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1 The Aryl Hydrocarbon Receptor: An Environmental Stress Sensor in Skin

1 The Aryl Hydrocarbon Receptor: An Environmental Stress Sensor in Skin

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16



Aryl Hydrocarbon Receptor (AhR) and Skin Barrier Function



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following ligand-dependent AhR activation. Additionally, the C-terminal

glutamine-rich transactivation domain (TAD) cooperates with co-activator proteins

for enhanced transcriptional gene expression (Rowlands et al. 1996).

In the unliganded state, AhR is retained in the cytoplasm as an inactive protein

complex consisting of an Hsp90 dimer, the chaperone prostaglandin E synthase 3

(PTGES3, p23), the immunophilin-like protein hepatitis B virus X-associated

protein 2 (XAP2/ARA9/AIP), and other factors including the tyrosine kinase

pp60src (Kazlauskas et al. 1999, 2001; Barouki et al. 2007). Hsp90 and p23 protect

AhR from ubiquitin-dependent proteolysis maintaining an AhR conformation that

is receptive to ligand binding. Consistent with AhR function as an Hsp90 client

protein, Hsp90 inhibitors suppress AhR-mediated activation of AhR target gene

expression causing pronounced down-regulation of AhR protein levels (Davis et al.

2015). After AhR ligands (such as dioxin, polycyclic aromatic hydrocarbons, and

endogenous tryptophan-derivatives) engage the receptor, a conformational change

exposes the NLS region critical for nuclear translocation. Nuclear AhR then

hetero-dimerizes with AhR nuclear translocator protein (Arnt; also termed HIF-1b)

via the PAS-A and PAS-B domains, and the AhR/Arnt heterodimer binds to DNA

via the xenobiotic response element (XRE) promoter sequence (-also termed DRE,

dioxin-response element; core DNA sequence: 5′-GCGTG-3′) located in the regulatory region of AhR-target genes.

The AhR ligand-binding domain exhibits a remarkable degree of promiscuity

concerning ligand binding, and the structure activity relationship underlying small

molecule AhR ligand activity remains poorly understood even though recent progress has been achieved based on in silico modeling and systematic site-directed

mutagenesis. Specifically, homology modeling of the mouse, rat, and human

AhR-ligand binding (LBD/PAS) domain was performed using the published

NMR-based PAS domain structure of human hypoxia-inducible factor 2a (HIF2a),

enabling virtual ligand docking (e.g. TCDD, FICZ, ITE) and screening of new AhR

ligands (such as the flavonoid pinocembrin), characterized further in cellular assays

measuring XRE-luciferase reporter transcriptional activation, AhR nuclear translocation, and AhR target gene expression (Bisson et al. 2009). Recently, the remarkable

species (e.g. human versus murine)-specificity of some AhR agonists (including the

microbiome-derived indole-type AhR agonists indole, 3-methylindole, and the

indole-dimer-derived indirubin) has been attributed to the bimolecular (2:1) stoichiometry between ligand and AhR-LBD, a finding that has been interpreted as a

molecular adaptation of the human AhR to sense microbiota-derived indoles as a

prerequisite of maintaining intestinal and cutaneous commensal homeostasis and

barrier function (Hubbard et al. 2015). In addition, comprehensive site-directed

AhR-LBD mutagenesis has identified specific amino acid residues within the

AhR-LBD, dictating selectivity of ligand binding, agonistic/antagonistic receptor

interaction, and Hsp90 engagement (Soshilov and Denison 2014) (Fig. 16.1).



