Year: 2010
Type: article
Abstract: As the largest and most exposed organ in the body, the skin has developed sophisticated and redundant mechanisms for cytoprotection against environmental insults, including oxidative and xenobiotic stresses and UV radiation. The antioxidant and detoxification pathways that provide this protection involve both enzymatic (glutathione reductases, catalase, dismutases, etc.) and non-enzymatic antioxidants (vitamin E, vitamin C, glutathione, etc.), which also help regulate skin cell death and differentiation. Nrf2 is an important transcriptional regulator of redox in the skin in response to oxidative and xenobiotic stresses. It is activated by many compounds, including sulforaphane, which is found in broccoli sprouts and other cruciferous vegetables. Belonging to a family of transcriptional regulators that include Nrf1 and 3, the factor heterodimerizes with Maf proteins, among others, to bind promoter elements called antioxidant response elements (ARE) and activates transcription. Genes regulated by Nrf2 include the antioxidant protein thioredoxin, enzymes involved in biosynthesis of the small antioxidant glutathione, reductases that ensure recycling of these antioxidants, transferases that utilize them to eliminate xenobiotics, and chaperones and degradative proteins that monitor and eliminate irreversibly damaged macromolecules, among others (Sykiotis and Bohmann, 2010). The role of Nrf2 in UVB- and DMBA/TPA-induced skin tumorigenesis has been explored using Nrf2 knockout and keratin 14-targeted dominant negative Nrf2 (K14-dnNrf2) mouse models. Both Nrf2 knockout and K14-dnNrf2 mice show increased incidence of papillomas in the two-stage carcinogenesis protocol compared to wild-type mice likely a result of reduced levels of antioxidant and detoxification enzymes (Beyer et al., 2007). Nrf2 knockout mice are more sensitive than wild-type mice to the pro-apoptotic and pro-inflammatory activities of UVB, generally believed to result from ROS induction, but surprisingly, UVB-induced tumorigenesis is similar in both genotypes (Kawachi et al., 2008). Paradoxically, short-term pre-activation of Nrf2 by plant-derived electrophilic compounds like sulforaphane (broccoli sprout extract) or avicins (Acacia Victoriae) protects against UVB-induced carcinogenesis, suggesting that there may be compensatory pathways that protect knockout mice from carcinogenesis after UVB-induced DNA damage (Beyer et al., 2007). In their paper, Schafer et al., 2010 present a new mechanism by which this redox-responsive factor protects the skin from UVB-induced insult. They utilize a constitutively active mutant of Nrf2 (caNrf2), targeted to basal keratinocytes of the mouse epidermis by the keratin 5 promoter, to specifically study Nrf2-regulated transcription upon UV-induced skin damage. Enhanced Nrf2 activity is evident in all layers of the epidermis as seen in upregulation of Nrf2-regulated genes. The mice exhibit mild skin hyperkeratosis, but expression of keratinocyte differentiation markers remains unaltered. Keratinocytes from caNrf2 mice exhibit low rates of apoptosis, in vitro and in the back skin, under basal conditions as well as upon UVB irradiation. This enhanced protection is likely a consequence of decreased levels of ROS and resultant ROS-induced DNA damage as evidenced by p53-positive cells. However, the extent of direct DNA damage by UVB, as measured by cyclobutyl pyrimidine dimers, is comparable with wild-type skin. Interestingly, the authors report a gradient of apoptosis in wild-type back skin in response to UVB, with the greatest number in the basal epidermal layer that progressively decreases through the lower and then upper suprabasal layers. The gradient inversely mirrors the expression and activity of Nrf2 in the epidermis, where suprabasal keratinocytes show the highest expression of Nrf2-responsive genes. Moreover, the authors show that the UVB-induced apoptotic gradient is dependent on Nrf2 activity, as the gradient diminishes in Nrf2 knockout mice. This is mainly a consequence of an increase in the number of apoptotic cells in the suprabasal layer, where Nrf2 activity is highest in wild-type skin. Basal cells in knockout skin also show an unexpected and unstudied decrease in apoptosis that begs the question whether the knockout skin now has more initiated cells. Conversely, keratin 5-driven caNrf2 mice also show a reduced apoptotic gradient because of the now comparable transcriptional activity in all the epidermal layers. The authors next targeted the constitutively active Nrf2 exclusively to suprabasal cells (K10caNrf2). The new transgenic mice continue to display hyperkeratosis, but surprisingly, the protection from UVB-induced apoptosis is similar to that observed when the