Figure 4. Role of MAP kinases in the modu- lation of keratin expression by SF. (A) Depen- dence of MAP kinase activation on alteration of glutathione levels by SF in skin. Mice were treated with either SF alone or SF in combina- tion with GME, and the activation status of MAP kinases in skin was assessed via ELISA. Data (mean SEM) are reported as fold change over treatment with SF alone (adjusted to unity). (B) Indirect immunoﬂuorescence for K17 and K16 in skin tissue sections after treat- ment with SF alone or in combination with inhibitors to p38 (SF SB), JNK (SF SP), or ERK (SF UO). (C) Indirect immunoﬂuores- cence for K16 in Nrf2 / mouse skin after treat- ment with SF alone, or in combination with in- hibitors to p38 (SF SB), JNK (SF SP), or ERK (SF UO). Sections were lightly stained with Hoechst dye to reveal nuclei. Arrows depict the dermo-epidermal interface. Epi, epidermis; hf, hair follicle. Bars, 100 m. Student’s t test: *p 0.05, **p 0.01, ***p 0.001. morph mutant mice extends our earlier study (Kerns et al., 2007). At ﬁrst glance, this ﬁnding may appear at odds with the report that the K6a gene contains a functional ARE and is robustly induced in the markedly hyperkeratotic esopha- geal epithelium of Keap1 mice (Wakabayashi et al., 2003). This said, SF has been shown to directly inhibit nu- A “Early” Effect of SF B “Late” Effect of SF SF MEK p38 GSH JNK AP-1 K17 GSH MEK ERK K16 AP-1 Nrf2 Figure 5. Model depicting the cascade of signaling events leading to keratin induction “early” (A) and “late” (B) after treatment of mouse epidermis with SF. (A) Within 3 h after SF exposure, a marked decrease in total glutathione levels occurs, coinciding in the activation of MAP kinases. This culminates in activation of AP-1, a transcription factor able to activate K17 expression. (B) Later on, SF can directly stimulate Nrf2 by alleviating its tethering to Keap1 in the cytoplasm. A portion of K16 induction is directly due to Nrf2 transcriptional activation. In addition, glutathione levels have re- bounded and exceed normal baseline levels, stimulating MEK and ERK signaling and leading to AP-1 activation and K16 induction. See Discussion for details. Vol. 21, December 1, 2010 SF and Keratin Expression in Skin clear factor B (NF- B; Heiss et al., 2001), a transcription factor required for K6 gene expression in at least some settings (alone or in combination for other factors such as AP-1; see Navarro et al., 1995; Ma et al., 1997). It could be, therefore, that NF- B inhibition prevents the induction of K6 gene expression after SF treatment. Additional studies are required to further probe this intriguing phenomenon. There is microarray-based evidence for SF-induced gene expression in the absence of Nrf2 in select cell models (Kraft et al., 2004; Hu et al., 2006), though to our knowledge this has not been shown for epidermal keratinocytes. The biochem- ical mechanism(s) involved have not been elucidated. The ﬁndings reported here bring forth several issues of interest. For instance, which genes other than K17 are turned on in an Nrf2-independent manner after keratinocyte exposure to SF and what is their relevance to the overall response mounted by the cell? In this regard several intermediate ﬁlaments genes are rapidly induced whenever the cells and tissues expressing them are subject to environmental challenges. For the K6/K16/K17 subgrouping of keratin genes, such ad- verse conditions include wounding, viral infection, UV ex- posure, and disease processes that result in aberrant differ- entiation within stratiﬁed epithelia (Weiss et al., 1984; Stoler et al., 1988; Jiang et al., 1994; Leigh et al., 1995; Paladini et al., 1996). In a few cases, the role of the IF protein being induced has been deﬁned and includes protection against apoptosis in a signal-speciﬁc manner (see Kim and Coulombe, 2007), stimulation of protein synthesis and cell growth (Kim et al., 2006), providing a phosphate sink for hyperactive, stress- related kinases (Ku and Omary, 2006), and regulation of reactive oxygen species (ROS)-induced and mitochondria- mediated death in hepatic cells (Mathew et al., 2008). The role that K17 might fulﬁll in a cellular context where gluta- thione levels are low is a question worth pursuing. Another issue of interest is the relationship between glutathione and K17 expression. Regulation of the balance between reduced and oxidized forms of glutathione (GSH/GSSG) in the cell, 4073