Transcription factor Nrf2 (encoded by Nfe2l2) regulates a battery of detoxifying and antioxidant genes, and Keap1 represses Nrf2 function. When we ablated Keap1, Keap1-deficient mice died postnatally, probably from malnutrition resulting from hyperkeratosis in the esophagus and forestomach. Nrf2 activity affects the expression levels of several squamous epithelial genes. Biochemical data show that, without Keap1, Nrf2 constitutively accumulates in the nucleus to stimulate transcription of cytoprotective genes. Breeding to Nrf2-deficient mice reversed the phenotypic Keap1 deficiencies. These experiments show that Keap1 acts upstream of Nrf2 in the cellular response to oxidative and xenobiotic stress.

In Japan, the number of patients with malignant skin tumors has increased year by year. The prognosis of patients with advanced malignant melanoma remains extremely poor, but that of patients in stage III has shown an improvement.

Ceramides are the major component of the stratum corneum, accounting for 30%-40% of stratum corneum lipids by weight, and are composed of at least seven molecular groups (designated ceramides 1-7). Stratum corneum ceramides, together with cholesterol and fatty acids, form extracellular lamellae that are responsible for the epidermal permeability barrier. Previous studies indicated that beta-glucocerebrosidase- and sphingomyelinase-dependent ceramide production from glucosylceramides and sphingomyelins, respectively, is important for epidermal permeability barrier homeostasis. A recent study indicated that sphingomyelins are precursors of two stratum corneum ceramide molecular groups (ceramides 2 and 5). In this study, we have examined the role of glucosylceramides in the generation of each of the seven stratum corneum ceramide molecular groups. First, the structures of various glucosylceramide species in human epidermis were determined by gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. The results indicate that total epidermal glucosylceramides are composed of six distinct molecular groups, glucosylceramides 1-6. Glucosylceramide 1 contains sphingenine and nonhydroxy fatty acids, glucosylceramide 2, phytosphingosine and nonhydroxy fatty acids, glucosylceramide 3, phytosphingosine with one double bond and nonhydroxy fatty acids, glucosylceramide 4, sphingenine and alpha-hydroxy fatty acids, glucosylceramide 5, phytosphingosine and alpha-hydroxy fatty acids, and glucosylceramide 6, phytosphingosine with one double bond and alpha-hydroxy fatty acids. The nonhydroxy fatty acids typically have 16-24-carbon-length chains, whereas alpha-hydroxy fatty acids are limited to 24-, 25-, and 26-carbon chains. The sphingosine bases are C18 or C20 chains. Next, acylglucosylceramides and glucosylceramides were treated with beta-glucocerebrosidase and the ceramides released were compared with stratum corneum ceramides. Ceramide moieties of acylglucosylceramides and glucosylceramides 1, 2, 4-6 correspond to stratum corneum ceramides 1-7. These results, together with those of our previous reports characterizing epidermal sphingomyelins, indicate that all ceramide species, including omega-hydroxy fatty-acid-containing ceramides, are derived from glucosylceramides, and fractions of ceramides 2 and 5 are from sphingomyelins. Furthermore, structural analysis of glucosylceramides revealed that human epidermal glycosphingolipids display a unique lipid profile that is rich in very long chain hydroxylated (alpha- and omega-hydroxy) fatty acids and phytosphingosine.

Aberrant methylation of promoter CpG islands (CGI) is involved in silencing of tumor suppressor genes and is also a potential cancer biomarker. Here, to identify CGIs aberrantly methylated in human melanomas, we did a genomewide search using methylation-sensitive representational difference analysis. CGIs in putative promoter regions of 34 genes (ABHD9,

Ultraviolet (UV) radiation is one of the most important environmental factors involved in the pathogenesis of skin aging and cancer. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species, and cellular antioxidants act to prevent the occurrence and reduce the severity of UV-induced skin disorders. Transcription factor NF-E2-related Factor 2 (Nrf2) and its cytoplasmic anchor protein Kelch-like-ECH-associated protein 1 (Keap1) are central regulators of the cellular antioxidant response. In this study, we investigated the effects of UV irradiation on the activation of Nrf2 in dermal fibroblasts. We found that UVA irradiation, but not UVB, causes nuclear translocation and accumulation of Nrf2 by a factor of 6.5 as compared with unirradiated controls. The nuclear accumulation of Nrf2 induced by UVA was enhanced by the photosensitizer hematoporphyrin. To evaluate the protective role of Nrf2 against UVA radiation, we examined UVA-induced apoptosis using dermal fibroblasts derived from nrf2 or keap1 gene knockout mice. Whereas disruption of nrf2 increased the number of apoptotic cells following UVA irradiation by 1.7-fold, disruption of keap1 decreased the apoptotic cell number by half as compared with wild-type controls. These findings thus demonstrate that the Nrf2-Keap1 pathway plays an important role in the protection of the skin against UVA irradiation.