Ligand Activation of the Aryl Hydrocarbon Receptor Upregulates Epidermal Uridine Diphosphate Glucose Ceramide Glucosyltransferase and Glucosylceramides.

Ligand activation of the aryl hydrocarbon receptor (AHR) accelerates keratinocyte differentiation and the formation of the epidermal permeability barrier. Several classes of lipids, including ceramides, are critical to the epidermal permeability barrier. In normal human epidermal keratinocytes, the AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, increased RNA levels of ceramide metabolism and transport genes: uridine diphosphate glucose ceramide glucosyltransferase (UGCG), ABCA12, GBA1, and SMPD1. Levels of abundant skin ceramides were also increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These included the metabolites synthesized by UGCG, glucosylceramides, and acyl glucosylceramides. Chromatin immunoprecipitation-sequence analysis and luciferase reporter assays identified UGCG as a direct AHR target. The AHR antagonist, GNF351, inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated RNA and transcriptional increases. Tapinarof, an AHR ligand approved for the treatment of psoriasis, increased UGCG RNA, protein, and its lipid metabolites hexosylceramides as well as increased the RNA expression of ABCA12, GBA1, and SMPD1. In Ahr-null mice, Ugcg RNA and hexosylceramides were lower than those in the wild type. These results indicate that the AHR regulates the expression of UGCG, a ceramide-metabolizing enzyme required for ceramide trafficking, keratinocyte differentiation, and epidermal permeability barrier formation.

CYP1B1 Augments the Mesenchymal, Claudin-Low, and Chemoresistant Phenotypes of Triple-Negative Breast Cancer Cells.

Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial-mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

Cutaneous Effects of In Utero and Lactational Exposure of C57BL/6J Mice to 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

To determine the cutaneous effects of in utero and lactational exposure to the AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), pregnant C57BL/6J mice were exposed by gavage to a vehicle or 5 µg TCDD/kg body weight at embryonic day 12 and epidermal barrier formation and function were studied in their offspring from postnatal day 1 (P1) through adulthood. TCDD-exposed pups were born with acanthosis. This effect was AHR-dependent and subsided by P6 with no evidence of subsequent inflammatory dermatitis. The challenge of adult mice with MC903 showed similar inflammatory responses in control and treated animals, indicating no long-term immunosuppression to this chemical. Chloracne-like sebaceous gland hypoplasia and cyst formation were observed in TCDD-exposed P21 mice, with concomitant microbiome dysbiosis. These effects were reversed by P35. CYP1A1 and CYP1B1 expression in the skin was increased in the exposed mice until P21, then declined. Both CYP proteins co-localized with LRIG1-expressing progenitor cells at the infundibulum. CYP1B1 protein also co-localized with a second stem cell niche in the isthmus. These results indicate that this exposure to TCDD causes a chloracne-like effect without inflammation. Transient activation of the AhR, due to the shorter half-life of TCDD in mice, likely contributes to the reversibility of these effects.

Commensal microbiota regulates skin barrier function and repair via signaling through the aryl hydrocarbon receptor.

The epidermis forms a barrier that defends the body from desiccation and entry of harmful substances, while also sensing and integrating environmental signals. The tightly orchestrated cellular changes needed for the formation and maintenance of this epidermal barrier occur in the context of the skin microbiome. Using germ-free mice, we demonstrate the microbiota is necessary for proper differentiation and repair of the epidermal barrier. These effects are mediated by microbiota signaling through the aryl hydrocarbon receptor (AHR) in keratinocytes, a xenobiotic receptor also implicated in epidermal differentiation. Mice lacking keratinocyte AHR are more susceptible to barrier damage and infection, during steady-state and epicutaneous sensitization. Colonization with a defined consortium of human skin isolates restored barrier competence in an AHR-dependent manner. We reveal a fundamental mechanism whereby the microbiota regulates skin barrier formation and repair, which has far-reaching implications for the numerous skin disorders characterized by epidermal barrier dysfunction.

Contributions of Nitric Oxide to AHR-Ligand-Mediated Keratinocyte Differentiation.