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16.2



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AhR Target Genes



The AhR gene battery comprises numerous genes involved in broad cellular biological functions. Nevertheless, AhR-mediated transcriptional changes are highly

ligand, cell and species specific (Flaveny et al. 2010; Dere et al. 2011). The classical

AhR target genes include the major phase one metabolizing cytochrome P450

monooxygenases (CYP1A1, CYP1A2, CYP2B1), aldehyde dehydrogenase-3

(ALDH3A1), and phase two enzymes including UDP-glucuronosyltransferase

(UGT1A1, UGT1A6), NADPH quinone oxidoreductase (NQO1), and

glutathione-S-transferase (GST). In addition, AhR is an important physiological

regulator of cellular homeostasis by controlling the expression of genes involved in

proliferation, differentiation, adhesion and matrix remodeling, as well as inflammation and immune responses as discussed below. Such genes include epidermal

barrier proteins (e.g. keratin 10, loricrin, pro-filaggrin), cell growth regulators (e.g.

transforming growth factors including TGF-a and TGF-b), and nuclear transcription factors (e.g. c-fos, c-jun) (van den Bogaard et al. 2013, 2015).

Activation of AhR is rapidly terminated by molecular pathways that negatively

regulate AhR. First, AhR is subject to feedback inhibition by mediating transcription of the gene encoding the AhR repressor (AhRR), which competes with

AhR for Arnt (Tsuchiya et al. 2003; Evans et al. 2008). Second, AhR undergoes

nuclear export engaging the NES, followed by ubiquitination and 26S

proteosomal-mediated degradation. Third, termination of AhR signaling is also

achieved through rapid ligand metabolism by CYP1A1-dependent turnover, a

mechanism effective for some ligands (FICZ) that are efficient CYP1A1 substrates

but not for others (e.g. TCDD) that are metabolically inert, potentially representing

the molecular basis of physiological versus pathological AhR engagement.



16.3



Exogenous and Endogenous AhR Ligands



Apart from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical AhR

ligand, numerous other planar hydrocarbons including halogenated aromatic

hydrocarbons (HAHs), polycyclic aromatic hydrocarbons (PAHs), and PAH-like

compounds display high AhR affinity upstream of AhR-dependent cellular signaling. Importantly, metabolically stable dioxin-like HAH ligands (including

TCDD, 2,3,7,8-tetrachlorodibenzofuran, and 3,3′,4,4′,5-pentachlorobiphenyl) cause

dramatic AhR-mediated toxic effects that seem to originate from supraphysiological, dysregulated, and persistent stimulation of AhR signaling. In contrast, metabolically susceptible high affinity AhR ligands [such as the carcinogenic

PAH 3-methylcholanthrene, the phytochemical b-naphthoflavone, and the

endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ)] initiate transient

AhR-dependent signaling and beneficial (or adverse) biological effects in the



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Aryl Hydrocarbon Receptor (AhR) and Skin Barrier Function



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absence of specific toxicity resulting from sustained AhR activation due to rapid

metabolic inactivation that attenuates pathological AhR signaling (Berghard et al.

1992; Kim et al. 2012).



16.3.1 Exogenous AhR Ligands

Exogenous ligands are synthetic (‘anthropogenic’) and natural compounds that are

not generated within or on the surface of the human body. This category includes

environmental pollutants (HAHs, PAHs and related compounds) (Safe 1990;

Denison et al. 1998; Poland and Knutson 1982). Members of the planar hydrophobic

HAH family are high-affinity AhR ligands including dibenzofurans, polyhalogenated dibenzo-p-dioxins, and biphenyls (Denison et al. 1998; Denison and

Heath-Pagliuso 1998). The prototypical and most potent HAH is TCDD (Poland and

Knutson 1982). In humans, TCDD has a half-life of 5-10 years due to its high

lipophilicity and resistance to metabolic degradation resulting in the sustained

induction of CYP1A1 (Aylward et al. 2005). Many studies have shown that TCDD

exposure can cause a diverse array of tissue-specific biological and toxicological

actions, most of which are mediated by AhR (Safe 1990, 1995; Devito 1994). The

members of the PAH include compounds such as benzo[a]pyrene (BaP), benzoflavones, 3-methylcholanthrene and benzanthracenes (Denison and Heath-Pagliuso

