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.

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.

Analyzing microarray data with transitive directed acyclic graphs.

Post hoc assignment of patterns determined by all pairwise comparisons in microarray experiments with multiple treatments has been proven to be useful in assessing treatment effects. We propose the usage of transitive directed acyclic graphs (tDAG) as the representation of these patterns and show that such representation can be useful in clustering treatment effects, annotating existing clustering methods, and analyzing sample sizes. Advantages of this approach include: (1) unique and descriptive meaning of each cluster in terms of how genes respond to all pairs of treatments; (2) insensitivity of the observed patterns to the number of genes analyzed; and (3) a combinatorial perspective to address the sample size problem by observing the rate of contractible tDAG as the number of replicates increases. The advantages and overall utility of the method in elaborating drug structure activity relationships are exemplified in a controlled study with real and simulated data.

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.

Potent protection against aflatoxin-induced tumorigenesis through induction of Nrf2-regulated pathways by the triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole.

Synthetic triterpenoid analogues of oleanolic acid are potent inducers of the phase 2 response as well as inhibitors of inflammation. We show that the triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), is a highly potent chemopreventive agent that inhibits aflatoxin-induced tumorigenesis in rat liver. The chemopreventive potency of CDDO-Im was evaluated by measuring inhibition of formation of putative preneoplastic lesions (glutathione S-transferase P positive foci) in the liver of rats exposed to aflatoxin B1. CDDO-Im produces an 85% reduction in the hepatic focal burden of preneoplastic lesions at 1 micromol/kg body weight and a >99% reduction at 100 micromol/kg body weight. CDDO-Im treatment reduces levels of aflatoxin-DNA adducts by approximately 40% to 90% over the range of 1 to 100 micromol/kg body weight. Additionally, changes in mRNA levels of genes involved in aflatoxin metabolism were measured in rat liver following a single dose of CDDO-Im. GSTA2, GSTA5, AFAR, and EPHX1 transcripts are elevated 6 hours following a 1 micromol/kg body weight dose of CDDO-Im. Microarray analysis using wild-type and Nrf2 knockout mice confirms that many phase 2 and antioxidant genes are induced in an Nrf2-dependent manner in mouse liver following treatment with CDDO-Im. Thus, low-micromole doses of CDDO-Im induce cytoprotective genes, inhibit DNA adduct formation, and dramatically block hepatic tumorigenesis. As a point of reference, oltipraz, an established modulator of aflatoxin metabolism in humans, is 100-fold weaker than CDDO-Im in this rat antitumorigenesis model. The unparalleled potency of CDDO-Im in vivo highlights the chemopreventive promise of targeting Nrf2 pathways with triterpenoids.

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.

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.

Analysis of the CYP1A1 mRNA dose-response in human keratinocytes indicates that relative potencies of dioxins, furans, and PCBs are species and congener specific.

Reports indicate that toxic equivalency factors (TEFs) based primarily on rodent data do not accurately predict in vitro human responsiveness to certain dioxin-like chemicals (DLCs). To investigate this in cells responsive to dioxins and relevant to chloracne, normal human epidermal keratinocytes were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and several DLCs, each with a TEF value of 0.1, representing three classes of congeners. We estimated half maximal effective concentration (EC50)-based donor-specific relative potency (REP) values for cytochrome P450 1A1 (CYP1A1) messenger RNA (mRNA) induction for TCDD, 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HxCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,6,7,8-hexachlorodibenzofuran (HxCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB 126). We also determined EC50-based population-level REP values (n = 4) for CYP1A1 mRNA induction for TCDD, HxCDF, and PCB 126. Furthermore, an alternative factor, the relative threshold factor (RTF) based on the low end (threshold) of the dose-response curve, was calculated. Our results demonstrated that HxCDF had a population-based REP value of 0.98, 9.8-fold higher than its assigned TEF value of 0.1. Conversely, PCB 126 had an REP value of 0.0027 and an RTF of 0.0022, 37-fold and 45-fold less than its assigned TEF of 0.1, respectively. The REP values for HxCDD and TCDF were 0.24 and 0.10, respectively, similar to their assigned value of 0.1. Therefore, although the DLCs tested in the current study all possessed the same assigned TEF value of 0.1, congener-specific differences in REPs and RTFs were observed for human keratinocytes. These congener-specific discrepancies are likely because of differences in interspecies factors that have yet to be defined.

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.

3H-1,2-dithiole-3-thione targets nuclear factor kappaB to block expression of inducible nitric-oxide synthase, prevents hypotension, and improves survival in endotoxemic rats.

Septicemia is a major cause of death associated with noncoronary intensive care. Systemic production of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) is a major cause of hypotension and poor organ perfusion seen in septic shock. Here, we show that pretreatment of F344 rats with the cancer chemoprotective agent 3H-1,2-dithiole-3-thione (D3T) blocks lipopolysaccharide (LPS)-mediated induction of hepatic iNOS and significantly reduces the associated serum levels of NO metabolites and enzyme markers of toxicity provoked by treatment with LPS. Immunohistochemical analysis shows that this protective effect is largely due to suppression of iNOS expression in hepatocytes. Importantly, pretreatment of animals with D3T blunts LPS-mediated hypotension and dramatically increases their survival. Inasmuch as iNOS expression can be regulated by nuclear factor (NF) kappaB, mechanistic studies show that D3T blocks NFkappaB nuclear translocation and DNA binding and that these effects are accompanied by changes in the levels of phospho-inhibitor of NFkappaB. In conclusion, this study identifies new drug classes and targets that may improve the prevention and treatment of septic shock, as well as chronic diseases associated with the NFkappaB and iNOS pathways.

Evaluation of the cancer chemopreventive potency of dithiolethione analogs of oltipraz.

Oltipraz and related dithiolethiones constitute an important class of chemopreventive agents that enhance the expression of carcinogen detoxication and antioxidant genes. Dose-response studies were undertaken to characterize the cancer chemopreventive activities of several dithiolethiones that are at least as active as oltipraz as inducers. Inhibition of formation of pre-neoplastic lesions and formation of DNA adducts in livers of rats exposed to aflatoxin B1 (AFB1) was monitored. In the tumorigenesis experiment, the dithiolethiones were orally gavaged 3 days/week for 3 successive weeks and at four doses ranging from 0.03 to 0.3 mmol/kg body wt. AFB1 was gavaged beginning 1 week after the start of the dithiolethiones and for two successive weeks. The burden of AFB1-induced putative pre-neoplastic lesions (glutathione S-transferase-placental isoform positive foci) was quantified by light microscopy. Reduction in AFB-DNA adduct burden was assessed 24 h following the first dose of AFB1. Both the parent 1,2-dithiole-3-thione (D3T) and its 5-tert-butyl derivative were more potent inhibitors than oltipraz against these endpoints, while two of the seven tested analogs were slightly less inhibitory. D3T, the most potent dithiolethione of this series, was examined by microarray analysis for induction of hepatic genes at an intermediate chemopreventive dose (0.1 mmol/kg). Transcript levels of eight genes, including two known to detoxify aflatoxin, namely, glutathione S-transferase A5 (GSTA5) and AFB1 aldehyde reductase (AFAR) were elevated. Western analysis indicated that induction of hepatic GSTA5 and AFAR were directly related to the dose of D3T. At the highest dose of D3T (0.3 mmol/kg), protein levels of GSTA5 and AFAR were induced by 7- and 27-fold, respectively. While efficacy in humans has yet to be tested, D3T is clearly more potent than oltipraz and serves as a useful molecular probe for determining the key events associated with protection by this class of agents.