Medical Hypotheses xxx (2015) xxx–xxx
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TCDD-induced activation of aryl hydrocarbon receptor regulates the skin stem cell population Chirag Mandavia ⇑ Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA
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Article history: Received 21 August 2014 Accepted 27 December 2014 Available online xxxx
a b s t r a c t The environmental toxin 2,3,7,8 tetrachlorodibenzo p-dioxin (TCDD) plays an important role in the development of chloracne. Chloracne is characterized by hyperkeratosis of the interfollicular squamous epithelium and metaplasia of sebaceous glands. Dysregulation of keratinocyte terminal differentiation leading to accelerated formation of the cornified envelope as a result of TCDD-mediated aryl hydrocarbon receptor (AHR) activation has been implicated as one of the molecular pathogenic mechanisms contributing to the development of chloracne. In addition, chloracne is characterized by altered skin stem cell characteristics, and it has been speculated that the phenotype of chloracne closely matches that of c-Myc overexpressing transgenic mice. Therefore, we sought to determine whether TCDD plays a role in regulation of the skin stem cell population. We have proposed in this report that TCDD may directly or indirectly (via AHR receptor cross-talk) upregulate c-Myc via epidermal growth factor receptor– extracellular signal regulated kinase (EGFR–ERK) axis stimulation, which may correspond with an increase in human epidermal stem cell activation and differentiation of EPSCs into keratinocytes, with eventual depletion of the epidermal stem cell compartment of the skin. Thus, TCDD may cause increased epidermal stem cell turnover during chloracne. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction TCDD is a major environmental pollutant and the most potent compound in the family of polychlorinated dibenzodioxins (PCDDs). It is classified as a Type 1 carcinogen by the International Agency for Cancer Research (IARC). This toxin has been implicated in a wide range of human toxicities, the most common manifestation of which is chloracne [1–3]. Chloracne is characterized by thickening of the epidermal layers and sebaceous cyst and comedone formation. The molecular pathogenic mechanism for chloracne has been well-studied by several groups, and is thought to involve an acceleration of barrier formation as a result of disordered keratinocyte terminal differentiation [3–6]. TCDD is a physiologic ligand for the aryl hydrocarbon receptor (AHR) and is thought to mediate its toxic actions through binding to this receptor [1,3–5,7]. The aryl hydrocarbon receptor (AHR) is a common nuclear receptor–transcription factor involved in xenobiotic metabolism [3,5,7]. The binding of TCDD on AHR results in AHR nuclear translocation and dimerization with aryl hydrocarbon receptor nuclear translocator (ARNT) to induce transcription of
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cytochrome P450 (CYP) genes [1,3,7]. Among these, CYP family members CYP1A1, CYP1A2 and CYP1B1 are thought to be most important for the metabolism of TCDD [1,3,8]. In addition to this early response, induction of the CYP450 system also involves a late-phase response characterized by upregulation of various antioxidant proteins and enzymes, that catalyze the conversion of Phase I metabolites, produced as a result of the early CYP induction, into Phase II metabolites [6,8,9]. The pathogenesis of TCDD-induced chloracne is also thought to cause an alteration in skin stem cell characteristics, in particular the epidermal stem cell population [2,10]. Furthermore, it has been proposed that the acne-form pustule and blister formation seen in TCDD-induced chloracne is similar to that observed in c-Myc over-expressing transgenic mice [2,10,11]. Skin stem cells occur in the basal layer of the epidermis and at the base of hair follicles. In particular, the epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer [12]. The follicular stem cells can give rise to both the hair follicle and to the epidermis [12,13]. Melanocyte stem cells are responsible for regeneration of melanocytes, a type of pigment cell, and do not contribute to the epidermis. In addition, it has been postulated that mesenchymal stem cells exist in the dermis and hypodermis. However, the role of these cells has not yet been determined [14].
http://dx.doi.org/10.1016/j.mehy.2014.12.023 0306-9877/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mandavia C. TCDD-induced activation of aryl hydrocarbon receptor regulates the skin stem cell population. Med Hypotheses (2015), http://dx.doi.org/10.1016/j.mehy.2014.12.023
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TCDD-mediated AHR activation is known to play a role in HSC cell lineage differentiation [10,11,15]. Thus, it seems likely that TCDD-mediated AHR activation would similarly be able to regulate the epidermal stem cell population, allowing activation and proliferation of quiescent epidermal stem cells and their eventual differentiation into keratinocytes, in response to the TCDD-mediated insult. Furthermore, chronic stimulation by TCDD could result in skin stem cell depletion as a result of the TCDD-induced constant stem cell turnover. Therefore, we sought to elicit the molecular mechanism by which the endogenous skin stem cell compartment would be regulated in response to TCDD treatment and AHR activation. c-Myc, a mammalian viral oncogene homolog, is one of the activators of the epidermal stem cell population [16]. The authors implicated both polycomb-dependent, as well as independent, functions of Cbx4 in regulation of epidermal stem cell activity and senescence [16]. AHR has been shown to constitutively repress c-myc transcription by binding to its promoter in mammary tumor cells [17]. The promoter of c-myc contains 6 AHR response elements and 2 NF-kappa B binding sites [17]. Interestingly however, TCDD was found to have no effect on this regulation. AHR also had no effect on c-myc transcription in normal cells. Separately, TCDD treatment during viral infection was found to upregulate AHR receptor and down-regulate bTERT and c-Myc in MDBK cells [18]. Regulation of c-Myc via growth factor signaling is well-defined [19]. Recent studies showed that TCDD mediates some of its effects via direct regulation of the epidermal growth factor