1998; Devito 1994; Poland and Knutson 1982). The PAHs are more metabolically

labile and have relatively lower binding affinity than HAHs towards AhR (Fraschini

et al. 1996). The AhR binding affinity of the PAH 3-methylcholanthrene is almost

equal to TCDD but rapid metabolic conversion results in only a transient induction

of CYP1A1 expression. Other classes of exogenous AhR agonists include

plant-derived phenolics including flavonoids (Ashida 2000; Ciolino et al. 1999) and

curcumin (Ciolino et al. 1998), carotenoids (Gradelet et al. 1997), and indol-3carbinol (I3C; Bjeldanes et al. 1991) that can reach skin by either topical application

or dietary intake. Interestingly, flavonoids and other related polyphenols have been

shown to have agonist/antagonist effects and may actually competitively block AhR

activation by dioxin-like contaminants. Additionally, pharmacological agents have

been reported to display AhR activity including the azole antifungal ketoconazole

and the proton pump inhibitor omeprazole (Tsuji et al. 2012; Jin et al. 2014).

Other AhR-directed FDA-approved drugs currently used for distinct therapeutic

indications include flutamide, leflunomide, nimodipine, mexiletine, and tranilast

(Hu et al. 2007; Jin et al. 2014).



16.3.2 Endogenous AhR Ligands

The cutaneous presence of endogenous AhR ligands supports a physiological role

of AhR in skin barrier structure and function. In skin, tryptophan is available in free



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form and also as a constituent of cellular protein, serving as the major precursor of

endogenous indolic AhR ligands. Key enzymes involved in the catabolism of free

tryptophan such as indoleamine 2,3-dioxygenase 1/2 (IDO1/2), tryptophan 2,3dioxygenase (TDO) and tryptophan hydroxylase are expressed in human skin cells

engaged in the formation of tryptophan metabolites including kynurenine, 3-hydroxyanthranilic acid (HA), picolinic acid, and quinolinic acid (Moffett and

Namboodiri 2003; Sheipouri et al. 2015). Tryptophan metabolites serve as precursors for the biosynthesis of niacin and nicotinamide adenine dinucleotide,

kynurenine, melatonin, and serotonin (Thomas and Stocker 1999; Sheipouri et al.

2012, 2015). IDO depletes tryptophan, a crucial factor in T-cell proliferation, and

AhR-KO mice display impaired IDO expression (Nguyen et al. 2010). The

IDO-encoding gene is under transcriptional control of AhR, and IDO expression is

impaired in murine AhR-deficient versus AhR-wt Langerhans cells (Jux et al.

2009). Kynurenines are known AhR agonists involved in the induction

AhR-dependent regulatory T cells (Tregs) from naïve T cells, suggesting an

immunosuppressive role that could potentially confer tolerance and may also play a

role in tumor immune evasion (DiNatale et al. 2010; Mezrich et al. 2010; Nguyen

et al. 2010; Opitz et al. 2011). In addition, the endogenous AhR ligand cinnabarinic

acid, another kynurenine pathway metabolite derived from tryptophan, produced by

the oxidation of 3-hydroxyanthranilic via non-enzymatic or enzymatic (such as

laccase- or ceruloplasmin-dependent) oxidation, has been demonstrated to stimulate

the differentiation of human and mouse T cells producing the Th17-associated

cytokine IL-22 (Lowe et al. 2014). Initially linked to IL-17 as a pro-inflammatory

cytokine, recent evidence suggests that IL-22 plays an independent immunoregulatory role in the context of non-hematopoietic cells, maintaining epithelial cell

homeostasis in mucosal tissues and serving a specific role in tissue repair following

inflammation. Other endogenous tryptophan metabolites with AhR-directed activity

are tryptamine, 5-hydroxy-tryptophan (Bittinger et al. 2003), and indigo and

indirubin (Spink et al. 2003; Adachi et al. 2001; Heath-Pagliuso et al. 1998);

however, indigo and indirubin are detectable in human urine only at picomolar

concentrations, well below the concentrations that would activate AhR-driven gene

expression (Adachi et al. 2001; Guengerich et al. 2004). Only a few other

tryptophan-derived endogenous AhR ligands have been identified such as

2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), a novel

nontoxic (i.e., non-chloracnegenic) AhR ligand, that efficiently suppresses T

cell-immunity in an experimental model of autoimmune uveitis, but the potential

physiological role of ITE in the context of skin barrier function remains largely

undefined (Song et al. 2002; Nugent et al. 2013; Forrester et al. 2014).

