The potential role of AhR/ARNT interactions in pathogenesis of TCDD-induced skin lesions. Despite clear evidence documenting the involvement of AhR/ARNT dimerization in TCDD toxicity in such target tissues as lungs, liver and thymus, the role of this dimer in mediating skin effects of TCDD action is still unclear.
First, the testing of different chloracnegens in hr mouse skin revealed no clear correlation between the level of chloracnegen affinity to the Ahr and the skin response. All tested compounds that did not bind the Ahr did not exhibit any skin effects. Nevertheless, some compounds with high affinity to the Ahr had little or no effect on hr mouse skin (233). Then, TCDD effects on potent regulators of epithelial homeostasis– the oncogenes c-fos and c-jun – are thought to be Ahr independent (163), suggesting that the skin effects of TCDD, at least in part, may also be Ahr independent.
On the other hand, in hr mice, the susceptibility to TCDD-induced epidermal hyperplasia strongly segregates with the AhRb1 allele (233) suggests direct involvement of AhR in the development of skin effects of TCDD toxicity at least in hr skin. The high level of AhR expression and its direct correlation with keratinocyte differentiation in human epidermis (166) support the possible involvement of AhR in chloracne pathogenesis also in humans. Yet, clear proof of this assumption is still lacking.
The crucial role of ARNT in several regulatory pathways implicated in response to the environmental insult (234–236) suggests that ARNT may be a key element of skin-environment communication, yet very little is known about the function(s) of ARNT in the skin (237). It was shown that ARNT is essential for such effects of TCDD action as thymic involution (238) and induction of xenobiotic-metabolizing enzymes including CYP1A1, CYP1A2 and UDP-glucuronosyltransferases (239), but its potential role in pathogenesis of TCDD-induced skin effects was never assessed. Nevertheless, essential role of many AhR/ARNT-dependent genes in the maintenance of skin homeostasis indirectly suggests that AhR/ARNT heterodimerization may be a prerequisite of chloracne pathogenesis as well.
Fig. 5 summarizes the potential mechanisms of AhR and ARNT involvement in the pathogenesis of chloracne. The downstream targets of AhR/ARNT activity include the complex of xenobiotic-metabolizing enzymes, PAI-2, IL-1β and other growth factors and cytokines (184,240) that play a role in the maintenance of skin homeostasis. TCDD-activated AhR may also compete for ARNT with other ARNT-binding factors (e.g. hypoxia inducible factor (Hif) 1α), thus reducing its availability for other interactions, not directly dependent on AhR activity. This pathway may suppress erythropoietin, VEGF, platelet-derived growth factor and glycolytic enzymes (155). The binding of TCDD with AhR may compete with as-yet unidentified endogenous AhR ligand. The ARNT-independent direct activation of the protein kinase cascade by TCDD/AhR complex (177) may also represent a molecular mechanism of chloracne induction because its downstream targets (e.g. EGFR, c-Myc, IL-1β, PAI-2, p53, MMP1, etc.) are known to be involved in regulating epithelial cell proliferation and differentiation (241–245).
Figure 5. Potential mechanisms of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) implication in molecular pathogenesis of chloracne. Red arrows – ARNT-mediated pathways; blue arrows – ARNT-independent pathways.
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Stem cell recruitment and lineage commitment as potential targets of TCDD activity in the skin. Among all existing attempts to provide an explanation for the puzzling features of chloracne, we wish to highlight the notion by Cunliffe et al. (6) who proposed that chloracne is determined by ‘the continuing modification of the metabolism of the pilosebaceous unit’ by the chloracnegen. Although this proposition was made long before the advent of the concept of HF stem cell, it invokes the existence of long-lived, stable cell population in the HF which can propagate the impact of the chloracnegen over several hair cycles (which normally last in humans about 3–5 years). Only stem cells fulfill these requirements. In itself, Cunliffe's prescient recognition of stable, long-living modification of the HF as the major cause of chloracne sheds little light on the pathomechanism of this disease. However, in conjunction with the HF stem cell concept, it provides a basis for a totally new approach to further explore the mechanisms of TCDD action upon skin.
According to the current concept, all epithelial structures in mammalian skin are maintained throughout the adult life by stem cells that not only generate daughter cells that undergo terminal differentiation along several pathways specific for every skin epithelial lineage but also self-renew (228,246). During the last decade, solid evidence has been obtained pointing at the bulge region of the HF as the primary niche for the skin stem cell population (247,248). There is reason to believe that the nature and location of stem cells may differ between HF types. Stem cells are normally quiescent or slow cycling [over 14 months in the mouse; (249)], but in response to certain stimuli like wounding or still mysterious hair cycle-inducing signals, they can be recruited into proliferation. Another set of still poorly understood signals determines the specific pattern of differentiation for resulting transit-amplifying cells. As a result, epidermal, SG or hair matrix progenitor cells are replenished by direct progeny of HF stem cell population (228).