receptor (EGFR), or indirectly via AHR–EGFR receptor cross-talk [3–5,20]. These findings led us to evaluate whether TCDD could stimulate c-Myc to activate the epidermal stem cell population. Hypotheses Our hypothesis is that TCDD-mediated AHR activation is able to allow transition of human epidermal stem cells (EPSCs) to an actively proliferating state via activation of c-Myc. Since this action would depend on stimulation of c-Myc via growth factors, we also seek to investigate if TCDD can activate c-Myc via the EGFR-ERK pathway. In addition, we want to determine if activation of c-Myc is able to allow not only activation of the EPSCs, but also their eventual differentiation into keratinocytes, and whether this will cause a depletion of the endogenous stem cell population over the long term. Evaluation of hypotheses In order to address these questions, we will use skin samples from TCDD-treated mice, as well as TCDD-treated normal human keratinocytes in culture, to determine if c-Myc is activated in response to TCDD. We plan to use AG1478, which is an EGFR-
specific inhibitor and PD98059, an ERK-specific inhibitor, to determine whether the EGFR-ERK axis is able to mediate this activation. In addition, we will treat normal human epidermal stem cells in culture with TCDD and test them for activation and differentiation into mature keratinocytes via immuno-staining for various markers. Specifically, we will look for proliferating cells in the basal layer via analyzing expression of keratin markers K5 or K14 and early differentiating cells in the spinous layer or late differentiating cells in the granular layer via expression of K1/K10 or filaggrin/ loricrin, respectively. In addition, we can analyze the epidermal stem cell depletion in culture via immuno-staining for markers such as CD200, follistatin, integrin alpha 6, after chronic treatment of cells with TCDD (Fig. 1). Our studies should demonstrate that TCDD-mediated AHR activation contributes to epidermal stem cell compartment activation, and eventual differentiation, to form new keratinocytes via c-Myc activation. We should also be able to show the mechanistic aspect governing c-Myc activation by TCDD. Using inhibitors specific for EGFR and ERK, we aim to show that activation of c-Myc by TCDD occurs through EGFR-ERK pathway. In addition, our report highlights and brings into focus a novel concept, whereby TCDD can induce depletion of the endogenous epidermal stem cell population, as a result of the constant stem cell turnover. Methods Materials, antibodies and reagents High-affinity AHR receptor skin hairless (SKH) mice will be purchased from Jackson Laboratories. Human keratinocytes and epidermal stem cells will be obtained from American Type Culture Collection (ATCC). All antibodies will be purchased from Cell Signaling. TCDD, inhibitors and reagents are from Sigma. Cell culture media and supplements are from ATCC. All other supplies are from Fisher Scientific. Sample treatments and collection SKH mice will be conditioned for 2 weeks per IACUC protocol and treated with a single acute dose of 30 mg per kg TCDD. Control mice will simultaneously be treated with an equal volume of 0.1% DMSO. After 24 h, mice will be euthanized by CO2 asphyxiation. Skin samples will be dissected and stored at 80 °C for downstream assays. Similarly, human keratinocytes and epidermal stem cells in culture will be grown to 100% confluence and treated with 10 nM TCDD in 0.1% DMSO for 6 h and 24 h prior to collection. Cells that are treated with 0.1% DMSO will be used as control. Whereas keratinocytes were collected at 24 h after treatment start, similar to mice, cultured epidermal stem cells will be allowed to differentiate in culture for 5, 10 or 15 days before collection. Wherever
Fig. 1. Experimental approach to define the role of TCDD-mediated AHR activation in epidermal stem cell activation and differentiation. Abbreviations: EGFR, epidermal growth factor receptor; ERK, extracellular signal regulated kinase; ±=, with or without inhibitors; c-Myc – V-myc myelocytomatosis viral oncogene homolog; Act, activation; Diff, differentiation; Depl, depletion; TCDD, 2,3,7,8 tetrachlorodibenzo p-dioxin. General note: mice were treated with an acute dose of 30 lg/kg TCDD, while keratinocytes were treated with 10 nM TCDD for 6 h or 24 h with or without inhibitors.
Please cite this article in press as: Mandavia C. TCDD-induced activation of aryl hydrocarbon receptor regulates the skin stem cell population. Med Hypotheses (2015), http://dx.doi.org/10.1016/j.mehy.2014.12.023
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inhibitors are employed, cells in culture will be treated with a 10-fold molar excess of EGFR-specific inhibitor AG1478 or ERKspecific inhibitor PD98059 for 1 h prior to TCDD treatment. Cells will be washed once with phosphate-buffered saline, collected by centrifugation and frozen down for subsequent experiments. Immunoblotting Human keratinocytes differentially exposed to various inhibitor treatments will be collected, as mentioned above, subjected to non-denaturing lysis and protein obtained as per standard protocols. Radio-immunoprecipitation Assay (RIPA) buffer will be used to make whole cell lysates. Protein amounts can be equalized using Bradford Assay. The phosphorylation patterns and protein content of the proteins c-Myc, EGFR and ERK will be tested by standard immunoblotting techniques using polyclonal rabbit antibodies against these proteins with cross-reactivity for both mouse and human isoforms. Beta-actin will be used for loading control. The protein content and phosphorylation data will be analyzed using ImageJ protein quantitation software developed by NCBI. Similarly, skin tissue will be lysed and protein content subjected to immunoblotting as described above. Human epidermal stem cells in culture can also be assayed for c-Myc phosphorylation and expression by immunoblotting, as described above, at 5, 10 and 15 days post-treatment to correlate c-Myc activation with epidermal stem cell maturation. Quantitative PCR Skin samples or cells, collected as mentioned above, will be subjected to non-denaturing lysis and total RNA obtained using the Trizol-based method previously described, as per standard protocol. cDNA will be synthesized using a commercially available qRT-PCR kit from Qiagen. Taqman probes from Applied Biosystems (ABI) will be generated to c-Myc, EGFR