescence detection reagents. Migration of the TAM67-FLAG fusion protein is indicated by an arrow. Gel loading was normalized based on β-actin level (not shown). C TAM67-FLAG is detected in the nucleus of suprabasal keratinocytes. TAM67-rTA mice were treated with or without doxycycline and frozen sections were incubated with mouse monoclonal anti-FLAG (Sigma, F1804) followed by HRP-conjugated sheep anti-mouse IgG (Amersham, NA931). The sections were not counterstained. As a second method of detection, paraffin-embedded sections were stained with FITC-conjugated anti-FLAG (Sigma, F1804). These are 1 μm confocal optical sections. The epidermis (Epi) and dermis are indicated and separated by a dotted line. The arrows identify nuclear TAM67-FLAG staining.

Fig. 2

Differentiation and proliferation in suprabasal TAM67 epidermis. A Suprabasal TAM67 expression increases keratinocyte proliferation. TAM67-rTA mice were treated with or without doxycycline for 12 d. The mice were shaved on day ten, injected with 100 μg BrdU per gram body weight on day twelve, and euthanized 2 h later. Paraffin-embedded sections were prepared for detection of BrdU (peroxidase staining) and Ki67 (Fluorescence staining). Arrows indicate BrdU- or Ki67-positive nuclei. The number of BrdU-positive and Ki67-positive nuclei were counted and expressed per unit length of epidermis in five random sections derived each of three mice per treatment group. This analysis indicated five times more BrdU-positive cells per unit length of epidermis in Dox treated versus not-treated TAM67-rTA mice. B Suprabasal TAM67 expression delays keratinocyte differentiation. Epidermis from TAM67-rTA mice, treated for 12 d with or without doxycycline, was embedded in paraffin and sections were stained to detect the indicated epitopes. These are 1 μm confocal optical sections. C TAM67 impact on marker protein level. Two TAM67-rTA littermates were treated with (+Dox) and two without (-Dox) doxycycline for 12 d and epidermal extracts were prepared and electrophoresed for detection of the indicated proteins.

Fig. 3

Tumor formation in suprabasal TAM67 expressing epidermis. A Reduced tumor formation in mice expressing suprabasal TAM67. TAM67-rTA mice were treated with or without a single topical application of 100 μg DMBA followed 1 week later by treatment with or without doxycycline and TPA for 22 weeks as outlined in Materials and Methods. Mice were observed weekly for tumor onset, number and size. Each treatment group included ten animals. The values are mean ± SEM. At 10 - 21 weeks, there were significantly more tumors in the DMBA + TPA group as compared to the DMBA + TPA (+Dox) group, p < 0.5 as compared using the t-test. B Reduced tumor size in suprabasal TAM67 mice. Tumor size was monitored using a caliper and the formula tumor volume = (length × Width²)/2. Only the DMBA/TPA treated (+/- Dox) groups are shown. Each treatment group includes ten animals and the values are mean ± SEM. Tumor size at 13 - 18 weeks was significantly greater in the DMBA/TPA group as compared to the DMBA + TPA (+Dox) group, p < 0.5 as compared using the t-test. C DMBA/TPA treatment produces benign papillomas. Tumors, derived from DMBA/TPA-treated TAM67-rTA mice, were collected and sectioned for H&E staining and immunofluorescence detection of TAM67-FLAG using anti-FLAG. The fluorescence picture shows a confocal 1 μm optical section. TAM67-FLAG is localized in tumor cell nuclei. Optical scanning of the tissue revealed that most tumor cells are TAM67-FLAG positive. The arrows indicate nuclear staining.

Fig. 4

Response to acute TPA treatment. A Suprabasal TAM67 positive epidermis resembles TPA-treated skin. TAM67-rTA mice were maintained on drinking water with or without doxycycline for 12 d followed by topical application of TPA as indicated. After 24 h of TPA treatment the tissue was harvested and sectioned for H&E staining or incubation with the indicated antibody. The fluorescent pictures are confocal 1 μm optical sections. B Acute TPA treatment increases proliferation in TAM67-positive epidermis. TAM67-rTA mice were maintained on dietary doxycycline for 12 d. The epidermis was then challenged with 10 μg TPA/100 μl acetone and tissue was harvested 24 h later for staining to Ki67.

Fig. 5

Lack of response in corneal epithelium. A/B High level expression of TAM67-FLAG in mouse corneal epithelium. TAM67-rTA mice were treated with or without doxycycline for 12 d and the epidermal and corneal epithelium were harvested for assay of TAM67-FLAG level and to prepare histological sections. The immunoblot results are normalized based on detection of β-actin (not shown).