Skin cancer, chloracne and hyperpigmentation have been associated with the exposure to environmental contaminants such as polychlorinated biphenyls, dioxins or polycyclic aromatic hydrocarbons. These compounds are xenobiotic high-affinity ligands for the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor with important physiological roles in, for example, the control of cell proliferation and inflammation. We show here that exposure of normal human melanocytes to the most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), results in activation of the AHR signaling pathway and an AHR-dependent induction of tyrosinase activity, the key enzyme of the melanogenic pathway. In accordance with the upregulation of tyrosinase enzyme activity, total melanin content was also elevated in TCDD-exposed melanocytes. Neither the induction of tyrosinase enzyme activity or of total melanin could be attributed to enhanced cell proliferation, but was rather due to the induction of tyrosinase and tyrosinase-related protein 2 gene expression. Thus, the AHR is able to modulate melanogenesis by controlling the expression of melanogenic genes.
The molecular mechanisms underlying xenobiotic-induced hyperpigmentation have not been established. In the present study, we propose that the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, which has mainly been associated with dioxin toxicity and detoxification of xenobiotic compounds, plays a role in pigment formation by regulating the expression of genes coding for enzymes of the melanogenic pathway. UV light, the major physiological stimulus for melanogenesis, activates the aryl hydrocarbon receptor via a specific bioactive signal molecule that is formed in response to light. Therefore, we propose a previously unknown role of this transcription factor that may be of significance for UV-induced tanning.
DNA damage induced by ultraviolet radiation or chemical mutagens activates the production of melanin (melanogenesis). The increase in pigment is part of adaptive responses, which also include DNA repair, anti-oxidant defenses and survival pathways, that are crucial for protection against UV-induced skin cancer and aging (Abdel-Malek et al., 2010; Park et al., 2009). Highly toxic environmental contaminants such as halogenated aromatic hydrocarbons (HAH) are associated with hyperpigmentation, chloracne and the development of skin cancer, without causing DNA damage and mutations (Schecter et al., 2006).
Polychlorinated biphenyls, dioxins and other polycyclic aromatic hydrocarbons (PAHs) are known to mimic endogenous activators of the aryl hydrocarbon receptor (AHR) signaling pathway (for review see Nebert and Dalton, 2006). The AHR is a ligand-activated transcription factor which, upon ligand binding, heterodimerizes with its partner protein ARNT (aryl hydrocarbon receptor nuclear translocator). First, the AHR/ARNT dimer binds to its cognate response elements (aryl hydrocarbon receptor response elements, AHRE) in the promoter region of target genes including members of the cytochrome P450 family of proteins involved in xenobiotic metabolism (reviewed in Kawajiri and Fujii-Kuriyama, 2007). Secondly, the AHR is the target of proteosomal degradation, a process that serves to control the activity of the receptor complex (Ma and Baldwin, 2000; Pollenz, 2002).
The transcriptional function of the AHR has been shown to be activated by UV light and by visible light via formation of the highly active tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ). The AHR has therefore been suggested to be a light sensor (Rannug et al., 1987; Wincent et al., 2009). In this context, the AHR has been shown to be involved in multiple homeostatic processes in the skin, including activation of UVB-related stress responses such as EGF receptor (EGFR) internalization (Fritsche et al., 2007) and cross-talk with the nuclear factor κB (Luecke et al., 2010). In the present work we documented the functioning AHR signaling in normal human melanocytes and human melanoma cells (FM55) and observed increased melanin production via the activated AHR. The data strongly suggest that the AHR is involved in the tanning response and that exposure to non-metabolizable potent AHR ligands such as the highly toxic dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can cause hyperpigmentation via this process.
For detailed materials and methods see Appendix S1.
Results and discussion
Normal human melanocytes express a functional AHR signaling pathway
To characterize the AHR pathway in melanocytes, AHR expression levels and the induction of AHR target genes after treatment with the prototypical AHR ligand and environmental contaminant TCDD were investigated. As shown by Western immunoblot in Figure 1A, the AHR was expressed in primary human melanocytes prepared from five different donors, although expression levels varied between donors. Treatment of normal melanocytes with 10 nM TCDD resulted in the activation of the AHR signaling pathway as characterized by ligand-induced receptor degradation (Figure 1B) and the upregulation of cytochromes P450 1A1 and 1B1 (CYP1A1 and CYP1B1), AHR repressor (AHRR) and AHR mRNA expression (Figure 1C). Induction of these genes following TCDD exposure for 3 days was abolished by co-treatment with the partial AHR antagonist 3′-methoxy-4′-nitroflavone (MNF) (Henry et al., 1999). These results clearly indicate that human melanocytes express a fully functional AHR signaling pathway.