and ERK, as mentioned above, and after equalization of the total amount of RNA, can be used in quantitative PCR to determine the transcriptional regulatory content of these molecules. GAPDH can be used as a control. The qPCR data will be analyzed using manufacturer-provided quantitation software, as per manufacturer’s protocol. Immuno-staining Organotypic cultures of human epidermal stem cells will be treated with TCDD and fixed with 4% formaldehyde for 15 min at room temperature. Briefly immuno-fluorescence assay (IF) will be performed using primary antibodies suitable for immunofluorescence to K5, K10, filaggrin or integrin alpha 6 obtained from Cell Signaling, following which the cells will be incubated for 30 min with fluorescently labeled secondary antibodies, also from Cell-Signaling, as per manufacturer’s protocol. DAPI will be used to stain nuclei. Samples will be fixed overnight and analyzed using a confocal microscope. Statistical analysis All data presented will be representative of at least 3 independent experiments. Comparisons between groups will be performed using the analysis of variance (ANOVA) test. Expected results
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inhibitors, as described above, and subject them to qPCR and Western blot to determine the activation of c-Myc, EGFR and ERK. We intend to use phosphorylation as a proxy for c-Myc, EGFR and ERK activation, while the total protein will be used to obtain a ratio of phosphorylated over total protein. In addition, we will use quantitative PCR to determine the amount of transcript corresponding to these proteins. We should see increased mRNA corresponding to c-Myc, ERK and EGFR in the TCDD-treated samples compared to control. Phosphorylation of EGFR, ERK and c-Myc will also be increased in response to acute dose of TCDD in mice, and at 6 h post TCDD-treatment in human keratinocytes in culture. In contrast, inhibition of both EGFR and ERK for 1 h prior to TCDD treatment in culture should down-regulate c-Myc phosphorylation and expression. Activation and differentiation of TCDD-treated human epidermal stem cells in culture into mature keratinocytes As described above, we will grow human epidermal stem cells in culture to 100% confluence before treating them with 10 nM TCDD for 6 h or 24 h. EPSCs will be allowed to differentiate in culture post TCDD treatment and collected at 5, 10 and 15 days post treatment to observe keratinocyte maturation. c-Myc activity will be tested in parallel to see if it corresponds to EPSC activation and differentiation. We hope to see increased intra-cellular mRNA and protein levels of c-Myc in TCDD-treated human epidermal stem cells compared to controls in cells treated for 6 h. Immunostaining for K5, K10 and loricrin should show increased expression of these keratinocyte differentiation markers after TCDD treatment for 6 h at days 5, 10 and 15 in organotypic cultures, indicating keratinocyte maturation. These data will indicate that c-Myc is activated after 6 h of TCDD treatment in human epidermal stem cells, similar to the response seen in normal human keratinocytes. Since the duration of c-Myc activation corresponds in EPSCs and keratinocytes, it is likely that this increase in c-Myc activity is mediated by TCDD influence on the EGFR-ERK axis, corresponding to our data in human keratinocytes. Depletion of the endogenous epidermal stem cell population after chronic (24 h) treatments of EPSCs with TCDD As mentioned above, we will treat epidermal stem cells in culture with TCDD at both 6 h and 24 h, and collect them at 5, 10 and 15 days post treatment. The 5-day collection samples that were subjected to long-term TCDD exposure (24 h) will not be assayed for c-Myc activity or keratinocyte activation or maturation markers, but rather immuno-stained for integrin alpha 6, which is a marker of epidermal stem cells. Even if we observe an increase in c-Myc activity and epidermal stem cell activation and maturation after TCDD treatment for 6 h in all samples collected, we should observe a decrease in the integrin alpha 6 expression after 24 h of TCDD treatment of EPSCs, indicating a depletion of the endogenous stem cell compartment after chronic treatment with TCDD. Taken together, these data will suggest that acute or shorterterm TCDD exposure leads to a transient upregulation of c-Myc via the EGFR-ERK axis, which serves to activate the quiescent epidermal stem cell compartment of the skin and their eventual differentiation into mature keratinocytes as a mechanism of combating the toxin-mediated injury, but eventually leads to a depletion of the endogenous EPSCs, if the TCDD exposure is long-term and chronic (Fig. 2).
c-Myc should be activated through the EGFR-ERK axis in response to TCDD treatment in human keratinocytes and skin samples from SKH mice
Discussion
We will treat high-affinity AHR receptor skin hairless mice and human keratinocytes in culture with TCDD, with or without
The role of TCDD-mediated AHR activation in the pathogenesis of chloracne is well-studied and involves increased keratinocyte
Please cite this article in press as: Mandavia C. TCDD-induced activation of aryl hydrocarbon receptor regulates the skin stem cell population. Med Hypotheses (2015), http://dx.doi.org/10.1016/j.mehy.2014.12.023
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Fig. 2. Proposed model for TCDD-mediated c-Myc Stimulation via the AHR pathway and regulation of epidermal stem cell activation and differentiation and eventual depletion: AHR constitutively suppresses c-Myc expression, keeping the epidermal stem cell cells in a quiescent state. TCDD serves to initially down-regulate c-Myc via the AHR–ARNT pathway, but after longer exposure, activates the EGFR-ERK axis either directly or via AHR–EGFR receptor mediated crosstalk, to up-regulate c-Myc, which leads to increased stem cell turnover. Thus, TCDD leads initially to epidermal stem cell compartment activation and proliferation and eventual differentiation into mature keratinocytes. Very long-term chronic TCDD exposure eventually leads to endogenous skin stem cell compartment depletion. (Abbreviations: AHR– ARNT – aryl hydrocarbon receptor–aryl hydrocarbon receptor nuclear translocator, EGFR-ERK – epidermal growth factor receptor–extracellular signal regulated kinase, c-Myc – V-myc myelocytomatosis viral oncogene homolog, TCDD – 2,3,7,8 tetrachlorodibenzo p-dioxin).