As we mentioned before, pathogenesis of TCDD-induced chloracne suggests the following stem cell-associated events (Fig. 6): (i) recruitment of stem cells into cycling and their active exit into transit-amplifying cell compartment that probably determine hyperplasia in the middle portion of chloracne follicles; (ii) elevated level of stem cell renewal that maintains the sufficient number of stem cells even under their active exit into proliferating compartment, thus probably determining the persistence of chloracne; (iii) the shift in the fate of transit-amplifying cells – differentiation along epidermal pathway at the expense of HF and sebaceous differentiation.
Figure 6. The hypothetical model for potential cellular and molecular mechanisms of chloracne development based on the model of role of c-Myc in skin stem cell control (222, 265). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)/aryl hydrocarbon receptor complex modulate c-Myc activity through one of the putative upstream c-Myc regulatory cascades (c-Src, Wnt, TGFβ). That results in the recruitment of stem cells into proliferation and in differentiation of their progeny (transit amplifying cells) mainly along epidermal lineage at the expense of follicular differentiation. Along with simultaneous suppression of sebaceous differentiation, this results in the formation of keratinizing comedones. TCDD action can also stimulate hair follicle stem cell renewal, thus determining chloracne persistence.
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Recently, it was shown that transient activation of a nuclear protooncogene c-Myc in mouse skin drives keratinocytes from the stem to the transit-amplifying compartment and stimulates differentiation along the epidermal and sebaceous lineage at the expense of hair-type differentiation (222,223,250). It was also found that the increased proliferation in the basal layer was not dependent on continuous activation of transgene. ‘Thus, transient activation of c-myc could result in sustained effects on the behaviour of keratinocytes’ (222,223). This model of c-Myc effects on HF stem cells is also supported by the finding that ablation of β-catenin, genetically upstream of c-Myc, prevents cycling of HFs by forcing stem cells into an epidermal fate (224).
Thus, the general scheme of c-Myc-dependent changes in the kinetics of HF stem cells fits the main features of TCDD-induced skin phenotype, while some noticeable differences also exist. For example, deregulation of c-Myc expression stimulates transit-amplifying cells to adopt epidermal and sebaceous fates in the expense of HF differentiation (222,250), although TCDD action stimulates epidermal differentiation at the expense of both, HF and sebaceous lineages. This difference suggests that if c-Myc pathway is a target of TCDD activity in the skin, its deregulation is not the only mechanism of chloracne pathogenesis. This proposition is also supported by partial, not complete, similarity between skin phenotype in transgenic mice overexpressing c-Myc (222) and chloracne-like symptoms in laboratory animals (83).
Currently, little is known about possible interactions between AhR/ARNT and c-Myc pathways. c-Myc is tightly regulated by growth factors and has been shown to be inhibited by the TGF-β2 signalling (251,252) which in turn is strongly suppressed by TCDD in vitro (191,192), suggesting potential positive influence of TCDD upon c-Myc expression. On the other hand, TCDD is a potent suppressor of c-Myc-induced apoptosis in Alb/c-Myc transgenic mice (253). In the liver of TCDD-treated guinea pigs, c-Myc DNA binding is downregulated (254). Treatment of 3T3-L1 preadipocyte cells in vitro (pretreated with TCDD) with antisense c-Myc oligonucleotides blocked the toxic effects of TCDD (255).
Another possible link between TCDD/AhR and c-Myc may be represented by c-Src/β-catenin pathway that plays an essential role in HF and skin biology (256). Although c-Src, a well-known target of TCDD action (177), does not directly participate in c-Myc phosphorylation, it phosphorylates tyrosine residues of β-catenin, leading to its extensive degradation (257). β-catenin, in turn, is a known upstream modulator of c-Myc expression (258–260). The role of β-catenin as a linker in the c-Src/c-myc signal transduction pathway in epithelial tissues is also confirmed by a significant increase in the tyrosine phosphorylation state of β-catenin in renal epithelial cells under c-Src activation (261).
Thus, existing data strongly suggest that c-Myc/Max and TCDD/AhR signalling pathways are linked together, but mechanisms of their interaction are still unclear.
According to our model of chloracne pathogenesis, TCDD not only induces the recruitment of stem cells into proliferation but also stimulates stem cell renewal, thus driving chloracne persistence. Although some genes involved in Wnt signalling, adhesion and transcriptional regulation have been proposed as possible candidates to play a role in self-renewal of HF stem cells (262), the mechanisms of this regulation remain largely unknown. Therefore, the possible role of TCDD/AhR pathway in the maintenance of stem cell population also remains obscure.
Thus, based on the data presented above and taking into account the scheme for the role of c-Myc in skin stem cell control (263), we propose that the primary mechanism of chloracne may be represented by TCDD-induced stimulation of keratinocytes to exit from the stem to the transit-amplifying cell compartment, resulting in hyperplasia. This exit is asymmetrical, with differentiation of resultant cells mainly along the epidermal lineage at the expense of pilosebaceous differentiation. We further suggest the AhR/c-Src/c-Myc cascade as the likely dioxin-induced signal transduction pathway leading to chloracne (Fig. 6). Predisposition of specific types of HFs to chloracne may relate to differential dynamics of stem cell activity in various types of follicles.
It is our hope that this model will provide a blueprint for future studies and that step-by-step testing of the assertions put forth in our review will lead to the disclosure of molecular and cellular pathways of chloracne development.