Activation of the aryl hydrocarbon receptor (AHR) in normal human epidermal keratinocytes (NHEKs) accelerates keratinocyte terminal differentiation through metabolic reprogramming and reactive oxygen species (ROS) production. Of the three NOS isoforms, NOS3 is significantly increased at both the RNA and protein levels by exposure to the very potent and selective ligand of the AHR, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Inhibition of NOS with the chemical N-nitro-l-arginine methyl ester (l-NAME) reversed TCDD-induced cornified envelope formation, an endpoint of terminal differentiation, as well as the expression of filaggrin (FLG), a marker of differentiation. Conversely, exposure to the NO-donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), increased the number of cornified envelopes above control levels and augmented the levels of cornified envelopes formed in response to TCDD treatment and increased the expression of FLG. This indicates that nitric oxide signaling can increase keratinocyte differentiation and that it is involved in the AHR-mediated acceleration of differentiation. As the nitrosylation of cysteines is a mechanism by which NO affects the structure and functions of proteins, the S-nitrosylation biotin switch technique was used to measure protein S-nitrosylation. Activation of the AHR increased the S-nitrosylation of two detected proteins of about 72 and 20 kD in size. These results provide new insights into the role of NO and protein nitrosylation in the process of epithelial cell differentiation, suggesting a role of NOS in metabolic reprogramming and the regulation of epithelial cell fate.

AHR Regulates Metabolic Reprogramming to Promote SIRT1-Dependent Keratinocyte Differentiation.

Activation of the transcription factor, AHR, in normal human epidermal keratinocytes increased AHR binding in the gene regions of the glucose transporter, SLC2A1, and the glycolytic enzyme, ENO1. This increased chromatin binding corresponded with AHR-dependent decreases in levels of SLC2A1 and ENO1 mRNA, protein, and activities. Studies of the ENO1 promoter showed activation of the AHR decreases the transcription of ENO1. Glycolysis was lowered by activation of the AHR as measured by decreases in glucose uptake and the production of pyruvate and lactate. Levels of ATP were also decreased. Downregulation of glucose metabolism, either by activation of the AHR, inhibition of glycolysis, inhibition of glucose transport, or inhibition of enolase, increased SIRT1 protein levels in normal human epidermal keratinocytes and the immortalized keratinocyte cell line, N/TERT-1. This increase in SIRT1 was abrogated by the addition of exogenous pyruvate. Moreover, keratinocyte differentiation in response to downregulation of glycolysis, either by activation of the AHR, inhibition of glucose transport, or inhibition of enolase, was dependent on SIRT1. These results indicate that regulation of glycolysis controls keratinocyte differentiation, and that activation of the AHR, by lowering the expression of SLC2A1 and ENO1, can determine this fate.

Breastfeeding duration modifies the effect of smoking during pregnancy on eczema from early childhood to adolescence.

BACKGROUND: Cigarette smoke contains compounds similar to coal tar, an ancient remedy of eczema. Some studies have reported protective effects of maternal gestational smoking on offspring eczema; however, others have shown no or increased risks. Similarly, studies linking breastfeeding duration and eczema have demonstrated contradictory findings. No study has yet investigated combined effects of these two factors on eczema. OBJECTIVE: Since tobacco compounds can pass to offspring via breast milk, we investigated their combined effects on eczema development from childhood to adolescence. METHODS: We obtained information regarding gestational smoking, exclusive breastfeeding duration, and eczema at ages 1-or-2, 4, 10, and 18 years from the Isle of Wight (IOW) birth cohort, UK. Using generalized estimating equations, we assessed the interaction of gestational smoking and residual exclusive breastfeeding duration (Resid-BF-duration, obtained by regressing the latter on maternal smoking) on eczema over time adjusting for confounders. For the three transition periods of 1-or-2 to 4 years, 4-10, and 10-18 years, we estimated risks of persistent, incident, and remitting eczema associated with the interaction using repeated measurements. RESULTS: If the mother smoked during gestation, longer Resid-BF-duration was associated with a lower risk of eczema, compared to if she did not smoke. The risk ratios (95% CI) if the mother smoked during gestation and exclusively breastfed for at least 3, 9, 15, 21 weeks are 0.7 (0.6, 1.7), 0.6 (0. 4, 0.9), 0.5 (0.3, 0.8), and 0.4 (0.2, 0. 8), respectively. Additionally, in all three transition periods, the risk of persistent eczema was lower with longer Resid-BF-duration if the mother smoked during gestation. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest a protective effect of gestational smoking combined with longer duration of exclusive breastfeeding on early-onset persistent eczema. Future studies should examine underlying biological mechanisms. Prolonged breastfeeding should be encouraged even if the mother smoked during gestation.