whole epidermis is targeted. Moreover, the increased cytoprotection is largely in the basal cells although Nrf2 activity and the inflammatory response remain unchanged, suggesting that a paracrine factor produced by suprabasal cells may provide protection. As Nrf2 regulates the expression of a number of genes encoding proteins involved in glutathione synthesis and cystine transport, the authors go on to test the small antioxidant, glutathione (GSH) as a candidate factor. Double transgenics of their K10caNrf2 and Gclm (glutamate cysteine ligase) knockout mice, the latter having a >80% reduction in GSH levels, fail to show enhanced protection in response to UVB compared to Gclm knockout mice, suggesting that active Nrf2 mediates protection through the GSH antioxidant pathway. In an effort to explore how suprabasal Nrf2 mediates protection in basal cells through GSH, the authors looked at the transport and synthesis of its components. Their results show that suprabasal cells have high levels of mRNA for cystine antiporter (xCT) and GSH exporter (Mrp3) and synthesis enzymes that are further increased when Nrf2 is overexpressed in these cells. The authors hypothesize that this may enhance availability of GSH and its precursor cysteine for uptake by basal keratinocytes, thus enhancing its antioxidant environment. Thus, the authors layout a mechanism, previously shown with astrocytes and neurons, whereby suprabasal activity of Nrf2 indirectly enhances production of GSH in basal cells which in turn protects the latter cell type from UVB-induced apoptosis (Figure 1). Modified Model of Schafer et al., 2010 of Nrf2-induced protection from UVB in the Mouse Epidermis. (Right) Schematic of mouse epidermis with basal layer and melanocytes (BL & MC), suprabasal layer (SL), granular layer (GL) and cornified layer (CL). Paracrine-mediated protection is represented by arrows for established (solid arrows) and hypothesized (dotted arrows) mechanisms. (Left) Nrf2-induced GSH-mediated cytoprotection of basal cells. Two step synthesis of GSH in both cell types by Gcl and Gss is shown. Nrf2-induced upregulation of cystine transporter Xc, GSH exporter Mrp in suprabasal cells and cysteine importer Asc in basal cells is represented by red block arrows. Possible regulation of thioredoxin (Trx) and thioredoxin reductase (TR1) by Nrf2, and of Gcl and Gss by NFκB are represented by dashed red arrows. While the study illuminates a novel mechanism for cytoprotection from UV in skin, it also raises new questions and leaves several avenues open for additional exploration. The authors do not examine caNrf2 mice for protection from ROS-induced DNA damage products like 8-hydroxy-2′-deoxyguanosine (8-OH-dG) as a positive readout for enhanced cytoprotection. These 8-OH-dG adducts have previously been reported to be high in UV-irradiated nrf2 knockout mice (Kawachi et al., 2008). The study does not address whether the upregulated antioxidant genes are direct transcriptional targets of Nrf2. Previous reports have implicated NF-κB as a direct transcriptional regulator of GSH synthesis enzymes in fibroblasts through IKKβ (Peng et al., 2010). This pathway may also be relevant in keratinocytes under basal and stress conditions (Figure 1). Moreover, thioredoxin, a known downstream effector of Nrf2-mediated cytoprotection (Figure 1), was not examined. Elevated thioredoxin could serve a compensatory role in Gclm knockout mice, where despite 80% reduction in GSH levels, the UV-induced apoptosis is comparable to that in wild-type mice. Strikingly, the authors provide results suggesting that human skin does not show the same gradient of UVB-induced apoptosis and speculate that this may be because of the presence of melanocytes (Figure 1). Indeed, melanin is known to physically scatter and absorb UV radiation, thus protecting the lower levels of the epidermis from damage, while also acting as a scavenger of ROS (Brenner and Hearing, 2008). Paradoxically, UV-induced degradation of melanin can also increase ROS levels that can in turn enhance UV-induced DNA damage (Brenner and Hearing, 2008). Thus, it will be important to analyze the cytoprotective contribution of the Nrf2 pathway from UV radiation in the human epidermis. This could be addressed using mouse models like the hepatocyte growth factor/scatter factor transgenic mice, where melanocytes localize in the epidermis representing a more humanized skin (Noonan et al., 2003). If the Nrf2 pathway proves to add a level of protection for UV damage in human epidermis, then ‘going green’ takes on additional meaning for human health.
Source: Pigment Cell & Melanoma Research
Authors: Mariam Malik, Stuart Yuspa
Institution: National Institutes of Health
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W1533626415