AHR activation by TCDD induces melanogenesis
In humans, TCDD exposure causes different kinds of skin lesions. Disturbance of heme biosynthesis leads to porphyria cutanea tarda-like effects that include photosensitivity and pigmentation on skin exposed to sunlight (Smith and Elder, 2010). TCDD exposure also results in the development of chloracne as well as hyperpigmentation, a pathological condition in which elevated melanin levels can be observed (Pastor et al., 2002). The amount of melanin produced by melanocytes is regulated via the expression and activity of the three melanogenic enzymes, tyrosinase (TYR), tyrosinase-related protein 1 and 2 (TYRP1 and TYRP2) located in the melanosomes. Tyrosinase, the key enzyme of the melanogenic pathway, catalyzes the rate-limiting first step in the biochemical pathway of melanin formation, the oxidation of l-tyrosine to 3,4-dihydroxyphenylalanine (l-Dopa) (for review see Park et al., 2009) and serves as a sensitive indicator of changes in the melanogenic pathway (Park et al., 1993).
To investigate whether the activation of the AHR pathway by dioxin directly affects melanogenesis in human melanocytes, we assessed tyrosinase enzyme activity and melanin content in exposed human cells in vitro. Tyrosinase enzyme activity was spectrophotometrically measured in melanocyte lysates as the formation of dopachrome from l-Dopa and normalized to protein levels. To allow comparison between cultures from different donors, tyrosinase activity was expressed as fold induction of the basal activity of that culture. After 3 days of TCDD exposure, tyrosinase enzyme activity was profoundly upregulated in melanocytes (Figure 2A, left panel). Co-treatment with 1 μM of the partial AHR antagonists MNF or α-naphthoflavone (αNF) abolished TCDD-induced tyrosinase activity, suggesting that the observed induction in tyrosinase activity by TCDD is mediated by the AHR (Figure 2A, right panel). High tyrosinase activity was also observed at day 5 in melanocytes continuously treated with 10 nM TCDD (Figure 2B). In these cells, tyrosinase activity was not only increased, but also total intracellular melanin was elevated up to threefold (Figure 2C, left panel). In addition, a significant increase in the melanin content was also observed in TCDD-exposed human FM55 melanoma cells (Figure 2C, right panel), confirming the upregulation of the melanogenic pathway following AHR activation in melanoma cells.
To exclude the possibility that the observed effects of AHR activation on melanogenesis were due to enhanced cell proliferation, we performed a WST-1 cell proliferation assay. No stimulation of cell proliferation was observed in melanocytes or FM55 melanoma cells (Figure 2D). Thus, the increase in neither tyrosinase activity or melanin could be attributed to an enhanced cell proliferation, confirming histopathological analyses which reported normal melanocyte numbers but increased pigment formation in dioxin-exposed skin (Pastor et al., 2002).
An increase in melanogenesis can be mediated by (i) the elevated expression of genes coding for melanogenic enzymes tyrosinase and tyrosinase-related proteins, (ii) the phosphorylation of serine residues in the cytoplasmic domain of tyrosinase by PKC-β (Park et al., 1993) and (iii) the stabilization of a multienzyme complex of tyrosinase and tyrosinase-related protein 1 (Wu and Park, 2003). The PKC-β inhibitor bisindolylmaleimide (50 nM) had no effect on TCDD-induced tyrosinase activity (data not shown), suggesting that TCDD treatment did not modulate tyrosinase enzyme activity via the PKC-β signaling pathway. Using promoter analysis, we noticed that tyrosinase and tyrosinase-related proteins have multiple AHREs in their promoter regions, introns and 3′ noncoding regions (Table 1) and might therefore serve as transcriptional targets of activated AHR. Indeed, following 3 days of TCDD treatment, the amounts of tyrosinase (TYR) and tyrosinase-related protein 2 (TYRP2) mRNA were on average upregulated twofold, whereas co-treatment with MNF abolished TCDD-induced expression (Figure 2E).