It is well known that tryptophan photolysis generates photoproducts that can

cause the AhR-dependent upregulation of Cyp expression in skin. These photoproducts are formed from tryptophan in response to UV and visible radiation

through non-enzymatic photooxidative mechanisms (including hydroxylation,

oxidative deamination, oxidative ring opening, condensation, and oxidative coupling) generating a range of potent AhR agonists including kynurenine,

indole-3-acetic acid (IAA), tryptamine, 1-(1H-indol-3-yl)-9H-pyrido[3,4-b]indole,



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6-formylindolo-[3,2-b]-carbazole (FICZ), indolo[3,2-b]carbazole (ICZ), 6,12-diformylindolo[3,2-b]carbazole (dFICZ), and the FICZ oxidation product indolo

[3,2-b]carbazole-6-carboxylic acid (CICZ) (Rannug et al. 1987; Helferich and

Denison 1991; Wei et al. 2000; Denison and Nagy 2003; Oberg et al. 2005; Fritsche

et al. 2007; Diani-Moore et al. 2011; Smirnova et al. 2016). Among these

tryptophan-derived photolytic products, FICZ displays extraordinary activity as an

AhR ligand with almost ten times higher AhR binding affinity than TCDD, suggesting a causative role of FICZ-induced AhR activity in the mediation of

UV-driven effects on skin (Rannug et al. 1987, 1995; Wincent et al. 2012)

(Fig. 16.2). More detailed biochemical analysis revealed that FICZ serves as an

exceptionally efficient substrate for CYP1A1, CYP1A2, and CYP1B1, and its

hydroxylated metabolites are substrates for the sulfotransferases SULT1A1,

SULT1A2, SULT1B1, and SULT1E1; sulfoconjugates of phenolic FICZ



solar

UV photons



Trp

microbial

metabolism



UVB

O2



1

FICZ



3



FICZ



UVA/VIS



2



LINE-1

LINE

1

genomic rearrangement



*

AhR Arnt



O2

FICZ*



CYP1A1 etc.

xenobiotic metabolism

carcinogen activation

immunomodulation



type I/II



photo-oxidative stress



?

skin photodamage& carcinogenesis

Fig. 16.2 Molecular pathways mediating FICZ-dependent effects in human skin downstream of

its formation as a cutaneous microbial metabolite or solar UVB-induced tryptophan photooxidation product; pathway 1: FICZ as an AhR-ligand causing AhR/Arnt-dependent upregulation of

target gene expression including CYP1A1; pathway 2: UVA-induced formation of FICZ

photoexcited states (FICZ*) followed by generation of reactive oxygen species (ROS) through

type I and II photosensitization reactions causing cutaneous photo-oxidative stress; suggested

pathway 3: FICZ-induced AhR-independent genomic reorganization through stimulation of

LINE-1 retrotransposition



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R. Justiniano and G.T. Wondrak



metabolites are present in human urine (Wincent et al. 2009). LC/MS/MS analysis

confirmed UVB-driven FICZ formation, followed by AhR nuclear translocation and

upregulation of CYP1A1 mRNA levels as demonstrated in HaCaT keratinocytes

(Fritsche et al. 2007), and inhibition of cytochrome P450-dependent clearance of

FICZ has been suggested as a physiological mechanism for AhR activation and

FICZ potentiation (Wei et al. 2000; Wincent et al. 2012). Indeed, elevated levels of

unmetabolized FICZ are observable as a result of pharmacological AhR (using 3′methoxy-4′-nitroflavone) or CYP inhibition (Bergander et al. 2004; Wincent et al.

2012). Remarkably, FICZ is also a fungal metabolite in cutaneous patient samples,

detectable in seborrheic dermatitis and pityriasis versicolor skin specimens

(Magiatis et al. 2013; Wincent et al. 2009).