Differential and Overlapping Effects of 20,23(OH)2D3 and 1,25(OH)2D3 on Gene Expression in Human Epidermal Keratinocytes: Identification of AhR as an Alternative Receptor for 20,23(OH)2D3.

A novel pathway of vitamin D activation by CYP11A has previously been elucidated. To define the mechanism of action of its major dihydroxy-products, we tested the divergence and overlap between the gene expression profiles of human epidermal keratinocytes treated with either CYP11A1-derived 20,23(OH)2D3 or classical 1,25(OH)2D3. Both secosteroids have significant chemical similarity with the only differences being the positions of the hydroxyl groups. mRNA was isolated and examined by microarray analysis using Illumina's HumanWG-6 chip/arrays and subsequent bioinformatics analyses. Marked differences in the up- and downregulated genes were observed between 1,25(OH)2D3- and 20,23(OH)2D3-treated cells. Hierarchical clustering identified both distinct, opposite and common (overlapping) gene expression patterns. CYP24A1 was a common gene strongly activated by both compounds, a finding confirmed by qPCR. Ingenuity pathway analysis identified VDR/RXR signaling as the top canonical pathway induced by 1,25(OH)2D3. In contrast, the top canonical pathway induced by 20,23(OH)2D3 was AhR, with VDR/RXR being the second nuclear receptor signaling pathway identified. QPCR analyses validated the former finding by revealing that 20,23(OH)2D3 stimulated CYP1A1 and CYP1B1 gene expression, effects located downstream of AhR. Similar stimulation was observed with 20(OH)D3, the precursor to 20,23(OH)2D3, as well as with its downstream metabolite, 17,20,23(OH)3D3. Using a Human AhR Reporter Assay System we showed marked activation of AhR activity by 20,23(OH)2D3, with weaker stimulation by 20(OH)D3. Finally, molecular modeling using an AhR LBD model predicted vitamin D3 hydroxyderivatives to be good ligands for this receptor. Thus, our microarray, qPCR, functional studies and molecular modeling indicate that AhR is the major receptor target for 20,23(OH)2D3, opening an exciting area of investigation on the interaction of different vitamin D3-hydroxyderivatives with AhR and the subsequent downstream activation of signal transduction pathways in a cell-type-dependent manner.

NRF2 regulates core and stabilizing circadian clock loops, coupling redox and timekeeping in Mus musculus.

Diurnal oscillation of intracellular redox potential is known to couple metabolism with the circadian clock, yet the responsible mechanisms are not well understood. We show here that chemical activation of NRF2 modifies circadian gene expression and rhythmicity, with phenotypes similar to genetic NRF2 activation. Loss of Nrf2 function in mouse fibroblasts, hepatocytes and liver also altered circadian rhythms, suggesting that NRF2 stoichiometry and/or timing of expression are important to timekeeping in some cells. Consistent with this concept, activation of NRF2 at a circadian time corresponding to the peak generation of endogenous oxidative signals resulted in NRF2-dependent reinforcement of circadian amplitude. In hepatocytes, activated NRF2 bound specific enhancer regions of the core clock repressor gene Cry2, increased Cry2 expression and repressed CLOCK/BMAL1-regulated E-box transcription. Together these data indicate that NRF2 and clock comprise an interlocking loop that integrates cellular redox signals into tissue-specific circadian timekeeping.

Pharmacogenomics of Chemically Distinct Classes of Keap1-Nrf2 Activators Identify Common and Unique Gene, Protein, and Pathway Responses In Vivo.