terminal differentiation and accelerated barrier formation [4–6]. It has been proposed that the phenotype of chloracne seen in TCDDmediated AHR activation is similar to that observed in c-Myc overexpressing transgenic mice [2]. Chloracne also induced alterations in skin stem cell characteristics, including increased stem cell cycling and self-renewal and a skewing of differentiation towards the epidermal pathway [2]. This led to the hypothesis that TCDDmediated AHR activation may regulate skin stem cell population. c-Myc is an important transcription factor involved in the regulation of many target genes in response to mitogenic signals. Its regulation via the MAPK/ERK pathway is well-defined. In addition, it plays an important role in epigenetic gene regulation due to its intrinsic histone acetyltransferase activity. It has been shown to be one of the four factors responsible for inducing reprogramming of somatic cells to a pluripotent stem-cell state [21]. It has been shown recently that pluripotent factor and polycomb group proteins repress AHR in murine embryonic stem cells, and that reversible Ahr repression holds the gene poised for expression and allows for a quick switch to activation [22]. In addition, it may indicate the presence of a regulatory feedback loop, in order to check excessive over-stimulation of AHR by c-Myc. Previous studies have implicated the AHR in hematopoietic cell lineage development [10,11,15]. These authors have shown that AHR has a role in stem cell cycling, regulation and quiescence [15], and loss of aryl hydrocarbon receptor activity is associated with premature hematopoietic stem cell exhaustion [11]. Under normal conditions, AHR is a negative regulator of c-Myc [17,18] and acts to inhibit cell proliferation. Since AHR is involved in detoxification and xenobiotic metabolism, TCDD-mediated stimulation of AHR results in mitochondrial oxidative stress [3], which may lead to downstream activation of c-Myc. Recently, the CYP1 family enzyme CYP1B1 was implicated in contributing to the cellular and mitochondrial oxidative stress produced as a result of TCDD-mediated AHR stimulation [3,23]. The authors showed that TCDD treatment up-regulated CYP1B1 levels and targeted it to mitochondria, where it caused oxidative stress by various mechanisms [23]. CYP1B1 has also been found to be overexpressed in several extra-hepatic tumor cell lines, and has been proposed to play a role in hormone-mediated tumor metabolism [24]. Sustained overexpression of CYP1B1 has been shown to cause DNA adduct formation upon exposure to benzo(a)pyrene (BaP) [25]. Since AHR stimulation by TCDD induces CYP1B1 and
may regulate c-Myc through CYP1B1-mediated oxidative stress, c-Myc may represent the underlying link between CYP1B1 overexpression and cancer. Moreover, TCDD is classified as a carcinogen, and addition of TCDD before BaP treatment in mice sensitized them to increased formation of DNA adducts and tumorigenesis [26,27]. TCDD itself has not been shown to cause DNA adduct formation leading to tumors, and is therefore considered as a tumor-promoting agent rather than a tumor-initiating agent. However, it is also possible that TCDD itself may possess tumor-initiating properties, either due to its ability to cause DNA adducts directly mediated by CYP1B1-generated oxidative stress, or as result of the downstream activation of c-Myc as a result of the oxidative stress-induced mitogenic signaling. All these data show a direct link between TCDD-mediated AHR expression and c-Myc activation, which formed the basis of our proposal to study skin stem cell cycling after TCDD exposure. We are interested in studying the molecular mechanism of skin stem cell activation and differentiation in response to TCDD treatment and AHR activation. c-Myc, an important epidermal stem cell activator and inhibitor of differentiation, has been shown to be down-regulated by TCDD-mediated AHR activation during infection, and its basal transcription was constitutively repressed by AHR binding to its promoter in mammary tumor cells. We intend to show that TCDD-mediated AHR activation in normal human skin cells up-regulated c-Myc through cross-talk via the EGFR-ERK pathway. This upregulation should correspond to an increase in human epidermal stem cell activation and differentiation of EPSCs into keratinocytes. Chronic treatment with TCDD should result in eventual depletion of the epidermal stem cell compartment of the skin. In conclusion, our studies will provide a mechanistic basis for the changes observed in epidermal stem cell characteristics underlying chloracne and the role of TCDD as a stimulator of keratinocyte differentiation. In addition, as c-Myc regulates a wide variety of genes and has been thought by some to regulate almost 15% of the genes in the human genome, we speculate that chromatin immunoprecipitation and sequencing (CHIP-seq) studies on c-Myc in skin samples treated with TCDD will serve as a useful diagnostic tool to identify the genes it regulates under these conditions, and will be essential for understanding the role of c-Myc in altering skin stem cell characteristics during TCDD-induced chloracne. Conflicts of interest statement The authors declare no conflicts of interest. Acknowledgements We thank Dr. Amy Abell, PhD, for her valuable advice, insight and helpful suggestions in the preparation of this manuscript. This work was supported, in part, by a research fellowship to Dr. Chirag Mandavia by the University of Memphis. References [1] Forrester AR, Elias MS, Woodward EL, Graham M, Williams FM, Reynolds NJ. Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down. J Dermatol Sci 2014;73(1):10–22. [2] Panteleyev AA, Bickers DR. Dioxin-induced chloracne–reconstructing the cellular and molecular mechanisms of a classic environmental disease. Exp Dermatol 2006;15(9):705–30. Review. [3] Kennedy LH, Sutter CH, Leon Carrion S, Tran QT, Bodreddigari S, Kensicki E, et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated production of reactive oxygen species is an essential step in the mechanism of action to accelerate human keratinocyte differentiation. Toxicol Sci 2013;132(1):235–49.
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C. Mandavia / Medical Hypotheses xxx (2015) xxx–xxx [4] Sutter CH, Bodreddigari S, Campion C, Wible RS, Sutter TR. 2,3,7,8Tetrachlorodibenzo-p-dioxin increases the expression of genes in the human epidermal differentiation complex and accelerates epidermal barrier formation. Toxicol Sci 2011;124(1):128–37. [5] Sutter CH, Yin H, Li Y, Mammen JS, Bodreddigari S, Stevens G, et al. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes. Proc Natl Acad Sci USA 2009;106(11):4266–71. [6] Schäfer M, Willrodt AH, Kurinna S, Link AS, Farwanah H, Geusau A, et al. Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice. EMBO Mol Med 2014;6(4):442–57. [7] Hao N, Whitelaw ML. The emerging roles of AhR in physiology and immunity. Biochem Pharmacol 2013;86(5):561–70. [8] Watson JD, Prokopec SD, Smith AB, Okey AB, Pohjanvirta R, Boutros PC. TCDD dysregulation of 13 AHR-target genes in rat liver. Toxicol Appl Pharmacol 2014;274(3):445–54. [9] Wang L, He X, Szklarz GD, Bi Y, Rojanasakul Y, Ma Q. The aryl hydrocarbon receptor interacts with nuclear factor erythroid 2-related factor 2 to mediate induction of NAD(P)H:quinoneoxidoreductase 1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch Biochem Biophys 2013;537(1):31–8. [10] Singh KP, Casado FL, Opanashuk LA, Gasiewicz TA. The aryl hydrocarbon receptor has a normal function in the regulation of hematopoietic and other stem/progenitor cell populations. Biochem Pharmacol 2009;77(4):577–87. [11] Singh KP, Bennett JA, Casado FL, Walrath JL, Welle SL, Gasiewicz TA. Loss of aryl hydrocarbon receptor promotes gene changes associated with premature hematopoietic stem cell exhaustion and development of a myeloproliferative disorder in aging mice. Stem Cells Dev 2014;23(2):95–106. [12] Blanpain C, Fuchs E. Epidermal stem cells of the skin. Annu Rev Cell Dev Biol 2006;22:339–73. [13] Plikus MV, Gay DL, Treffeisen E, Wang A, Supapannachart RJ, Cotsarelis G. Epithelial stem cells and implications for wound repair. Semin Cell Dev Biol 2012;23(9):946–53. [14] Rajasekhar VK, Vemuri MC. Molecular insights into the function, fate, and prospects of stem cells. Stem Cells 2005;23(8):1212–20. [15] Gasiewicz TA, Singh KP, Bennett JA. The Ah receptor in stem cell cycling, regulation, and quiescence. Ann N Y Acad Sci 2014;1310(1):44–50. [16] Luis NM, Morey L, Mejetta S, Pascual G, Janich P, Kuebler B, et al. Regulation of human epidermal stem cell proliferation and senescence requires polycombdependent and -independent functions of Cbx4. Cell Stem Cell 2011;9(3): 233–46.
