SF and Keratin Expression in Skin Figure 2. Role of glutathione in SF-mediated alterations in keratin expression. (A) Time-dependent changes in cellular levels of glutathione in skin after exposure to SF. Reported data (mean SEM) are from three independent experiments. (B and C) Modulation of glutathione levels in the skin of 7-wk-old SKH-1 mice that received four i.p. injections of 50 M BSO or 5 M GME on consecutive days and were harvested 24 h after the ﬁnal treatment. Data (mean SEM) are expressed as fold change relative to mice injected with PBS. (D) Indirect immunoﬂuorescence for keratins 16 and 17 (as indicated in the top left corner) on frozen sections prepared from mice treated with PBS (Cont), BSO, or GME as described for B and C. Sections were lightly stained with Hoechst dye to reveal nuclei. Arrows depict the dermo-epidermal interface. Small bright dots are staining artifacts. Epi, epidermis; hf, hair follicle. Bar, 100 m. (E and F) Quantitation of fold-change in immunoﬂuorescence, relative to control, in the epidermis of 7-wk-old SKH-1 mice after BSO or GME treatment, as described for B and C. Student’s t test: *p 0.05, **p 0.01, ***p 0.001. expression as shown by immunoﬂuorescence (Figure 2, D and E; Supplemental Table S1c). Likewise, systemic treat- ment with GME signiﬁcantly raised glutathione levels in the backskin of SKH-1 hairless mice to 167 7% relative to vehicle-treated control (Figure 2C; Supplemental Table S2c). The epidermis of the GME-treated mice was immunoreactive for K16 but showed no K17 signal beyond hair follicles (Figure 2, D and F; Supplemental Table S1c), where K17 is constitu- tively expressed (McGowan and Coulombe, 1998a,b). Systemic treatment with GME partially prevented the de- crease in glutathione levels 2 h after treatment of skin tissue with SF, creating an opportunity to assess whether this drop plays a role in K17 and/or K16 induction. Glutathione levels in dorsal skin of SKH-1 mice treated with both GME and SF is 83 6% of vehicle-treated mice, compared with 50 11% when treated with SF alone (Figure 3A; Supplemental Table S2d). Remarkably, no induction of K17 was observed in the epidermis of mice treated with both SF and GME (Figure 3B; Supplemental Figure S2A and Supplemental Table S1d). In contrast, the induction of K16 was unaltered in this context (Figure 3B). Next we wanted to test whether the SF-medi- ated induction of K16 treatment shows a dependency on the late-onset rise in the glutathione levels, in the absence of Nrf2. In SKH-1 mice, systemic treatment of SKH-1 mice with BSO partly prevents the rise in glutathione levels 24 h after SF treatment. Glutathione levels were 74 1% of vehicle-treatment, compared with 121 6% seen with SF treatment alone, in such mice (Figure 3C; Supplemental Table S2e). When examined in Nrf2 / SKH-1 mouse epi- dermis, induction of K16 is markedly attenuated after com- bined treatment with SF and BSO relative to SF alone (Figure 3D, Supplemental Figure S2B and Supplemental Table S1e). Together these results suggest that the modulation of total glutathione levels, in either direction, is poised to play a key role in the SF-mediated induction of K16 and K17 in skin tissue. Vol. 21, December 1, 2010 SF-induced Keratin Expression Involves MAP Kinase Activation Previous studies have implicated the MAP kinases p38, ERK, and JNK in SF-mediated chemoprevention (e.g., Cho et al., 2005; Hwang et al., 2005; Jo et al., 2007). In addition, glutathione has been shown to modulate several MAP ki- nase cascades, including ERK, p38, and JNK (Limon-Pa- checo et al., 2007). Each of these MAP kinases converge on AP-1, a transcription factor that regulates the differential expression of K6 among other keratin genes (Navarro et al., 1995; Ma et al., 1997), raising the possibility that they could be involved in the SF-induced, Nrf2-independent expression of K17 and/or K16. We treated SKH1- mice with 1 mol SF (or vehicle control) and measured the levels of total and phosphorylated forms of key MAP kinases using ELISA. At 2 h after SF treatment, the time at which glutathione levels are decreased (Figure 2A), there were statistically signiﬁcant in- creases in phospho-p38 ( 1.2-fold) and phospho-MEK1/2 ( 2.2-fold), an increase in phospho-SAP/JNK ( 1.7-fold), and little if any change in phospho-ERK1/2 (Figure 4A, data expressed relative to vehicle-treated skin; see Supplemental Table S3a for corresponding absolute values and p values). Compared with controls, systemic GME treatment reduced the activation of phospho-p38 (by 45%) and phospho- MEK1/2 (by 56%) at 2 h after SF treatment (Figure 4A; Supplemental Table S3a). At 24 h after SF treatment, a time at which glutathione levels are slightly above normal (Figure 2A), the levels of phospho-p38 ( 0.93-fold), phospho-SAP/JNK ( 1.03-fold), phospho-MEK1/2 ( 1.2-fold), and phospho- ERK1/2 ( 0.90-fold) are similar to levels observed of vehicle- treated mice (Supplemental Table S3b). These ﬁndings suggest that the transient activation of p38 and MEK1/2 kinases shortly after skin keratinocyte exposure to SF is partly due to the depletion in glutathione levels, whereas JNK activation occurs independent of the glutathione status. 4071