Molecular Biology of the Cell Vol. 21, 4068 – 4075, December 1, 2010 Differential Modulation of Keratin Expression by Sulforaphane Occurs via Nrf2-dependent and -independent Pathways in Skin Epithelia Michelle Kerns,* Daryle DePianto,* Masayuki Yamamoto,† and Pierre A. Coulombe*‡§ *Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205; †Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan; and Departments of ‡Biological Chemistry and §Dermatology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205 Submitted February 23, 2010; Revised July 27, 2010; Accepted September 28, 2010 Monitoring Editor: Robert Goldman Treatment with the natural chemical sulforaphane (SF) ameliorates skin blistering in keratin 14 (K14)-deﬁcient mice, correlating with the induction of K16 and K17 in the basal layer of epidermis (Kerns et al., PNAS 104:14460, 2007). Here we address the basis for the SF-mediated K16 and K17 induction in mouse epidermis in vivo. As expected, induction of K16 partly depends on the transcription factor Nrf2, which is activated by SF exposure. Strikingly, K17 induction occurs independently of Nrf2 activity and parallels the decrease in glutathione occurring shortly after epidermal exposure to SF. Pharmacological manipulation of glutathione levels in mouse epidermis in vivo alters K17 and K16 expression in the expected manner. We present ﬁndings suggesting that select MAP kinases participate in mediating the Nrf2- and glutathione-dependent alterations in K16 and K17 levels in SF-treated epidermis. These ﬁndings advance our under- standing of the effect of SF on gene expression in epidermis, point to a role for glutathione in mediating some of these effects, and establish that SF induces the expression of two contiguous and highly related genes, K16 and K17, via distinct mechanisms. INTRODUCTION Diets rich in cruciferous vegetables correlate with a reduced risk of developing various cancers in the human population (Talalay and Fahey, 2001; Hayes et al., 2008; Zhang et al., 2009). The general mechanism accounting for this beneﬁt is thought to involve the inhibition and activation of phase 1 and phase 2 metabolic enzymes, respectively, causing a deactivation and/or hastened disposal of potential carcinogens (Kobayashi and Yamamoto, 2006; Myzak and Dashwood, 2006; Tan and Spivack, 2009). Isolation and identiﬁcation of the isothiocya- nate sulforaphane (SF) as the chemopreventive agent in a precursor form, in broccoli sprout extracts (Zhang et al., 1992) paved the way for the discovery of the Nrf2-Keap1- ARE pathway of transcriptional regulation, which mediates these effects at the molecular level (Talalay and Fahey, 2001; Kobayashi and Yamamoto, 2006). Nrf2, a cap’n’collar bZIP family transcription factor (Moi et al., 1994), activates gene This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10 – 02– 0153) on October 6, 2010. Address correspondence to: Pierre A. Coulombe (coulombe@ jhsph.edu). © 2010 M. Kerns et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc- sa/3.0). 4068 expression after binding to antioxidant response elements (AREs) located in or near the promoter region of its target genes (McMahon et al., 2001; Wakabayashi et al., 2004). Nrf2 is normally sequestered in the cytoplasm by the cysteine- rich Keap1 protein, a substrate adaptor protein for a Cul3- dependent ubiquitin ligase complex that targets Nrf2 for proteasome-mediated degradation (Zhang and Gordon, 2004). SF and other isothiocyanates allow for Nrf2 trans- location to the nucleus (and Nrf2-dependent transcrip- tion) by reacting with thiol groups in Keap1, inactivating it (Dinkova-Kostova et al., 2002). The full extent of the transcriptional program regulated by SF or Nrf2-Keap1-ARE and its biological implications and therapeutic potential ap- pear underestimated at present. We reported that treatment with SF markedly reduces the skin blistering and morbidity associated with a keratin 14 (K14) deﬁciency in neonatal mice (Kerns et al., 2007). Nor- mally, K14 copolymerizes with its obligatory partner protein K5 to yield 10-nm intermediate ﬁlaments that, once orga- nized into a pan-cytoplasmic network, provide crucial struc- tural support in basal keratinocytes of epidermis and related stratiﬁed epithelia (Coulombe et al., 2009). Partial or com- plete loss of either K5 or K14 function (e.g., through mutations or dominantly acting missense alleles) renders basal keratinocytes fragile and accounts for epidermolysis bullosa simplex, a rare inherited bullous disease in which the basal layer of epidermis, and occasionally related surface epithelia, rupture readily in response to trivial mechanical trauma (Fine et al., 2008; Coulombe et al., 2009). The beneﬁt afforded by SF treatment in K14 neonatal mice likely involves the induction of K16 and K17, two keratins highly