Carrie Hayes Sutter

EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes

Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell differentiation, as measured by the formation of cornified envelopes. We show that this acceleration is mediated by the AHR; also, that dioxin increases the expression of several genes known to be regulated by ARNT, which have critical roles in the cornification and epidermal barrier function of the skin. Importantly, we demonstrate that all of these responses are opposed by ligand-activation of the EGF receptor (R), an important regulator of keratinocyte cell fate. In the CYP1A1 enhancer, EGFR activation prevents recruitment of the p300 coactivator, although not affecting the binding of the AHR or ARNT. The total cellular level of p300 protein does not decrease, and overexpression of p300 relieves EGFR-mediated repression of transcription, indicating that p300 is a critical target for the repression of the AHR complex by EGFR signaling. These results provide a mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin is able to disrupt epidermal homeostasis and identify EGFR signaling as a regulator of the AHR. This signaling may modulate the incidence and severity of chloracne and be of therapeutic relevance to human poisonings by dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Increases the Expression of Genes in the Human Epidermal Differentiation Complex and Accelerates Epidermal Barrier Formation

Chloracne is commonly observed in people exposed to dioxins, yet the mechanism of toxicity is not well understood. The pathology of chloracne is characterized by hyperkeratinization of the interfollicular squamous epithelium, hyperproliferation and hyperkeratinization of hair follicle cells as well as a metaplastic response of the ductular sebum secreting sebaceous glands. In vitro studies using normal human epidermal keratinocytes to model interfollicular human epidermis demonstrate a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated acceleration of differentiation and increase in gene expression of several prodifferentiation genes, including filaggrin (FLG). Here, we demonstrated that the TCDD-activated aryl hydrocarbon receptor (AHR) bound a small fragment of DNA upstream of the transcriptional start sites of the FLG gene, containing one of two candidate xenobiotic response elements (XREs). Reporter assays using the promoter region of FLG containing the two putative XREs indicated that the increase in this messenger RNA (mRNA) was due to TCDD-mediated enhanced transcription, which was lost when both XREs were mutated. As FLG is part of the human epidermal differentiation complex (EDC) found on chromosome 1, we measured mRNAs from an additional 18 EDC genes for their regulation by TCDD. Of these genes, 14 were increased by TCDD. Immunoblot assays demonstrated that the proteins of FLG as well as that of another prodifferentiation gene, small proline rich protein 2, were increased by TCDD. In utero exposure to TCDD accelerated the formation of the epidermal barrier in the developing mouse fetus by approximately 1 day. These results indicate that the epidermal permeability barrier is a functional target of the TCDD-activated AHR.

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.

Chloracne is commonly observed in humans exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); yet, the mechanism of toxicity is not well understood. Using normal human epidermal keratinocytes, we investigated the mechanism of TCDD-mediated enhancement of epidermal differentiation by integrating functional genomic, metabolomic, and biochemical analyses. TCDD increased the expression of 40% of the genes of the epidermal differentiation complex found on chromosome 1q21 and 75% of the genes required for de novo ceramide biosynthesis. Lipid analysis demonstrated that eight of the nine classes of ceramides were increased by TCDD, altering the ratio of ceramides to free fatty acids. TCDD decreased the expression of the glucose transporter, SLC2A1, and most of the glycolytic transcripts, followed by decreases in glycolytic intermediates, including pyruvate. NADH and Krebs cycle intermediates were decreased, whereas NAD(+) was increased. Mitochondrial glutathione (GSH) reductase activity and the GSH/glutathione disulfide ratio were decreased by TCDD, ultimately leading to mitochondrial dysfunction, characterized by decreased inner mitochondrial membrane potential and ATP production, and increased production of the reactive oxygen species (ROS), hydrogen peroxide. Aryl hydrocarbon receptor (AHR) antagonists blocked the response of many transcripts to TCDD, and the endpoints of decreased ATP production and differentiation, suggesting regulation by the AHR. Cotreatment of cells with chemical antioxidants or the enzyme catalase blocked the TCDD-mediated acceleration of keratinocyte cornified envelope formation, an endpoint of terminal differentiation. Thus, TCDD-mediated ROS production is a critical step in the mechanism of this chemical to accelerate keratinocyte differentiation.

