M. Kerns et al. Figure 1. Nrf2-independent modulation of keratin expression in skin topically treated with SF. (A) Indirect immunoﬂuorescence for K6, K16, and K17 (as indicated in the top left corner) in frozen sections prepared from adult SKH-1/Nrf2 / mouse skin treated either with vehicle (Cont, control) or SF. Sections were lightly stained with Hoechst dye to reveal nuclei. Arrows depict the dermo-epidermal interface. Epi, epidermis; hf, hair follicle. Bar, 100 m. (B) Quantiﬁcation of fold-change in immunoﬂuorescence signal in the epidermis of SF-treated adult SKH-1/Nrf2 / mice, relative to treated controls (see A). (C) Quantiﬁcation of fold-change in immunoﬂuorescence signal in the epidermis of Nrf2 / mice in the C57/Bl6 background, relative to treated controls (see D). (D) Pattern of keratin expression in 8-d-old control and SF-treated Nrf2 / mice in the C57/Bl6 background. Bar, 100 m. (E) Western immunoblots depicting levels of K16 and 17 antigens (proteins) in response to control or SF treatment of adult Nrf2 / mice. Actin was used as a loading control. Student’s t test: *p 0.05, **p 0.01, ***p 0.001. Relative to vehicle treatment, Nrf2 mice topically treated with 1 mol SF consistently showed a signiﬁcant increase of K17 and K16 immunoreactivity in the epidermis (Figure 1, A and B; see Supplemental Table S1a for numer- ical data and p values) and Western blotting (Figure 1E). A minimal impact was seen on K6 expression (Figure 1, A and B). The epidermis of SF-treated Nrf2 mice in the SKH-1 background was thicker than in controls. Epidermal thick- ening was previously observed after chronic SF treatment of SKH-1 hairless mice (Kerns et al., 2007). A similar change in keratin expression was observed after SF treatment of Nrf2 pups in the C57BL6 strain background (e.g., Figure 1, C and D; Supplemental Table S1b), indicating that this out- come was not a function of the genetic background. We examined the mRNA levels for select genes in back- skin tissue of adult C57BL6 mice carrying a hypomorphic Keap1 allele. Previous studies of these mice showed en- hanced Nrf2-dependent transcription in several tissues (Os- burn et al., 2008). Relative to wild type, Keap1 mRNA levels were reduced to 60% in Keap1 mutant skin, after normal- ization to HPRT (hypoxanthine phosphoribosyltransferase) levels (Supplementary Figure S1A). K16 and K6 mRNAs were elevated 2.1- and 1.4-fold, respectively, in Keap1 mutant skin. K17 mRNA, in contrast, remained unaltered, whereas the K14 mRNA was slightly decreased (Supple- mental Figure S1A). An increase in K6 and K16 immunore- activity, but not K17, was detected in Keap1 mutant epider- mis (Supplemental Figure S1B). The ﬁndings garnered from Nrf2 and Keap1 mutant mice suggest that the induction of K16 in SF-exposed epidermal keratinocytes proceeds through Nrf2-dependent and -independent mechanisms, whereas K17 induction occurs independently from Nrf2. Although not a primary concern in this study, the issue of how the type I-to-type II keratin ratio is maintained after SF treatment is of interest. Preliminary studies revealed modest 4070 increases in the steady-state levels of K5 and K1, but no change in K6 antigens, in extracts prepared from SF-treated skin (data not shown). Follow-up studies are required to address this issue in full. Correlation of Glutathione Levels and Induction of Keratins by SF in Skin Epidermis In cultured hepatocytes, SF directly conjugates with GSH, an antioxidant with a critical role in the cellular response to stress, resulting in lowered GSH levels 30 min after treat- ment (Zhang, 2000), and a subsequent elevation of GSH levels 24 h after treatment (Ye and Zhang, 2001). Similar ﬁndings were recently reported in SF-treated mouse embry- onic ﬁbroblasts (Higgins et al., 2009). We tested whether SF impacts glutathione levels in a similar manner in mouse skin tissue. Total glutathione levels were measured at 1.5, 2, 5.5, and 24 h after topical treatment of SKH-1 hairless mice with 1 mol SF or vehicle. There was no signiﬁcant change in glutathione levels at 1.5 h after SF treatment. At 2 h, gluta- thione levels had fallen to 53 11% of those seen in control- treated skin. This was followed by a rise in glutathione levels to 117 17 and 121 06% of controls by 5.5 and 24 h, respectively (Figure 2A; see Supplemental Table S2a for absolute values and statistics). Next, we tested whether the modulation of glutathione levels impacts K16 and K17 expression in mouse skin epi- dermis. Female SKH-1 hairless mice were treated systemi- cally (see Materials and Methods) with BSO, an inhibitor of glutathione synthesis, or GME, a form of glutathione readily taken up by cells. Total glutathione levels were measured in skin samples. Systemic treatment with BSO of SKH-1 hair- less mice lowered glutathione levels in the backskin to 51 4% of vehicle-treated control (Figure 2B; Supplemental Table S2b) and was associated with a signiﬁcant increase in K17 and a smaller, but statistically signiﬁcant increase in K16 Molecular Biology of the Cell