Recently, in addition to light-dependent photooxidative or microbial biosynthetic formation of cutaneous FICZ, three light-independent oxidative pathways of

FICZ formation with potential physiological relevance have been identified

dependent on the intermediate formation of indole-3-acetaldehyde, the key chemical precursor of all pathways leading from tryptophan to FICZ: (i) nonenzymatic

tryptophan oxidation by the endogenous oxidant H2O2, (ii) nonenzymatic oxidative

conversion of indole-3-pyruvate formed enzymatically from tryptophan through

L-amino acid oxidase, (iii) enzymatic oxidative deamination of tryptamine via

mitochondrial monoamine oxidase. Due to the non-enzymatic nature of the reaction, formation of FICZ from tryptophan has the potential to produce a complex

mixture of indole derivatives, some of which are CYP1A1 inhibitors (including

tryptamine and melatonin), thereby potentially enhancing FICZ bioavailability

through blockade of oxidative metabolism (Heath-Pagliuso et al. 1998; Chang et al.

2010; Smirnova et al. 2016).



16.4



The Skin Microbiome as a Source of Cutaneous

AhR Agonists



Recent research indicates that the commensal cutaneous microbiome is a rich source

of AhR-directed small molecules. Indeed, various species of human skin yeasts (e.g.

Malassezia) generate tryptophan metabolites displaying AhR activity including

indirubin, tryptanthrin, malassezin, FICZ, and ICZ (indole [3,2-b]-carbazole)

(Fig. 16.2). Although the biochemical mechanism underlying the biosynthesis of

theses compounds currently remains undetermined, it is evident that these AhR

ligands have cutaneous functions influencing immunoregulation and barrier quality.

For example, the fungus-derived metabolite malassezin induces AhR mediated

apoptosis in melanocytes consistent with the molecular scenario relevant to long

lasting depigmented plaques, a characteristic of pityriasis versicolor (Kramer et al.

2005), and absence of sunburn in pityriasis versicolor macules has been attributed to

the indole pityriacitrin, which acts as a potent UV filter (Larangeira de Almeida and

Mayser 2006). Other microbial indoles display various biological functions



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including the ultrapotent AhR ligand FICZ, whose AhR-dependent functions

include pro- and anti-inflammatory effects and skin barrier modulation (Quintana

et al. 2008; Wincent et al. 2009; Luecke et al. 2010; Prochazkova et al. 2011;

Wincent et al. 2012; Gaitanis et al. 2012; Mexia et al. 2015). The potential

immune-suppressive capacity of cutaneous microbiome-derived AhR ligands has

been substantiated by experimental evidence indicating that these ligands impede

Toll-like receptor-induced dendritic cell (DC) maturation and DC-induction of

T-cell proliferation, representing an AhR-controlled mechanism of suppressed

surveillance of microbial infections that might facilitate tolerance towards cutaneous

microbial colonization (Vlachos et al. 2012).

Malassezia yeasts comprise the majority of the microbial flora of healthy skin,

and these commensal fungi have also been implicated in the pathogenesis of skin

diseases including seborrheic dermatitis, pityriasis versicolor, atopic dermatitis and

psoriasis (Nakabayashi et al. 2000; Sugita et al. 2002; Gao et al. 2010; Gaitanis

et al. 2012; Jagielski et al. 2014). Indeed, skin extracts from seborrheic dermatitis

and pityriasis versicolor patient lesions indicate high quantities of Malassezia

furfur-derived indoles (Gaitanis et al. 2008, 2012; Magiatis et al. 2013; Mexia et al.

2015), and it seems reasonable to postulate that the increased presence of these

AhR-directed indoles alters local immune function relevant to pityriasis versicolor

lesions and chronic inflammation, as seen in atopic dermatitis and seborrheic dermatitis (Gupta et al. 2004; Kuo et al. 2013).

