c-Fos-dependent induction of the small ras-related GTPase Rab11a in skin carcinogenesis

Abstract

Malignant transformation of mouse skin by tumor promoters and chemical carcinogens, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), is a multistage process leading to the formation of squamous cell carcinomas. It has been shown that mice lacking the AP-1 family member c-Fos exhibit an impaired transition from benign to malignant skin tumors. Here, we demonstrate enhanced expression of the small Ras-related GTPase Rab11a after short-term TPA treatment of mouse back skin. Expression of Rab11a in vivo and in vitro critically depended on c-Fos, because TPA application to the back skin of c-Fos-deficient mice and to mouse embryonic fibroblasts did not induce Rab11a mRNA or protein expression. Moreover, dexamethasone, which is a potent inhibitor of AP-1-mediated transactivation that exhibits anti-inflammatory and anti-tumor promoting activities, inhibited TPA-induced expression of Rab11a. Within the Rab11a gene promoter, we identified a functional AP-1 binding element that exhibited elevated c-Fos binding activity after TPA treatment of keratinocytes. Enhanced expression was not restricted to chemically induced mouse skin tumors but was also found in tumor specimens derived from patients with epithelial skin tumors. These data identify Rab11a as a novel, tumor-associated c-Fos/AP-1 target and may point to an as yet unrecognized function of Rab11a in the development of skin cancer.

Figure 1

TPA-induced expression of Rab11a mRNA and protein in mouse back skin. A: Back skin of wild-type mice was treated with acetone (lane 1, co) or TPA (lane 2, TPA). Animals were sacrificed after 6 hours and total RNA was isolated. The level of Rab11a transcripts was visualized by Northern blot analysis. Rehybridization with an 18S-rRNA-specific probe was performed serving as a loading control for RNA quantity and quality. B: PMK-R3 mouse keratinocytes were treated with acetone (lane 1, co) or TPA (lane 2, TPA). Cells were harvested for RNA preparation 6 hours thereafter and analyzed as described in A. C: Paraformaldehyde-fixed and paraffin-embedded sections (6 μm) of acetone- and TPA-treated mouse back skin was analyzed by indirect immunofluorescence using a monoclonal antibody raised against Rab11. Staining was performed using a Cy3-labeled secondary antibody and H33342 for nuclear staining (blue signal). Specific signals were visualized by immunofluorescence microscopy. Expression of Rab11a protein (signal in red) was detectable in keratinocytes and dermal fibroblasts 6 hours after TPA application and was even stronger after 12 hours. Only basal expression was visible in skin of acetone-treated animals (0 hours). Blocking of the antibody signal with a blocking peptide served as a control for specificity (12 h_co). The dashed line indicates the border between epidermis and dermis. Scale bar, 25 μm.

Figure 2

Impaired TPA-induced expression of Rab11a mRNA and protein by co-treatment with dexamethasone and in the absence of c-Fos. Back skin of wild-type (fos^+/+) or c-Fos-deficient mice (fos^−/−) was treated with acetone (co), TPA (TPA), or TPA together with dexamethasone (T/D). Animals were sacrificed after 6 hours and back skin was used for total RNA isolation or was fixed in paraformaldehyde and embedded in paraffin. A: Expression of Rab11a mRNA and 18S-rRNA was determined as described in Figure 1. Results of two independent experiments were quantified and the relative fold induction is shown. B: Expression of Rab11a and Rab11b mRNA was ana-lyzed in wild-type (fos^+/+) and c-Fos-deficient mice (fos^−/−) after treatment with acetone (co), TPA (TPA), TPA and dexamethasone (T/D), or dexamethasone alone (Dex). Expression of Hprt served as internal control and relative expression in acetone-treated skin was set to one. The bars represent the mean value of three independent experiments and the SD is indicated. C: Sections (6 μm) of mouse back skins were analyzed by indirect immunofluorescence using a specific monoclonal antibody. Staining was performed using a Cy3-labeled secondary antibody and H33342 for nuclear staining (blue signal). Specific signals were visualized by immunofluorescence microscopy. Expression of Rab11a protein (red signal) was detectable in TPA-treated skin of wild-type animals (fos^+/+ + TPA) but not in skin of TPA-treated mice lacking c-Fos (fos^−/− + TPA). The dashed line indicates the border between epidermis and dermis. Scale bar, 25 μm.

Figure 3

Rab11a is a direct c-Fos target gene. A: Schematic representation of the mouse Rab11a promoter region. Three potential TRE elements and their position according to the transcriptional start site are shown. B:In vitro protein-binding assay with nuclear extract of control (−) and TPA-treated (+) PMK-R3 cells using primers containing the identified TRE elements. The classical TRE element of mouse collagenase-3 (TRE) was used as a positive control for AP-1 complex formation and an octamer probe (Oct) served as control for quality and quantity of nuclear extracts. C: ChIP analysis was performed with extracts of control (−) and TPA-treated (+) PMK-R3 cells using a c-Fos-specific antibody. Immunoprecipitated material was amplified with primers located within the mouse Rab11a promoter region that were specific for TRE1, TRE2, and TRE3 or with primers of the coding region (Rab11a) as a negative control. The material used for co-immunoprecipitation was amplified with primers specific for TRE1 as a control for quality and quantity. D: Western immunoblot with protein extracts of acetone-treated (−) and TPA-treated (+) wild-type (fos^+/+) and c-Fos-deficient (fos^−/−) MEFs. Expression of β-actin served as control for quality and quantity of protein extracts.

Figure 4

Expression of Rab11a mRNA and protein in chemically induced papillomas and SCC. A: Expression of Rab11a mRNA was analyzed by RQ-PCR as described in Figure 2B using biopsies taken from mouse skin 6 hours after TPA treatment (TPA) or from chemically induced mouse skin tumors such as papillomas (Pap) and SCC. B: ³⁵S-UTP-labeled anti-sense riboprobe of Rab11a was hybridized to sections of biopsies taken from chemically induced mouse skin tumors. Expression of Rab11a mRNA was detectable in neoplastic keratinocytes of both benign papilloma (Pap) and SCC. Sections are representative for three independent experiments. ³⁵S-UTP-labeled sense riboprobe served as controls for specificity and are shown as inlets in Pap_DF and SCC_DF. Indirect immunofluorescence analysis using a monoclonal antibody raised against Rab11 and parallel sections of the skin tumors revealed protein expression at the same area (SCC_α-Rab11). Staining was performed using a Cy3-labeled secondary antibody (red signal) and H33342 for nuclear staining (blue signal). Incubation without primary antibody served as control for specificity (SCC_co). Specific signals were visualized by immunofluorescence microscopy. Pap_BF and SCC_BF are bright-field images and Pap_DF and SCC_DF are dark-field images. Arrows indicate specific signals within the tissue section. Scale bar, 100 μm.

Figure 5

Expression of Rab11a protein in human skin tumors. Indirect immunofluorescence analysis of Rab11a protein level on human biopsies was done using a specific monoclonal antibody. The specimens represent skin (A, B), two SCCs (C, D), one keratoacanthoma (E), and one BCC (F). Staining was performed using a Cy3-labeled secondary antibody and H33342 for nuclear staining (blue signal). B: Treatment with secondary antibody alone served as control. Specific signals were visualized by immunofluorescence microscopy. Scale bar, 50 μm.