The Kelch-like erythroid-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) signaling pathway is the subject of several clinical trials evaluating the effects of Nrf2 activation on the prevention of cancer and diabetes and the treatment of chronic kidney disease and multiple sclerosis. 3H-1,2-dithiole-3-thione (D3T) and 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) are representative members of two distinct series of Nrf2 chemical activators. Previous reports have described activator-specific effects on Nrf2-dependent gene regulation and physiologic outcomes. Here we used a robust chemical genomics approach to characterize expression profiles between D3T and CDDO-Im in livers from wild-type and Nrf2-null mice. At equally efficacious doses in wild-type mice, 406 genes show common RNA responses to both treatments. These genes enriched the Nrf2-regulated pathways of antioxidant defense and xenobiotic metabolism. In addition, 197 and 745 genes were regulated uniquely in response to either D3T or CDDO-Im, respectively. Functional analysis of the D3T-regulated set showed a significant enrichment of Nrf2-regulated enzymes involved in cholesterol biosynthesis. This result was supported by Nrf2-dependent increases in lanosterol synthase and CYP51 protein expression. CDDO-Im had no effect on cholesterol biosynthesis regardless of the dose tested. However, unlike D3T, CDDO-Im resulted in Nrf2-dependent elevation of peroxisome proliferator α and Kruppel-like factor 13, as well as the coactivator peroxisome proliferator γ coactivator 1ß, together indicating regulation of ß-oxidation and lipid metabolic pathways. These findings provide novel insights into the pharmacodynamic action of these two activators of Keap1-Nrf2 signaling. Although both compounds modify Keap1 to affect canonical cytoprotective gene expression, additional unique sets of Nrf2-dependent genes were regulated by each agent with enrichment of selective metabolic pathways.

Loss of C-5 Sterol Desaturase Activity Results in Increased Resistance to Azole and Echinocandin Antifungals in a Clinical Isolate of Candida parapsilosis.

Among emerging non-albicans Candida species, Candida parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatments for such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting susceptibility to azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate, including upregulation of ergosterol biosynthesis pathway genes ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, and UPC2 Whole-genome sequencing revealed that the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate's allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins.

Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification.

The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

Role of EGF receptor ligands in TCDD-induced EGFR down-regulation and cellular proliferation.

In cultures of normal human epidermal keratinocytes (NHEKs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces the expression of the epidermal growth factor receptor ligands transforming growth factor-α (TGF-α) and epiregulin (EREG). TCDD also down-regulates EGF receptors (EGFR), suggesting that decreases in signaling contribute to the effects of TCDD. In this study, we treated post-confluent NHEKs with 10 nM TCDD and assessed its effects on EGFR binding, EGFR ligand secretion, basal ERK activity, and proliferation. TCDD caused time-dependent deceases in [(125)I]-EGF binding to levels 78% of basal cell values at 72 h. Amphiregulin (AREG) levels increased with time in culture in basal and TCDD-treated cells, while TGF-α and epiregulin (EREG) secretion were stimulated by TCDD. Inhibiting EGFR ligand release with the metalloproteinase inhibitor batimastat prevented EGFR down-regulation and neutralizing antibodies for AREG and EREG relieved receptor down-regulation. In contrast, neutralizing TGF-α intensified EGFR down-regulation. Treating NHEKs with AREG or TGF-α caused rapid internalization of receptors with TGF-α promoting recycling within 90 min. EREG had limited effects on rapid internalization or recycling. TCDD treatment increased ERK activity, a response reduced by batimastat and the neutralization of all three ligands indicating that the EGFR and its ligands maintain ERK activity. All three EGFR ligands were required for the maintenance of total cell number in basal and TCDD-treated cultures. The EGFR inhibitor PD1530305 blocked basal and TCDD-induced increases in the number of cells labeled by 5-ethynyl-2'-deoxyuridine, identifying an EGFR-dependent pool of proliferating cells that is larger in TCDD-treated cultures. Overall, these data indicate that TCDD-induced EGFR down-regulation in NHEKs is caused by AREG, TGF-α, and EREG, while TGF-α enhances receptor recycling to maintain a pool of EGFR at the cell surface. These receptors are required for ERK activity, maintenance of total cell number, and stimulating the proliferation of a small subset cells.