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TCDD-induced activation of aryl hydrocarbon receptor regulates the skin stem cell population Chirag Mandavia ⇑ Department of Biological Sciences, University of Memphis, Memphis, TN 38152, USA
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Article history: Received 21 August 2014 Accepted 27 December 2014 Available online xxxx
a b s t r a c t The environmental toxin 2,3,7,8 tetrachlorodibenzo p-dioxin (TCDD) plays an important role in the development of chloracne. Chloracne is characterized by hyperkeratosis of the interfollicular squamous epithelium and metaplasia of sebaceous glands. Dysregulation of keratinocyte terminal differentiation leading to accelerated formation of the cornified envelope as a result of TCDD-mediated aryl hydrocarbon receptor (AHR) activation has been implicated as one of the molecular pathogenic mechanisms contributing to the development of chloracne. In addition, chloracne is characterized by altered skin stem cell characteristics, and it has been speculated that the phenotype of chloracne closely matches that of c-Myc overexpressing transgenic mice. Therefore, we sought to determine whether TCDD plays a role in regulation of the skin stem cell population. We have proposed in this report that TCDD may directly or indirectly (via AHR receptor cross-talk) upregulate c-Myc via epidermal growth factor receptor– extracellular signal regulated kinase (EGFR–ERK) axis stimulation, which may correspond with an increase in human epidermal stem cell activation and differentiation of EPSCs into keratinocytes, with eventual depletion of the epidermal stem cell compartment of the skin. Thus, TCDD may cause increased epidermal stem cell turnover during chloracne. Ó 2015 Elsevier Ltd. All rights reserved.
Introduction TCDD is a major environmental pollutant and the most potent compound in the family of polychlorinated dibenzodioxins (PCDDs). It is classified as a Type 1 carcinogen by the International Agency for Cancer Research (IARC). This toxin has been implicated in a wide range of human toxicities, the most common manifestation of which is chloracne [1–3]. Chloracne is characterized by thickening of the epidermal layers and sebaceous cyst and comedone formation. The molecular pathogenic mechanism for chloracne has been well-studied by several groups, and is thought to involve an acceleration of barrier formation as a result of disordered keratinocyte terminal differentiation [3–6]. TCDD is a physiologic ligand for the aryl hydrocarbon receptor (AHR) and is thought to mediate its toxic actions through binding to this receptor [1,3–5,7]. The aryl hydrocarbon receptor (AHR) is a common nuclear receptor–transcription factor involved in xenobiotic metabolism [3,5,7]. The binding of TCDD on AHR results in AHR nuclear translocation and dimerization with aryl hydrocarbon receptor nuclear translocator (ARNT) to induce transcription of
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cytochrome P450 (CYP) genes [1,3,7]. Among these, CYP family members CYP1A1, CYP1A2 and CYP1B1 are thought to be most important for the metabolism of TCDD [1,3,8]. In addition to this early response, induction of the CYP450 system also involves a late-phase response characterized by upregulation of various antioxidant proteins and enzymes, that catalyze the conversion of Phase I metabolites, produced as a result of the early CYP induction, into Phase II metabolites [6,8,9]. The pathogenesis of TCDD-induced chloracne is also thought to cause an alteration in skin stem cell characteristics, in particular the epidermal stem cell population [2,10]. Furthermore, it has been proposed that the acne-form pustule and blister formation seen in TCDD-induced chloracne is similar to that observed in c-Myc over-expressing transgenic mice [2,10,11]. Skin stem cells occur in the basal layer of the epidermis and at the base of hair follicles. In particular, the epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin and form a protective layer [12]. The follicular stem cells can give rise to both the hair follicle and to the epidermis [12,13]. Melanocyte stem cells are responsible for regeneration of melanocytes, a type of pigment cell, and do not contribute to the epidermis. In addition, it has been postulated that mesenchymal stem cells exist in the dermis and hypodermis. However, the role of these cells has not yet been determined [14].