Protection Against Aflatoxin B1-Induced Cytotoxicity by Expression of the Cloned Aflatoxin B1-Aldehyde Reductases Rat AKR7A1 and Human AKR7A3

The reduction of the aflatoxin B 1 (AFB 1) dialdehyde metabolite to its corresponding mono and dialcohols, catalyzed by aflatoxin B 1-aldehyde reductase (AFAR, rat AKR7A1, and human AKR7A3), is greatly increased in livers of rats treated with numerous chemoprotective agents. Recombinant human AKR7A3 has been shown to reduce the AFB 1-dialdehyde at rates greater than those of the rat AKR7A1. The activity of AKR7A1 or AKR7A3 may detoxify the AFB 1-dialdehyde, which reacts with proteins, and thereby inhibits AFB 1-induced toxicity; however, direct experimental evidence of this hypothesis was lacking. Two human B lymphoblastoid cell lines, designated pMF6/1A2/AKR7A1 and pMF6/1A2, were genetically engineered to stably express AKR7A1 and/or cytochrome P4501A2 (1A2). The pMF6/1A2/AKR7A1 cells were refractory to the cytotoxic effects of 3 ng/mL AFB 1, in comparison to pM6/1A2 cells, which were more sensitive. Diminished protection occurred at higher concentrations of AFB 1 in pMF6/1A2/AKR7A1 cells, suggesting that additional factors were influencing cell survival. COS-7 cells were transfected with either vector control, rat AKR7A1, or human AKR7A3, and the cells were treated with AFB 1-dialdehyde. There was a 6-fold increase in the dialdehyde LC 50, from 66 microM in vector-transfected cells to 400 microM in AKR7A1-transfected cells, and an 8.5-fold increase from 35 microM in vector-transfected cells to 300 microM in AKR7A3-transfected cells. In both cases, this protective effect of the AFAR enzyme was accompanied by a marked decrease in protein adducts. Fractionation of the cellular protein showed that the mitochondria/nuclei and microsomal fractions contained the highest concentration of protein adducts. The levels of human AKR7A3 and AKR7A2 were measured in 12 human liver samples. The expression of AKR7A3 was detectable in all livers and lower than those of AKR7A2 in 11 of the 12 samples. Overall, these results provide the first direct evidence of a role for rat AKR7A1 and human AKR7A3 in protection against AFB 1-induced cytotoxicity and protein adduct formation.

Chemical genomics of cancer chemopreventive dithiolethiones

3H-1,2-dithiole-3-thione (D3T) and its analogues 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione (OLT) and 5-tert-butyl-3H-1,2-dithiole-3-thione (TBD) are chemopreventive agents that block or diminish early stages of carcinogenesis by inducing activities of detoxication enzymes. While OLT has been used in clinical trials, TBD has been shown to be more efficacious and possibly less toxic than OLT in animals. Here, we utilize a robust and high-resolution chemical genomics procedure to examine the pharmacological structure–activity relationships of these compounds in livers of male rats by microarray analyses. We identified 226 differentially expressed genes that were common to all treatments. Functional analysis identified the relation of these genes to glutathione metabolism and the nuclear factor, erythroid derived 2-related factor 2 pathway (Nrf2) that is known to regulate many of the protective actions of dithiolethiones. OLT and TBD were shown to have similar efficacies and both were weaker than D3T. In addition, we identified 40 genes whose responses were common to OLT and TBD, yet distinct from D3T. As inhibition of cytochrome P450 (CYP) has been associated with the effects of OLT on CYP expression, we determined the half maximal inhibitory concentration (IC50) values for inhibition of CYP1A2. The rank order of inhibitor potency was OLT ≫ TBD ≫ D3T, with IC50 values estimated as 0.2, 12.8 and >100 μM, respectively. Functional analysis revealed that OLT and TBD, in addition to their effects on CYP, modulate liver lipid metabolism, especially fatty acids. Together, these findings provide new insight into the actions of clinically relevant and lead dithiolethione analogues.

