related to K14 both in primary structure and properties (Kerns et al., 2007). In this setting, K16 and especially K17 are thought to act as surrogate keratins which, owing to their ability to copolymerize efﬁciently with partner K5, compen- sate for the loss of K14 protein (Kerns et al., 2007; Coulombe et al., 2009). Broccoli sprout extracts rich in SF, the natural product form in which known amounts of the latter can be safely delivered to humans (Shapiro et al., 2006; Cornblatt et al., 2007), represents a form of therapy worth considering for epidermolysis bullosa simplex and related conditions (Kerns et al., 2007; Coulombe et al., 2009). Several issues must be addressed in preparation for clinical trials testing this idea. Here, we investigate the mechanisms that account for the induction of K16 and K17 expression in mouse skin keratin- ocytes treated with SF in vivo. MATERIALS AND METHODS Animals, Antibodies, and Chemicals Studies involving mice were approved by the Johns Hopkins University Institutional Animal Care and Use Committee. SKH-1 hairless mice were purchased (Charles River Laboratories, Wilmington, MA). Nrf2 / and hy- pomorphic Keap1 mice were maintained as described (Chan et al., 1996; Osburn et al., 2008). Antibodies used included rabbit polyclonals against K6, K16, and K17 (P.A.C. laboratory) and a mouse monoclonal anti-actin (Sigma, St. Louis, MO). Fluorochrome-conjugated goat anti-rabbit antibodies were from Kirkegaard & Perry Laboratories (Gaithersburg, MD). SF [1-isothiocya- nato-4(R,S)-(methylsulﬁnyl)butane] was obtained from LKT Laboratories (St. Paul, MN). Solutions of SF in jojoba oil (MP Biomedicals, Solon, OH) were prepared on the day of application. Glutathione monoethyl ester, c-Jun N- terminal kinase (JNK) II inhibitor SP60015, p38 inhibitor SB202190, and ex- tracellular signal–regulated kinase (ERK)1/2 inhibitor UO126 were from Cal- biochem (San Diego, CA). Mitogen-activated protein (MAP) kinase inhibitors were diluted in 1:250 acetone:oil and made fresh before each application. l-Buthionine-sulfoximine was from Sigma (St. Louis, MO). Mouse Studies Seven-week-old female Nrf2 / SKH-1 hairless were topically treated with 1 mol SF in 100 l jojoba oil once a day for 4 d and killed on day 5. For keratin expression studies mice were treated four times, 48 h apart, and killed 24 h after the last treatment. Neonatal Nrf2 / mice in the C57BL/6 background were treated with 1 mol of SF in 100 l jojoba oil daily on postnatal day 5 (P5), P6, and P7 and killed on P8, and their dorsal skin was harvested for study. Seven-week-old SKH-1 female hairless mice were given four i.p. injec- tions of either 50 mol l-buthionine-sulfoximine (BSO) in 100 l jojoba oil or 5 mol glutathione monoethyl ester (GME) in 100 l jojoba oil, whereas a subset received a topical treatment of 1 mol SF in 100 l jojoba oil on the second, third, and fourth day. On the ﬁfth day dorsal skin was harvested for analysis. Seven-week-old female SKH-1 hairless mice and Nrf2 / SKH-1 hairless mice were treated with 10 M SB20219, 100 M SP600125, 100 M UO126, or vehicle control (100 l of acetone:oil 1:250) 30 min before and 30 min after topical treatment with 1 mol SF in 100 l jojoba oil daily for 2 d and were killed on day 3. Five-week-old female hypomorphic Keap1 C57BL/6 mice were killed, and tissue was analyzed as described. Biochemical Analyses RNA was extracted from full-thickness skin of wild-type C57BL/6 and hy- pomorphic Keap1 C57BL/6 mice using TRIzol according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). After DNAse treatment (RNase free DNase kit; Qiagen, Valencia, CA), 1 g of total RNA from each phenotype was reverse-transcribed (Advantage RT-for PCR kit; Clontech, Mountain View, CA). First-strand cDNA was used for PCR analysis of gene expression using Invitrogen Taq. Target mRNAs and oligonucleotide primer sets are described in Kerns et al. (2007). Intensity of electrophoresed, ethidium bro- mide–stained PCR products was quantiﬁed using the NIH ImageJ freeware (http://rsb.info.nih.gov/ij/). For protein analysis, dorsal skin was harvested from control and treated Nrf2 / SKH-1 hairless mice, and total protein was extracted (Paladini and Coulombe, 1999). Protein concentrations were ob- tained by using a Bradford Assay Kit (Bio-Rad, Hercules, CA). Equal amounts of protein (10 –20 g) were resolved on 8.5% SDS/PAGE gels and blotted onto nitrocellulose. Bound primary antibodies were detected