Table 1. Identification of candidate transcription binding sites
Conserved AHRE motifs
Gene (genomic sequence)
In the 5′ region
In the total extracted sequence
Total genomic DNA sequences covering 5′ to the translational start codons and 5 kb 3′ to the last exons of the human tyrosinase (TYR ), tyrosinase-related protein 1 (TYRP1) and tyrosinase-related protein 2 (TYRP2 ) sequences were extracted from the indicated human genomes and analyzed for conserved motifs using the clc main workbench program. The number of AHRE (aryl hydrocarbon receptor response element consensus sequence GCGTG (Fujisawa-Sehara et al., 1987)) motifs in the 5′ region and in the total sequences are indicated.
Supporting the early reports of hyperpigmentation in Yusho and Yu-Cheng victims (Hashiguchi et al., 1987; Hsu et al., 1985; Kikuchi, 1984; Lu and Wu, 1985; Urabe and Asahi, 1985) and observations in exposed experimental animals (Iwata et al., 1981; Zodrow and Tanguay, 2003), our data clearly suggest the AHR as a novel modulator of melanogenesis in response to xenobiotics. The mechanism described here points to a direct effect of AHR activation on the melanogenic pathway. However, melanin production in melanocytes is highly regulated by signals from surrounding keratinocytes, dermal fibroblast and Langerhans cells (Archambault et al., 1995). For example, keratinocyte-derived factors such as endothelin 1, proopiomelanocortin (POMC), prostaglandins and leukotrienes (reviewed in Park et al., 2009) can regulate melanogenesis in a paracrine fashion. Especially, since the AHR can influence cytochrome P450-dependent synthesis and degradation of numerous eicosanoids (Nebert and Karp, 2008), it cannot be excluded that AHR activation can also contribute indirectly to the stimulation of melanin production in melanocytes.
The UVB-derived tryptophan photoproduct and suggested physiological AHR ligand FICZ increases tyrosinase activity
Activation of the AHR signaling pathways occurs not only after xenobiotic exposure, but also after stimulation with UVB irradiation. UVB, the major physiological stimulus for melanogenesis, has been shown to activate the AHR and induce the expression of the well characterized AHR target gene CYP1A1 in human skin (Katiyar et al., 2000) and keratinocytes (Wei et al., 1999). AHR activation by UVB is mediated via the formation of the tryptophan-derived photoproduct FICZ (Fritsche et al., 2007; Rannug et al., 1987, 1995).
As expected, FICZ (10 nM) induced the expression of CYP1A1 and CYP1B1 mRNA in normal human melanocytes after 3 h (Figure 3A). Tyrosinase activity was also upregulated after 24 h in melanocytes treated with 100 nM FICZ (Figure 3B), although the effect was weaker and transient when compared to TCDD due to the efficient catabolic breakdown of FICZ by AHR-controlled CYP1 enzymes (Bergander et al., 2004; Wincent et al., 2009). Like TCDD, FICZ had no effect on melanocyte proliferation (Figure 3C). As we could show a regulatory role of AHR signaling in xenobiotic-induced melanogenesis, we suggest, based on these data, that UVB-activated AHR may partly contribute to the melanogenic response of the skin to UV irradiation. In support of a role of the AHR in facultative pigmentation, AHR-deficient mice were shown to have a lower UVB-induced tyrosinase activity in the epidermis, to respond to UVB with less melanocyte proliferation and to tan significantly less than wild-type mice (Jux et al., in press). The AHR is not needed for constitutive pigmentation in mice, as AHR-deficient mice have black skin and fur.
In summary, our results suggest the AHR as a novel regulator of melanogenesis in human melanocytes. By modulating melanogenic gene expression, leading to changes in tyrosinase enzyme activity and melanin formation, the AHR may mediate xenobiotic effects on skin pigmentation.
We would like to thank Dr Amir Sherif for providing the foreskin tissue samples. This study was supported by grants from the Swedish Research Council for Environment, Agricultural Science and Spatial Planning (FORMAS) and from the Deutsche Forschungsgemeinschaft SFB 728 and the BMU, Bonn, Germany.