Yap1 activation by H2O2 or thiol-reactive chemicals elicits distinct adaptive gene responses

The yeast Saccharomyces cerevisiae transcription factor Yap1 mediates an adaptive response to oxidative stress by regulating protective genes. H(2)O(2) activates Yap1 through the Gpx3-mediated formation of a Yap1 Cys303-Cys598 intramolecular disulfide bond. Thiol-reactive electrophiles can activate Yap1 directly by adduction to cysteine residues in the C-terminal domain containing Cys598, Cys620, and Cys629. H(2)O(2) and N-ethylmaleimide (NEM) showed no cross-protection against each other, whereas another thiol-reactive chemical, acrolein, elicited Yap1-dependent cross-protection against NEM, but not H(2)O(2). Either Cys620 or Cys629 was sufficient for activation of Yap1 by NEM or acrolein; Cys598 was dispensable for this activation mechanism. To determine whether Yap1 activated by H(2)O(2) or thiol-reactive chemicals elicits distinct adaptive gene responses, microarray analysis was performed on the wild-type strain or its isogenic single-deletion strain Δyap1 treated with control buffer, H(2)O(2), NEM, or acrolein. Sixty-five unique H(2)O(2) and 327 NEM and acrolein Yap1-dependent responsive genes were identified. Functional analysis using single-gene-deletion yeast strains demonstrated that protection was conferred by CTA1 and CTT1 in the H(2)O(2)-responsive subset and YDR042C in the NEM- and acrolein-responsive subset. These findings demonstrate that the distinct mechanisms of Yap1 activation by H(2)O(2) or thiol-reactive chemicals result in selective expression of protective genes.

Transgenic Expression of Aflatoxin Aldehyde Reductase (AKR7A1) Modulates Aflatoxin B1 Metabolism but not Hepatic Carcinogenesis in the Rat

In both experimental animals and humans, aflatoxin B(1) (AFB(1)) is a potent hepatic toxin and carcinogen against which a variety of antioxidants and experimental or therapeutic drugs (e.g., oltipraz, related dithiolethiones, and various triterpenoids) protect from both acute toxicity and carcinogenesis. These agents induce several hepatic glutathione S-transferases (GST) as well as aldo-keto reductases (AKR) which are thought to contribute to protection. Studies were undertaken in transgenic rats to examine the role of one inducible enzyme, AKR7A1, for protection against acute and chronic actions of AFB(1) by enhancing detoxication of a reactive metabolite, AFB(1) dialdehyde, by reduction to alcohols. The AFB(1) dialdehyde forms adducts with protein amino groups by a Schiff base mechanism and these adducts have been theorized to be at least one cause of the acute toxicity of AFB(1) and to enhance carcinogenesis. A liver-specific AKR7A1 transgenic rat was constructed in the Sprague-Dawley strain and two lines, AKR7A1(Tg2) and AKR7A1(Tg5), were found to overexpress AKR7A1 by 18- and 8-fold, respectively. Rates of formation of AFB(1) alcohols, both in hepatic cytosols and as urinary excretion products, increased in the transgenic lines with AKR7A1(Tg2) being the highest. Neither line offered protection against acute AFB(1)-induced bile duct proliferation, a functional assessment of acute hepatotoxicity by AFB(1), nor did they protect against the formation of GST-P positive putative preneoplastic foci as a result of chronic exposure to AFB(1). These results imply that the prevention of protein adducts mediated by AKR are not critical to protection against AFB(1) tumorigenicity.