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Please cite this article in press as: Mandavia C. TCDD-induced activation of aryl hydrocarbon receptor regulates the skin stem cell population. Med Hypotheses (2015), http://dx.doi.org/10.1016/j.mehy.2014.12.023
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TCDD-mediated AHR activation is known to play a role in HSC cell lineage differentiation [10,11,15]. Thus, it seems likely that TCDD-mediated AHR activation would similarly be able to regulate the epidermal stem cell population, allowing activation and proliferation of quiescent epidermal stem cells and their eventual differentiation into keratinocytes, in response to the TCDD-mediated insult. Furthermore, chronic stimulation by TCDD could result in skin stem cell depletion as a result of the TCDD-induced constant stem cell turnover. Therefore, we sought to elicit the molecular mechanism by which the endogenous skin stem cell compartment would be regulated in response to TCDD treatment and AHR activation. c-Myc, a mammalian viral oncogene homolog, is one of the activators of the epidermal stem cell population [16]. The authors implicated both polycomb-dependent, as well as independent, functions of Cbx4 in regulation of epidermal stem cell activity and senescence [16]. AHR has been shown to constitutively repress c-myc transcription by binding to its promoter in mammary tumor cells [17]. The promoter of c-myc contains 6 AHR response elements and 2 NF-kappa B binding sites [17]. Interestingly however, TCDD was found to have no effect on this regulation. AHR also had no effect on c-myc transcription in normal cells. Separately, TCDD treatment during viral infection was found to upregulate AHR receptor and down-regulate bTERT and c-Myc in MDBK cells [18]. Regulation of c-Myc via growth factor signaling is well-defined [19]. Recent studies showed that TCDD mediates some of its effects via direct regulation of the epidermal growth factor receptor (EGFR), or indirectly via AHR–EGFR receptor cross-talk [3–5,20]. These findings led us to evaluate whether TCDD could stimulate c-Myc to activate the epidermal stem cell population. Hypotheses Our hypothesis is that TCDD-mediated AHR activation is able to allow transition of human epidermal stem cells (EPSCs) to an actively proliferating state via activation of c-Myc. Since this action would depend on stimulation of c-Myc via growth factors, we also seek to investigate if TCDD can activate c-Myc via the EGFR-ERK pathway. In addition, we want to determine if activation of c-Myc is able to allow not only activation of the EPSCs, but also their eventual differentiation into keratinocytes, and whether this will cause a depletion of the endogenous stem cell population over the long term. Evaluation of hypotheses In order to address these questions, we will use skin samples from TCDD-treated mice, as well as TCDD-treated normal human keratinocytes in culture, to determine if c-Myc is activated in response to TCDD. We plan to use AG1478, which is an EGFR-
specific inhibitor and PD98059, an ERK-specific inhibitor, to determine whether the EGFR-ERK axis is able to mediate this activation. In addition, we will treat normal human epidermal stem cells in culture with TCDD and test them for activation and differentiation into mature keratinocytes via immuno-staining for various markers. Specifically, we will look for proliferating cells in the basal layer via analyzing expression of keratin markers K5 or K14 and early differentiating cells in the spinous layer or late differentiating cells in the granular layer via expression of K1/K10 or filaggrin/ loricrin, respectively. In addition, we can analyze the epidermal stem cell depletion in culture via immuno-staining for markers such as CD200, follistatin, integrin alpha 6, after chronic treatment of cells with TCDD (Fig. 1). Our studies should demonstrate that TCDD-mediated AHR activation contributes to epidermal stem cell compartment activation, and eventual differentiation, to form new keratinocytes via c-Myc activation. We should also be able to show the mechanistic aspect governing c-Myc activation by TCDD. Using inhibitors specific for EGFR and ERK, we aim to show that activation of c-Myc by TCDD occurs through EGFR-ERK pathway. In addition, our report highlights and brings into focus a novel concept, whereby TCDD can induce depletion of the endogenous epidermal stem cell population, as a result of the constant stem cell turnover. Methods Materials, antibodies and reagents High-affinity AHR receptor skin hairless (SKH) mice will be purchased from Jackson Laboratories. Human keratinocytes and epidermal stem cells will be obtained from American Type Culture Collection (ATCC). All antibodies will be purchased from Cell Signaling. TCDD, inhibitors and reagents are from Sigma. Cell culture media and supplements are from ATCC. All other supplies are from Fisher Scientific. Sample treatments and collection SKH mice will be conditioned for 2 weeks per IACUC protocol and treated with a single acute dose of 30 mg per kg TCDD. Control mice will simultaneously be treated with an equal volume of 0.1% DMSO. After 24 h, mice will be euthanized by CO2 asphyxiation. Skin samples will be dissected and stored at 80 °C for downstream assays. Similarly, human keratinocytes and epidermal stem cells in culture will be grown to 100% confluence and treated with 10 nM TCDD in 0.1% DMSO for 6 h and 24 h prior to collection. Cells that are treated with 0.1% DMSO will be used as control. Whereas keratinocytes were collected at 24 h after treatment start, similar to mice, cultured epidermal stem cells will be allowed to differentiate in culture for 5, 10 or 15 days before collection. Wherever
Fig. 1. Experimental approach to define the role of TCDD-mediated AHR activation in epidermal stem cell activation and differentiation. Abbreviations: EGFR, epidermal growth factor receptor; ERK, extracellular signal regulated kinase; ±=, with or without inhibitors; c-Myc – V-myc myelocytomatosis viral oncogene homolog; Act, activation; Diff, differentiation; Depl, depletion; TCDD, 2,3,7,8 tetrachlorodibenzo p-dioxin. General note: mice were treated with an acute dose of 30 lg/kg TCDD, while keratinocytes were treated with 10 nM TCDD for 6 h or 24 h with or without inhibitors.
