SF SF + GME K17 epi K16 hf *** *** K16 Nrf2-/- SF SF + BSO Figure 3. SF-induced ﬂuctuations in glutathione levels by SF play a role in keratin induction. (A) Fold-change in total glutathione levels (mean SEM from three experiments) measured in female SKH-1 hairless mouse skin tissue at 2 h after treatment with vehicle alone (adjusted to 1), SF, or SF combined with GME. (B) Indirect immunoﬂuorescence for K17 and K16 in skin tissue sections from SKH-1 mice after three treatments with SF or SF in combination with GME. (C) Fold-change in total glutathione levels (mean SEM from three experiments) measured in female SKH-1 hairless mouse skin tissue at 24 h after treatment with vehicle alone (adjusted to 1), SF, or SF combined with BSO. (D) Indirect immunoﬂuorescence for K16 in skin tissue sections from Nrf2 / mice after three treatments with SF or SF in combination with GME. In A and C, Student’s t test: *p 0.05, **p 0.01, ***p 0.001. For B and D, sections were lightly stained with Hoechst dye to reveal nuclei. Arrows depict the dermo- epidermal interface. Epi, epidermis; hf, hair follicle. Bars, 100 m. To directly test the role of these MAP kinases in the induction of K17 and K16 by SF, SKH-1 hairless mice were topically treated with inhibitors of p38 (100 l of SB20219 at 10 M), JNK (100 l of SP600125 at 100 M), or ERK1/2 (100 l of UO126 at 100 M) at 30 min before and 30 min after topical treatment with 1 mol SF formulated in jojoba oil. These inhibitors have been used topically in mice by many other groups (Jaffee et al., 2000; Takanami-Ohnishi et al., 2002; Bachelor et al., 2005; Lee et al., 2007). An ELISA assay 4072 M. Kerns et al. B K17 K16 A C D 1.2 G ot 1 T of0.8 e ng0.6 a ch0.4 ol0.2 d F 0 1.4 G ot1.2 ofT 1 e0.8 g an0.6 ch0.4 old0.2 F 0 K16 Nrf2-/- Vehicle SF 2 hrs SF + GME 2hrs hf epi Vehicle SF 24 hrs SF + BSO epi was used to conﬁrm the effectiveness of treatment with these inhibitors (Supplemental Figure S3, Supplemental Table S3c). Signiﬁcantly reduced immunoreactivity for K17 oc- curred in the epidermis of mice treated with SF and any of these inhibitors, compared with mice treated with SF alone (Figure 4B, Supplemental Figure S4A, Supplemental Table S1f). On the other hand, only UO treatment reduced K16 immunoreactivity that arises after SF treatment (Figure 4B, Supplemental Figure S4B, Supplemental Table S1f). When ERK1/2 is inhibited, we ﬁnd that both the Nrf2-dependent and -independent pathways are both blocked and the SF- mediated induction of K16 is lost. In the absence of Nrf2 signaling all inhibitor treatments are able to attenuate induc- tion of K16 gene expression (Figure 4C, Supplemental Figure S4C, Supplemental Table S1g). DISCUSSION The main ﬁndings of our study are as follows. First, we identify a gene, namely K17, that is induced in an Nrf2- independent manner in skin epidermis treated with SF. Sec- ond, we show that SF exposure causes a transient decrease in total glutathione levels in mouse skin. Such a decrease in glutathione has been reported for select cell lines in culture (Zhang et al., 2000; Higgins et al., 2009). Third, we show that K17 induction closely parallels the drop in glutathione levels that follows treatment of skin with SF and that this process also involves the activation of MAP kinases. The latter are known to converge on AP-1, a transcription factor already known to participate in the regulation of K6 (Navarro et al., 1995; Ma et al., 1997) and K16 (Wang and Chang, 2003) in other biological settings. Fourth, we show that a topical treatment that reduces glutathione levels induces K17 ex- pression in mouse epidermis, independently of SF exposure. Finally, we show that the SF-mediated epidermal induc- tion of K16 and K17, two neighboring keratin genes that exhibit a high degree of sequence homology and an over- lapping regulation (McGowan and Coulombe, 1998b), occurs through distinct mechanisms in SF-treated keratinocytes (i.e., it entails a differential involvement of Nrf2, glutathione, and MAP kinases). Our ﬁndings suggest a temporal sequence of molecular events that ultimately results in the stepwise activation of select keratin genes in SF-treated epidermis (Figure 5). Early on (e.g., 3 h; Figure 5A), SF induces K17, coinciding with the transient drop in glutathione levels and its associated impact on p38 kinase activity. K17 induction may also in- volve SF’s ability to activate JNK kinase, which, like other MAP kinases including p38, can activate AP-1 and (presum- ably) contribute to activating K17 gene transcription. Later on (e.g., 24 h; Figure 5B), glutathione levels have re- bounded, and activated Nrf2 may at this point represent the main mechanism through which SF impacts gene expression in epidermal keratinocytes. Activation of Nrf2 could also result from the MEK-mediated activation of ERK kinase, in addition to Keap1 inactivation, at this later time (Kang et al., 2007). Once activated, Nrf2 and AP-1 can each, or tandemly, activate K16 gene transcription (Figure 5B). This model could apply to the regulation of additional genes after ex- posure to SF. The lack of uniformity with which SF impacts the expres- sion of K17, K16, and the K6 isoforms in adult mouse epi- dermis (Kerns et al., 2007) was unexpected. The type II K6a and/or K6b are usually cotranscribed with the type I K16 in adult epithelia and sometimes with K17 as well (McGowan and Coulombe, 1998a; Smith et al., 1998). Our ﬁnding that K6a and K6b are not signiﬁcantly induced in Keap1 hypo- Molecular Biology of the Cell