EGFR regulation of epidermal barrier function

Keratinocyte terminal differentiation is the process that ultimately forms the epidermal barrier that is essential for mammalian survival. This process is controlled, in part, by signal transduction and gene expression mechanisms, and the epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Using microarray analysis of a confluent cell density-induced model of keratinocyte differentiation, we identified 2,676 genes that are regulated by epidermal growth factor (EGF), a ligand of the EGFR. We further discovered, and separately confirmed by functional assays, that EGFR activation abrogates all of the known essential processes of keratinocyte differentiation by 1) decreasing the expression of lipid matrix biosynthetic enzymes, 2) regulating numerous genes forming the cornified envelope, and 3) suppressing the expression of tight junction proteins. In organotypic cultures of skin, EGF acted to impair epidermal barrier integrity, as shown by increased transepidermal water loss. As defective epidermal differentiation and disruption of barrier function are primary features of many human skin diseases, we used bioinformatic analyses to identify genes that are known to be associated with skin diseases. Compared with non-EGF-regulated genes, EGF-regulated genes were significantly enriched for skin disease genes. These results provide a systems-level understanding of the actions of EGFR signaling to inhibit keratinocyte differentiation, providing new insight into the role of EGFR imbalance in skin pathogenesis.

CYP1B1 Is Not a Major Determinant of the Disposition of Aromatase Inhibitors in Epithelial Cells of Invasive Ductal Carcinoma

CYP1B1 and CYP19 (aromatase) have been shown to be expressed in breast tumors. Both enzymes are efficient estrogen hydroxylases, indicating the potential for overlapping substrate and inhibitor specificity. We measured the inhibition properties of aromatase inhibitors (AIs) against CYP1B1-catalyzed hydroxylation of 17β-estradiol (E2) to determine whether CYP1B1 affects the disposition of AIs. In addition, we estimated the frequency of coexpression of these enzymes in breast tumor epithelium. Immunohistochemical analyses of CYP19 and CYP1B1 in a panel of 29 cases of invasive ductal carcinoma of the breast showed epithelial cell staining for CYP19 in 76% and for CYP1B1 in 97% of the samples. Statistical analysis showed no significant correlation (0.33) for positive expression of CYP19 and CYP1B1 (p > 0.07). CYP1B1 inhibition was determined for two steroidal inhibitors: formestane and exemestane and five nonsteroidal inhibitors: aminoglutethimide, fadrozole, anastrozole, letrozole, and vorozole. Of the seven compounds tested, only vorozole exhibited inhibition of CYP1B1 activity with IC50 values of 17 and 21 μM for 4-hydroxy estradiol and 2-hydroxy estradiol, respectively. The estimated Ki values of vorozole for E2 4- and 2-hydroxylation were 7.26 and 6.84 μM, respectively. Spectrophotometric studies showed that vorozole was a type II inhibitor of CYP1B1. This study shows that with the exception of vorozole, the aromatase inhibitors are selective for CYP19 relative to CYP1B1. Thus, although both CYP19 and CYP1B1 are expressed in a high percentage of breast cancers, CYP1B1 is not a major determinant of the disposition of AIs.

Genotyping human cytochrome: P450 1B1 variants

Publisher Summary This chapter describes the methods used in laboratory to genotype the four common single nucleotide polymorphisms found in the coding region of human cytochrome P450 1B1 (CYP1B1). Human cytochrome P450 1B1 (CYP1B1) was first isolated by differential hybridization as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-responsive cDNA clone from a human keratinocyte cell line treated with TCDD. Initial characterization of the human CYP1B1 gene described the DNA sequence of a 12-kb genomic clone corresponding to the entire 5.1-kb CYP1B1 cDNA and containing 3.0 kb of upstream DNA. Comparison of these sequences revealed the location of three exons (371, 1044, and 3707 bp) and two introns (390 and 3032 bp), with the CYP1B1 open reading frame spanning exons 2 and 3. Genotyping by restriction fragment length polymorphism analysis will soon be replaced by more rapid, high-throughput assays based on electrochemical or fluorescent detection methods, including oligonucleotide microarrays. In the interim, the procedures described in the chapter should facilitate CYP1B1 genotyping of banked DNA samples.