Please cite this article in press as: Mandavia C. TCDD-induced activation of aryl hydrocarbon receptor regulates the skin stem cell population. Med Hypotheses (2015), http://dx.doi.org/10.1016/j.mehy.2014.12.023
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inhibitors are employed, cells in culture will be treated with a 10-fold molar excess of EGFR-specific inhibitor AG1478 or ERKspecific inhibitor PD98059 for 1 h prior to TCDD treatment. Cells will be washed once with phosphate-buffered saline, collected by centrifugation and frozen down for subsequent experiments. Immunoblotting Human keratinocytes differentially exposed to various inhibitor treatments will be collected, as mentioned above, subjected to non-denaturing lysis and protein obtained as per standard protocols. Radio-immunoprecipitation Assay (RIPA) buffer will be used to make whole cell lysates. Protein amounts can be equalized using Bradford Assay. The phosphorylation patterns and protein content of the proteins c-Myc, EGFR and ERK will be tested by standard immunoblotting techniques using polyclonal rabbit antibodies against these proteins with cross-reactivity for both mouse and human isoforms. Beta-actin will be used for loading control. The protein content and phosphorylation data will be analyzed using ImageJ protein quantitation software developed by NCBI. Similarly, skin tissue will be lysed and protein content subjected to immunoblotting as described above. Human epidermal stem cells in culture can also be assayed for c-Myc phosphorylation and expression by immunoblotting, as described above, at 5, 10 and 15 days post-treatment to correlate c-Myc activation with epidermal stem cell maturation. Quantitative PCR Skin samples or cells, collected as mentioned above, will be subjected to non-denaturing lysis and total RNA obtained using the Trizol-based method previously described, as per standard protocol. cDNA will be synthesized using a commercially available qRT-PCR kit from Qiagen. Taqman probes from Applied Biosystems (ABI) will be generated to c-Myc, EGFR and ERK, as mentioned above, and after equalization of the total amount of RNA, can be used in quantitative PCR to determine the transcriptional regulatory content of these molecules. GAPDH can be used as a control. The qPCR data will be analyzed using manufacturer-provided quantitation software, as per manufacturer’s protocol. Immuno-staining Organotypic cultures of human epidermal stem cells will be treated with TCDD and fixed with 4% formaldehyde for 15 min at room temperature. Briefly immuno-fluorescence assay (IF) will be performed using primary antibodies suitable for immunofluorescence to K5, K10, filaggrin or integrin alpha 6 obtained from Cell Signaling, following which the cells will be incubated for 30 min with fluorescently labeled secondary antibodies, also from Cell-Signaling, as per manufacturer’s protocol. DAPI will be used to stain nuclei. Samples will be fixed overnight and analyzed using a confocal microscope. Statistical analysis All data presented will be representative of at least 3 independent experiments. Comparisons between groups will be performed using the analysis of variance (ANOVA) test. Expected results
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inhibitors, as described above, and subject them to qPCR and Western blot to determine the activation of c-Myc, EGFR and ERK. We intend to use phosphorylation as a proxy for c-Myc, EGFR and ERK activation, while the total protein will be used to obtain a ratio of phosphorylated over total protein. In addition, we will use quantitative PCR to determine the amount of transcript corresponding to these proteins. We should see increased mRNA corresponding to c-Myc, ERK and EGFR in the TCDD-treated samples compared to control. Phosphorylation of EGFR, ERK and c-Myc will also be increased in response to acute dose of TCDD in mice, and at 6 h post TCDD-treatment in human keratinocytes in culture. In contrast, inhibition of both EGFR and ERK for 1 h prior to TCDD treatment in culture should down-regulate c-Myc phosphorylation and expression. Activation and differentiation of TCDD-treated human epidermal stem cells in culture into mature keratinocytes As described above, we will grow human epidermal stem cells in culture to 100% confluence before treating them with 10 nM TCDD for 6 h or 24 h. EPSCs will be allowed to differentiate in culture post TCDD treatment and collected at 5, 10 and 15 days post treatment to observe keratinocyte maturation. c-Myc activity will be tested in parallel to see if it corresponds to EPSC activation and differentiation. We hope to see increased intra-cellular mRNA and protein levels of c-Myc in TCDD-treated human epidermal stem cells compared to controls in cells treated for 6 h. Immunostaining for K5, K10 and loricrin should show increased expression of these keratinocyte differentiation markers after TCDD treatment for 6 h at days 5, 10 and 15 in organotypic cultures, indicating keratinocyte maturation. These data will indicate that c-Myc is activated after 6 h of TCDD treatment in human epidermal stem cells, similar to the response seen in normal human keratinocytes. Since the duration of c-Myc activation corresponds in EPSCs and keratinocytes, it is likely that this increase in c-Myc activity is mediated by TCDD influence on the EGFR-ERK axis, corresponding to our data in human keratinocytes. Depletion of the endogenous epidermal stem cell population after chronic (24 h) treatments of EPSCs with TCDD As mentioned above, we will treat epidermal stem cells in culture with TCDD at both 6 h and 24 h, and collect them at 5, 10 and 15 days post treatment. The 5-day collection samples that were subjected to long-term TCDD exposure (24 h) will not be assayed for c-Myc activity or keratinocyte activation or maturation markers, but rather immuno-stained for integrin alpha 6, which is a marker of epidermal stem cells. Even if we observe an increase in c-Myc activity and epidermal stem cell activation and maturation after TCDD treatment for 6 h in all samples collected, we should observe a decrease in the integrin alpha 6 expression after 24 h of TCDD treatment of EPSCs, indicating a depletion of the endogenous stem cell compartment after chronic treatment with TCDD. Taken together, these data will suggest that acute or shorterterm TCDD exposure leads to a transient upregulation of c-Myc via the EGFR-ERK axis, which serves to activate the quiescent epidermal stem cell compartment of the skin and their eventual differentiation into mature keratinocytes as a mechanism of combating the toxin-mediated injury, but eventually leads to a depletion of the endogenous EPSCs, if the TCDD exposure is long-term and chronic (Fig. 2).
c-Myc should be activated through the EGFR-ERK axis in response to TCDD treatment in human keratinocytes and skin samples from SKH mice
Discussion
We will treat high-affinity AHR receptor skin hairless mice and human keratinocytes in culture with TCDD, with or without
The role of TCDD-mediated AHR activation in the pathogenesis of chloracne is well-studied and involves increased keratinocyte
Please cite this article in press as: Mandavia C. TCDD-induced activation of aryl hydrocarbon receptor regulates the skin stem cell population. Med Hypotheses (2015), http://dx.doi.org/10.1016/j.mehy.2014.12.023
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Fig. 2. Proposed model for TCDD-mediated c-Myc Stimulation via the AHR pathway and regulation of epidermal stem cell activation and differentiation and eventual depletion: AHR constitutively suppresses c-Myc expression, keeping the epidermal stem cell cells in a quiescent state. TCDD serves to initially down-regulate c-Myc via the AHR–ARNT pathway, but after longer exposure, activates the EGFR-ERK axis either directly or via AHR–EGFR receptor mediated crosstalk, to up-regulate c-Myc, which leads to increased stem cell turnover. Thus, TCDD leads initially to epidermal stem cell compartment activation and proliferation and eventual differentiation into mature keratinocytes. Very long-term chronic TCDD exposure eventually leads to endogenous skin stem cell compartment depletion. (Abbreviations: AHR– ARNT – aryl hydrocarbon receptor–aryl hydrocarbon receptor nuclear translocator, EGFR-ERK – epidermal growth factor receptor–extracellular signal regulated kinase, c-Myc – V-myc myelocytomatosis viral oncogene homolog, TCDD – 2,3,7,8 tetrachlorodibenzo p-dioxin).