Importantly, beyond the established fungal origin, AhR-directed

tryptophan-derived and other metabolites can also originate from bacteria residing in human skin. Indeed, AhR has been identified as an intracellular pattern

recognition receptor for virulent factors including phenazine (originating from

Pseudomonas aeruginosa) and the naphthoquinone phthiocol (originating from

Mycobacterium tuberculosis), indicating a role for AhR in immune modulation

downstream of primary bacterial infections (Moura-Alves et al. 2014). Likewise,

Lactobacilli convert tryptophan into indole-3-aldehyde (3-IAld) which has

anti-inflammatory effects thought to originate from AhR-mediated induction of

IDO1 (Zelante et al. 2013).



16.5



FICZ Functions that Occur Independent

of AhR Ligand Activity



Recent research indicates that FICZ assumes additional biological functions that

might impact skin barrier function in response to environmental stressors without

involvement of AhR signaling (Fig. 16.2).



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16.5.1 FICZ: A Nanomolar Endogenous

UVA-Photosensitizer

Our own research has demonstrated that FICZ acts as a nanomolar photosensitizer

potentiating UVA-induced oxidative stress in skin cells, human epidermal skin

models, and murine skin, irrespective of AhR ligand activity (Park et al. 2015; Syed

and Mukhtar 2015). Photosensitization, a phototoxic mechanism downstream of

photon absorption by chromophores present in the human skin is a key mechanism

of UV-induced oxidative stress (Wondrak et al. 2006). In human HaCaT and primary epidermal keratinocytes, photodynamic induction of apoptosis was elicited by

the combined action of solar-simulated UVA [or visible (blue light)] photons and

FICZ, whereas exposure to the isolated action of light (UVA/visible) or FICZ did

not impair viability. In a human epidermal tissue reconstruct, FICZ/UVA

co-treatment caused pronounced phototoxicity inducing keratinocyte cell death,

and FICZ photodynamic activity was also substantiated in a murine skin exposure

model. Array analysis revealed pronounced potentiation of cellular heat shock,

endoplasmic reticulum stress, and oxidative stress response gene expression

observed only upon FICZ/UVA co-treatment. FICZ photosensitization caused

intracellular oxidative stress, and comet analysis revealed introduction of

formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions

suppressible by antioxidant cotreatment. Interestingly, FICZ but not its deformylated analogue ICZ, a potent agonist of equal AhR affinity as FICZ, displayed

photosensitizer activity, consistent with the carbonyl group-associated triplet state

being the ultimate structural determinant of FICZ-associated UVA- and blue

light-induced phototoxicity.

Taken together, these data provide evidence that the endogenous AhR ligand

FICZ displays nanomolar photodynamic activity representing a molecular mechanism of UVA-induced photooxidative stress potentially operative in human skin. It

may be hypothesized that FICZ serves as an endogenous photosensitizer, a role

similar to that attributed before to other endogenous chromophores such as riboflavin (vitamin B2) and protoporphyrin IX, but the precise mechanism and photobiological relevance of FICZ-induced photo-oxidative stress operative in solar

UV-exposed human skin remains to be elucidated. It also remains to be seen if

FICZ-induced photo-oxidative stress may facilitate its generation from tryptophan,

representing an autocatalytic mechanism that would be consistent with recent

observations on FICZ excited state chemistry and non-enzymatic,

non-photochemical FICZ formation originating from hydrogen peroxide-induced

oxidation of tryptophan (Park et al. 2015; Smirnova et al. 2016).



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16.5.2 FICZ: An Endogenous Activator of LINE-1

retrotransposition

Recent experimental evidence indicates that FICZ may cause genomic reorganization through stimulation of retrotransposition, a remarkable activity that impacts

genomic integrity without AhR involvement. FICZ-induced retrotransposition of

the long interspersed nucleotide element-1 (LINE-1) can be observed at picomolar

concentrations in human HuH-7 and HeLa cells. LINE-1 is a genetic non-LTR

retrotransposon comprising about 17 % of the human genome, of which 80–100

copies are competent as mobile elements. LINE-1 contains an internal polymerase

II promoter and ORF1 (encoding a 40-kDa basic RNA-binding protein with RNA

chaperone activity) and ORF2 (encoding a 150-kDa protein with endonuclease and

reverse transcriptase activities), involved in L1 cDNA genomic integration. LINE-1