via enhanced chemi- luminescence (Amersham Pharmacia Biotech, Pittsburgh, PA). For total glu- tathione measurements, full-thickness dorsal skin was ﬂash-frozen in liquid nitrogen and pulverized into a ﬁne powder. A total of 10 volumes of 5% 5-sulfosalicylic acid solution was added to 0.1– 0.3 g of tissue powder. Sam- ples were then homogenized with 3 ml PTFE pestle in glass tube, stored at 4°C Vol. 21, December 1, 2010 SF and Keratin Expression in Skin for 10 min, and then centrifuged at 10,000 g at 4°C for 10 min to remove the denatured protein precipitate. The samples were analyzed using a commer- cial kit (Sigma) based on the original method of Sedlak and Lindsay (1968), a kinetic assay in which GSH (reduced glutathione) continuously reduces 5,5 - dithiobis(2-nitrobenzoic acid) (DTNB) to 5-thio-2-nitrobenzoic acid (TNB), and the resulting GSSG (oxidized glutathione) is recycled by glutathione reductase and NADPH. TNB was spectrophotometrically measured at 412 nm. The levels of total and phosphorylated MAP kinases and their targets were analyzed using a commercial Sandwich ELISA kit from Cell Signaling (St. Louis, MO). Morphological Analyses Five-micrometer sections were obtained from surgically harvested back skin tissue samples, embedded, and frozen in OCT (Sakura, Torrance, CA). For indirect immunoﬂuorescence, they were thawed, postﬁxed with neutral buffer Formalin for 10 min at room temperature, and incubated with primary antibodies to K17, K16, and K6. Bound primary antibodies were revealed by incubation with FITC-conjugated secondary antibodies (Kirkegaard & Perry Laboratories). The samples were also stained with Hoechst (Sigma) stain following the manufacturer’s instructions. Quantitation of Immunoﬂuorescence Micrographs were recorded using a Zeiss AxioPlan2 upright microscope equipped with a Zeiss HRC high-resolution digital camera (Thornwood, NY). Control and experimental skin samples were examined consecutively, and micrographs of stained preparations were recorded using the same exposure conditions. Immunoﬂuorescence intensity was determined by measuring the mean pixel density of immunoﬂuorescence in three 25- m2 areas of epidermis per stained preparation of skin tissue, using Zeiss’ Axiovision software. Each section selected was representative of its experimental group, and the 25- m2 areas were selected from predetermined sites of epidermis (center, left, and right of the section) to foster unbiased sampling. Statistical Analyses Results were expressed as means comparison between groups. p RESULTS SEM. The Student’s t test was used for 0.05 was considered statistically signiﬁcant. Nrf2 Plays a Partial Role in Keratin Regulation by SF in Skin Epidermis The Nrf2-Keap-ARE pathway represents the best character- ized mechanism to account for the SF-dependent transcrip- tional induction of speciﬁc genes (Talalay and Fahey, 2001; Kobayashi and Yamamoto, 2006). Several AREs matching the consensus for Nrf2 binding can be found in the area of the mouse and human genome that houses the clustered and highly conserved K14, K16, and K17 genes (data not shown). Recent studies have shown that K6, a polymerization part- ner for K16/K17 in vivo (McGowan and Coulombe, 1998b), and K16 are bona ﬁde Nrf2 target genes (Wakabayashi et al., 2003; Endo et al., 2008). To examine the role of the Nrf2- Keap-ARE pathway in SF-mediated induction of K16 and K17 expression in the epidermis, studies were conducted in a mouse strain carrying a Nrf2 mutation (Wakabayashi et al., 2003), and another one in which Nrf2-dependent tran- scription is enhanced secondary to a Keap1 hypomorph allele (Osburn et al., 2008). The Nrf2 allele was transferred to the SKH-1 hairless strain (which is euthymic and immuno- competent; Benavides et al., 2009) to augment the ease and consistency of SF topical delivery. A dosage of 1 mol SF, applied to the middorsum of mice, was used throughout this study because it rescues the skin fragility characteristic of K14 mouse skin without signiﬁcantly impacting prolif- eration or apoptosis in vivo (Kerns et al., 2007). Because K16 and especially K17 are robustly expressed in hair follicles under normal conditions, whereas the epidermis represents the exclusive interest of our study, the primary mean of sample analysis used was indirect immunoﬂuorescence us- ing well-characterized primary antibodies coupled with software-based ﬂuorescence quantitation (see Materials and Methods). 4069