terminal differentiation and accelerated barrier formation [4–6]. It has been proposed that the phenotype of chloracne seen in TCDDmediated AHR activation is similar to that observed in c-Myc overexpressing transgenic mice [2]. Chloracne also induced alterations in skin stem cell characteristics, including increased stem cell cycling and self-renewal and a skewing of differentiation towards the epidermal pathway [2]. This led to the hypothesis that TCDDmediated AHR activation may regulate skin stem cell population. c-Myc is an important transcription factor involved in the regulation of many target genes in response to mitogenic signals. Its regulation via the MAPK/ERK pathway is well-defined. In addition, it plays an important role in epigenetic gene regulation due to its intrinsic histone acetyltransferase activity. It has been shown to be one of the four factors responsible for inducing reprogramming of somatic cells to a pluripotent stem-cell state [21]. It has been shown recently that pluripotent factor and polycomb group proteins repress AHR in murine embryonic stem cells, and that reversible Ahr repression holds the gene poised for expression and allows for a quick switch to activation [22]. In addition, it may indicate the presence of a regulatory feedback loop, in order to check excessive over-stimulation of AHR by c-Myc. Previous studies have implicated the AHR in hematopoietic cell lineage development [10,11,15]. These authors have shown that AHR has a role in stem cell cycling, regulation and quiescence [15], and loss of aryl hydrocarbon receptor activity is associated with premature hematopoietic stem cell exhaustion [11]. Under normal conditions, AHR is a negative regulator of c-Myc [17,18] and acts to inhibit cell proliferation. Since AHR is involved in detoxification and xenobiotic metabolism, TCDD-mediated stimulation of AHR results in mitochondrial oxidative stress [3], which may lead to downstream activation of c-Myc. Recently, the CYP1 family enzyme CYP1B1 was implicated in contributing to the cellular and mitochondrial oxidative stress produced as a result of TCDD-mediated AHR stimulation [3,23]. The authors showed that TCDD treatment up-regulated CYP1B1 levels and targeted it to mitochondria, where it caused oxidative stress by various mechanisms [23]. CYP1B1 has also been found to be overexpressed in several extra-hepatic tumor cell lines, and has been proposed to play a role in hormone-mediated tumor metabolism [24]. Sustained overexpression of CYP1B1 has been shown to cause DNA adduct formation upon exposure to benzo(a)pyrene (BaP) [25]. Since AHR stimulation by TCDD induces CYP1B1 and
may regulate c-Myc through CYP1B1-mediated oxidative stress, c-Myc may represent the underlying link between CYP1B1 overexpression and cancer. Moreover, TCDD is classified as a carcinogen, and addition of TCDD before BaP treatment in mice sensitized them to increased formation of DNA adducts and tumorigenesis [26,27]. TCDD itself has not been shown to cause DNA adduct formation leading to tumors, and is therefore considered as a tumor-promoting agent rather than a tumor-initiating agent. However, it is also possible that TCDD itself may possess tumor-initiating properties, either due to its ability to cause DNA adducts directly mediated by CYP1B1-generated oxidative stress, or as result of the downstream activation of c-Myc as a result of the oxidative stress-induced mitogenic signaling. All these data show a direct link between TCDD-mediated AHR expression and c-Myc activation, which formed the basis of our proposal to study skin stem cell cycling after TCDD exposure. We are interested in studying the molecular mechanism of skin stem cell activation and differentiation in response to TCDD treatment and AHR activation. c-Myc, an important epidermal stem cell activator and inhibitor of differentiation, has been shown to be down-regulated by TCDD-mediated AHR activation during infection, and its basal transcription was constitutively repressed by AHR binding to its promoter in mammary tumor cells. We intend to show that TCDD-mediated AHR activation in normal human skin cells up-regulated c-Myc through cross-talk via the EGFR-ERK pathway. This upregulation should correspond to an increase in human epidermal stem cell activation and differentiation of EPSCs into keratinocytes. Chronic treatment with TCDD should result in eventual depletion of the epidermal stem cell compartment of the skin. In conclusion, our studies will provide a mechanistic basis for the changes observed in epidermal stem cell characteristics underlying chloracne and the role of TCDD as a stimulator of keratinocyte differentiation. In addition, as c-Myc regulates a wide variety of genes and has been thought by some to regulate almost 15% of the genes in the human genome, we speculate that chromatin immunoprecipitation and sequencing (CHIP-seq) studies on c-Myc in skin samples treated with TCDD will serve as a useful diagnostic tool to identify the genes it regulates under these conditions, and will be essential for understanding the role of c-Myc in altering skin stem cell characteristics during TCDD-induced chloracne. Conflicts of interest statement The authors declare no conflicts of interest. Acknowledgements We thank Dr. Amy Abell, PhD, for her valuable advice, insight and helpful suggestions in the preparation of this manuscript. This work was supported, in part, by a research fellowship to Dr. Chirag Mandavia by the University of Memphis. References [1] Forrester AR, Elias MS, Woodward EL, Graham M, Williams FM, Reynolds NJ. Induction of a chloracne phenotype in an epidermal equivalent model by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on aryl hydrocarbon receptor activation and is not reproduced by aryl hydrocarbon receptor knock down. J Dermatol Sci 2014;73(1):10–22. [2] Panteleyev AA, Bickers DR. Dioxin-induced chloracne–reconstructing the cellular and molecular mechanisms of a classic environmental disease. Exp Dermatol 2006;15(9):705–30. Review. [3] Kennedy LH, Sutter CH, Leon Carrion S, Tran QT, Bodreddigari S, Kensicki E, et al. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated production of reactive oxygen species is an essential step in the mechanism of action to accelerate human keratinocyte differentiation. Toxicol Sci 2013;132(1):235–49.
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