retrotransposition depends on AhR nuclear translocator-1 (Arnt1), and FICZ is

thought to stimulate the interaction of the LINE-1-encoded ORF1 and Arnt1,

recruiting ORF1 to chromatin downstream of activation of mitogen-activated protein kinase. Prior research has demonstrated the activation of human LINE-1

retrotransposition by benzo(a)pyrene, an ubiquitous environmental carcinogen,

requiring DNA adduction after cytochrome P450-catalyzed oxidation of the parent

hydrocarbon (Stribinskis and Ramos 2006), and a causative involvement of LINE-1

reverse transcriptase (encoded by ORF-2) has been substantiated in UV-induced

transformation of cutaneous keratinocytes (Banerjee et al. 2005). However, the

molecular mechanism underlying FICZ-induced LINE-1 retrotransposition and its

potential role in the modulation of genomic adaptations to UV and other environmental stressors remain to be elucidated in more detail. In the context of

genomic rearrangement as a result of LINE-1 retrotransposition in response to DNA

damage, it is interesting that LINE-1 retrotransposition can be induced by oxidative

DNA damage in human neuroblastoma cells exposed to hydrogen peroxide, and

LINE-1 hypomethylation induced by ROS is mediated via depletion of

S-adenosylmethionine, suggesting a mechanistic link between oxidative stress and

LINE-1 driven insertional mutagenesis and genomic instability that may also be

operative in human skin cells under environmental oxidative stress (Giorgi et al.

2011; Carreira et al. 2014; Kloypan et al. 2015). Based on the recent emergence of

FICZ as a potent sensitizer of photo-oxidative stress via ROS formation upstream of

oxidative (8-oxo-dG-mediated) genomic damage (Park et al. 2015), it is tempting to

speculate that FICZ-driven AhR-independent LINE-1 transposition may also be

impacted by FICZ-induced photo-oxidative stress, a hypothesis to be tested by

future experimentation.



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R. Justiniano and G.T. Wondrak



AhR: Cutaneous Functions and Therapeutic

Opportunities



AhR is highly expressed in barrier organs such as skin, lung, and gut consistent

with its role as a critical environmental stress sensor and orchestrator of environmental and endogenous stress responses (Esser and Rannug 2015;

Haarmann-Stemmann et al. 2015).



16.6.1 AhR and Epidermal Barrier Function

Recent reports have demonstrated a critical role of AhR in epidermal differentiation,

skin barrier formation and homeostasis (Sutter et al. 2011; van den Bogaard et al.

2013, 2015). Earlier studies have reported that AhR deficient mice display abnormalities in keratinocyte terminal differentiation suggesting a physiological role of

AhR in skin morphogenesis (Loertscher et al. 2002). Using murine and human skin

models it has recently been shown that AhR activation is required for normal

keratinocyte differentiation as evidenced by impaired epidermal stratification

resulting from AhR inactivation during human skin equivalent development

(van den Bogaard EH et al. 2015). Comparative transcriptomic analysis between

AhR(−/−) and AhR(+/+) murine keratinocytes indicated a significant enrichment of

differentially expressed genes linked to epidermal differentiation, and AhR(−/−)

keratinocytes showed a significant reduction in terminal differentiation gene and

protein expression, an observation mimicked by pharmacological AhR antagonism

using drug-like small molecule modulators (including GNF351, CH223191,

SGA360). Likewise, monolayer cultured primary human keratinocytes subjected to

pharmacological AhR antagonism also display an impaired terminal differentiation

program. Based on these observations, pharmacological AhR activation employing

synthetic or endogenous AhR ligands has now emerged as a novel therapeutic

strategy targeting disturbed epidermal differentiation, a key clinical feature associated with numerous skin pathologies.



