Nrf transcription factors in keratinocytes are essential for skin tumor prevention but not for wound healing

Abstract

The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice. The functionality of the transgene product was verified in vivo using mice doubly transgenic for dnNrf2 and an Nrf2-responsive reporter gene. Surprisingly, no abnormalities of the epidermis were observed in dnNrf2-transgenic mice, and even full-thickness skin wounds healed normally. However, the onset, incidence, and multiplicity of chemically induced skin papillomas were strikingly enhanced, whereas the progression to squamous cell carcinomas was unaltered. We provide evidence that the enhanced tumorigenesis results from reduced basal expression of cytoprotective Nrf target genes, leading to accumulation of oxidative damage and reduced carcinogen detoxification. Our results reveal a crucial role of Nrf-mediated gene expression in keratinocytes in the prevention of skin tumors and suggest that activation of Nrf2 in keratinocytes is a promising strategy to prevent carcinogenesis of this highly exposed organ.

FIG. 1.

Expression and functionality of dnNrf2 in the skin of transgenic mice. (A) Left panel: 20 μg total cellular RNA from the skin of dnNrf2 transgenic mice (lines 3 and 16) and of wild-type littermates was analyzed by RNase protection assay for the expression of endogenous Nrf2 and dnNrf2 using a riboprobe that can distinguish between RNAs encoding the wild-type and the mutant proteins. One thousand counts per minute of the hybridization probe was loaded in the lane labeled “probe” and used as a size marker. Twenty micrograms of tRNA was used as a negative control. Middle panel: protein lysates from the skin of dnNrf2 transgenic mice (lines 3 and 16) and of wild-type littermates were analyzed by Western blotting for the presence of the dnNrf2 protein. Lysates from COS-1 cells transiently transfected with the transgene construct were used as a positive control. The sensitivity of the blot is insufficient to detect the endogenous Nrf2. Right panel: immunofluorescence analysis of tail skin of transgenic mice using antibodies against Nrf2 (red) and pankeratin (green). The transgene construct is shown above. (B) Alkaline phosphatase (upper panels) and hematoxylin and eosin (lower panels) staining of hyperproliferative back skin of ARE-hPAP (left panels) or ARE-hPAP/K14-dnNrf2 (right panels) double-transgenic mice topically treated with tBHQ. The staining in double-transgenic animals reveals the specific inhibition of Nrf2 action in the epidermis. D, dermis, E, epidermis, HF, hair follicle. (C) Primary keratinocytes from dnNrf2 transgenic mice and wild-type littermates were treated with tBHQ (50 μM) (5h) or DMSO (5hc) for 5 h. Ten μg total RNA was analyzed by RPA for the expression of γ-GCS_h, GST-Ya, HO-1, and GAPDH.

FIG. 2.

Normal wound healing in K14-dnNrf2 mice and ARE activation in murine skin wounds. (A) Full-thickness excisional wounds were made on the back of wild-type (wt) and K14-dnNrf2 transgenic (tg) mice. Sections from the middle of 1-day, 5-day, and 13-day wounds were stained with hematoxylin and eosin. (B) Wounds were made on the back of ARE reporter mice and wild-type littermates. Sections from the middle of 5-day wounds were stained for hPAP activity. Keratinocytes were visualized by keratin 14 staining (green). Note the hPAP staining (red) of cells in the granulation tissue (indicated by arrows) and the lack of hPAP activity in the hyperproliferative epithelium. CL, clot; D, dermis; E, epidermis; ES, eschar; HE, hyperproliferative epithelium; G, granulation tissue. The dashed white line indicates the border of the wound area.

FIG. 3.

Enhanced tumor susceptibility of K14-dnNrf2 transgenic mice. Back skin of transgenic mice and wild-type littermates was treated once topically with DMBA in acetone. Subsequently, the animals were treated weekly with TPA in acetone for 20 weeks. (A) Tumor incidence. The number of animals with papillomas was determined every week. The graph shows the percentages of animals with papillomas (n = 28 per genotype). (B) Tumor multiplicity. The number of papillomas per mouse was determined every week. The graph represents the averages of 28 animals per genotype. Error bars indicate the standard errors of the means. (C) Representative DMBA/TPA-treated wild-type or transgenic mice are shown. (D) Sections from papillomas from transgenic animals and wild-type littermates were stained with hematoxylin and eosin. Papillomas from both genotypes show characteristic cellular atypisms and inflammatory infiltrates. D, dermis; E, epidermis; P, papilloma. Stainings of tumor sections with an antibody against cleaved caspase 3 are shown above the histological pictures (left picture, middle of the tumor; right picture, edge of the tumor).

FIG. 4.

Inhibition of Nrf2-mediated gene expression does not affect the frequency of malignant conversion of skin tumors. Progression from papillomas to squamous cell carcinomas was monitored in nine wild-type and nine transgenic mice. (A) The ratio of carcinomas to papillomas at week 15 after the last TPA treatment as well as the frequency of malignant conversion is indicated. (B) Sections from squamous cell carcinomas of a wild-type mouse and a transgenic mouse were stained with hematoxylin and eosin. The tumors were identified as squamous cell carcinomas by a histopathologist. S, stroma; T, tumor cells.

FIG. 5.

Lack of ARE activation by DMBA and TPA during the development of skin papillomas. ARE reporter mice were subjected to the two-stage skin carcinogenesis protocol. Sections from DMBA- and DMBA/TPA-treated skin were analyzed by alkaline phosphatase staining to determine ARE reporter activity (upper panels) or stained with hematoxylin and eosin (lower panels). D, dermis; E, epidermis; HF, hair follicle.

FIG. 6.

Reduced expression of cytoprotective target genes and enhanced oxidative stress in skin and papillomas of dnNrf2 transgenic mice. (A) Samples of 20 μg total RNA from nontumorigenic skin (pools of five biopsy samples per genotype) and from papillomas (pools of five biopsy samples per genotype) of wild-type and transgenic mice were analyzed by RPA for the presence of NQO1, γ-GCS_h, IL-6, IL-1β, Nrf2, and GAPDH mRNAs. Twenty micrograms of tRNA was used as a negative control. One thousand cpm of the hybridization probes was loaded in the lanes labeled “probe” and used as a size marker. Densitometric quantification of each RNase protection assay result (normalized to GAPDH) is shown on the right side. The strongest signal was arbitrarily set as 100. (B) Thirteen micrograms of total protein from untreated skin, from nontumorigenic skin which was treated for 20 weeks with DMBA/TPA, and from papillomas of wild-type and transgenic animals was analyzed for the presence of oxidized proteins by Oxyblot analysis. (C) Twenty micrograms of total protein from DMBA-treated skin of wild-type and transgenic mice and 7.5 μg total protein from skin, which was treated once with DMBA and once with TPA were analyzed for the presence of oxidized proteins by Oxyblot analysis. Staining of the same membrane with an antibody directed against β-actin served as a loading control in panel B. Staining with an antibody against lamin A served as a loading control in panel C.

FIG. 7.

Model for skin tumor prevention by Nrf transcription factors. The carcinogen DMBA causes mutations in the DNA of keratinocytes, including a critical mutation in the ha-ras proto-oncogene (30). Detoxification of DMBA is at least in part achieved by the Nrf targets NQO1 and GST-π (21, 45). On the other hand, DMBA as well as TPA triggers the generation of ROS, which further enhance the rate of mutagenesis. In addition, ROS enforce the activity of TPA as a tumor-promoting agent (48). Nrf transcription factors allow ROS detoxification through positive regulation of ROS-detoxifying enzymes, such as γ-GCS and NQO1.