16.6.2 Lessons from TCDD-Induced Chloracne

Dysregulated expression of epidermal barrier proteins is thought to contribute to

various cutaneous pathologies, and TCDD-induced overexpression of these genes

may therefore cause detrimental effects including impairment of skin barrier

function. Indeed, toxicity studies have reported that long-term exposure to TCDD

causes chloracne, a severe acne-like condition characterized by aberrant epidermal

hyperproliferation and hyperkeratinization involving the interfollicular squamous

epithelium and hair follicles, as well as a metaplastic response of sebaceous glands



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(Loertscher et al. 2001; Sutter et al. 2009, 2011). Chloracne is clinically characterized by widespread dissemination of epidermal and dermal cysts with severe

atrophy of the sebaceous glands, and TCDD-induced transcriptional repression of

genes involved in sebum lipid metabolism may underlie sebaceous gland-directed

adverse effects (Saurat and Sorg 2010). Consequences of TCDD exposure are

exacerbated by the toxicant’s lipophilicity and metabolic inertness attributed to

polyhalogenation, preventing (or at least attenuating) enzymatic oxidative bioconversion and deactivation, resulting in a prolonged biological half-life of TCDD in

humans that exceeds one year. However, patho-mechanistic aspects of AhR

engagement underlying the chloracne phenotype remain poorly understood. It has

recently been shown that induction of a chloracne phenotype achieved in an epidermal equivalent model by TCDD depends on AhR activation and is not reproduced by AhR knockdown (Forrester et al. 2014). Indeed, when human epidermal

equivalents were treated with TCDD or two AhR-directed non-chloracnegens

[b-naphthoflavone (b-NF) and ITE], ligand-induced CYP1A1 and AhR degradation

did not correlate with their chloracnegenic potential, and only TCDD induced a

chloracne-like phenotype, whereas b-NF or ITE did not.

In a politically motivated assassination attempt using TCDD as a single toxicant

in 2004, the victim, former Ukrainian president Viktor Yushchenko, was hospitalized displaying TCDD serum levels 50,000-fold above average levels in the general

population (Sorg et al. 2009), a singular incident of TCDD-specific exposure different from mass exposure scenarios where victims of Agent Orange (Vietnam war;

Poland et al. 1976), industrial accidents (Seveso, Italy; Reggiani 1978), and environmental disasters (Yusho, Japan; Kuratsune et al. 1971) were exposed to a mixture

of chemicals including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs). Interestingly,

two TCDD metabolites (2,3,7-trichloro-8-hydroxydibenzo-p-dioxin and

1,3,7,8-tetrachloro-2-hydroxydibenzo-p-dioxin) were identified in the patient’s

feces, blood serum, and urine. Biopsies of cutaneous lesions revealed high concentrations of TCDD that surpassed serum levels tenfold at eleven months post

poisoning, accompanied by highly altered AhR-regulated gene expression indicative

of sustained AhR signaling in skin (Saurat et al. 2012).

Importantly, it is now firmly established that the epidermal barrier is a functional

target of the TCDD-activated AhR (Sutter et al. 2011). TCDD controls the

expression of genes in the human epidermal differentiation complex (EDC) locus, a

chromosomal region and gene complex spanning 1.9 Mbp (1q21) comprising over

fifty genes encoding proteins involved in the terminal differentiation and cornification of epidermal keratinocytes underlying epidermal barrier function (Mischke

et al. 1996). EDC gene expression is controlled by various transcription factors such

as KLF4, GATA3, GRHL3, Nrf2, and AhR/Arnt (Kypriotou et al. 2012). The

proteins encoded by EDC genes are functionally interrelated, representing members

of three evolutionarily distinct gene families: (i) the cornified envelope precursor

family [e.g. involucrin (IVL), loricrin (LOR), and various small proline-rich

(SPRRs) and late cornified envelope proteins (LCEs)], (ii) the S100 protein family

[e.g. psoriasin (S100A7), calgranulin A (S100A8), and calgranulin B (S100A9),



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1 The Aryl Hydrocarbon Receptor: An Environmental Stress Sensor in Skin

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