Effects of in utero exposure of C57BL/6J mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin on epidermal permeability barrier development and function.

BACKGROUND: Development of the epidermal permeability barrier (EPB) is essential for neonatal life. Defects in this barrier are found in many skin diseases such as atopic dermatitis. OBJECTIVE: We investigated the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development and function of the EPB. METHODS: Timed-pregnant C57BL/6J mice were gavaged with corn oil or TCDD (10 µg/kg body weight) on gestation day 12. Embryos were harvested on embryonic day (E) 15, E16, E17, and postnatal day (PND) 1. RESULTS: A skin permeability assay showed that TCDD accelerated the development of the EPB, beginning at E15. This was accompanied by a significant decrease in transepidermal water loss (TEWL), enhanced stratification, and formation of the stratum corneum (SC). The levels of several ceramides were significantly increased at E15 and E16. PND1 histology revealed TCDD-induced acanthosis and epidermal hyperkeratosis. This was accompanied by disrupted epidermal tight junction (TJ) function, with increased dye leakage at the terminal claudin-1-staining TJs of the stratum granulosum. Because the animals did not have enhanced rates of TEWL, a commonly observed phenotype in animals with TJ defects, we performed tape-stripping. Removal of most of the SC resulted in a significant increase in TEWL in TCDD-exposed PND1 pups compared with their control group. CONCLUSIONS: These findings demonstrate that in utero exposure to TCDD accelerates the formation of an abnormal EPB with leaky TJs, warranting further study of environmental exposures, epithelial TJ integrity, and atopic disease.

Complete protection against aflatoxin B(1)-induced liver cancer with a triterpenoid: DNA adduct dosimetry, molecular signature, and genotoxicity threshold.

In experimental animals and humans, aflatoxin B1 (AFB1) is a potent hepatic toxin and carcinogen. The synthetic oleanane triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a powerful activator of Keap1-Nrf2 signaling, protects against AFB1-induced toxicity and preneoplastic lesion formation (GST-P-positive foci). This study assessed and mechanistically characterized the chemoprotective efficacy of CDDO-Im against AFB1-induced hepatocellular carcinoma (HCC). A lifetime cancer bioassay was undertaken in F344 rats dosed with AFB1 (200 µg/kg rat/day) for four weeks and receiving either vehicle or CDDO-Im (three times weekly), one week before and throughout the exposure period. Weekly, 24-hour urine samples were collected for analysis of AFB1 metabolites. In a subset of rats, livers were analyzed for GST-P foci. The comparative response of a toxicogenomic RNA expression signature for AFB1 was examined. CDDO-Im completely protected (0/20) against AFB1-induced liver cancer compared with a 96% incidence (22/23) observed in the AFB1 group. With CDDO-Im treatment, integrated level of urinary AFB1-N(7)-guanine was significantly reduced (66%) and aflatoxin-N-acetylcysteine, a detoxication product, was consistently elevated (300%) after the first AFB1 dose. In AFB1-treated rats, the hepatic burden of GST-P-positive foci increased substantially (0%-13.8%) over the four weeks, but was largely absent with CDDO-Im intervention. The toxicogenomic RNA expression signature characteristic of AFB1 was absent in the AFB1 + CDDO-Im-treated rats. The remarkable efficacy of CDDO-Im as an anticarcinogen is established even in the face of a significant aflatoxin adduct burden. Consequently, the absence of cancer requires a concept of a threshold for DNA damage for cancer development.

Epigenetic analysis of neurocognitive development at 1 year of age in a community-based pregnancy cohort.

Multiple studies show that molecular genetic changes and epigenetic modifications affect the risk of cognitive disability or impairment. However, the role of epigenetic variation in cognitive development of neurotypical young children remains largely unknown. Using data from a prospective, community-based study of mother-infant pairs, we investigated the association of DNA methylation patterns in neonatal umbilical cord blood with cognitive and language development at 1 year of age. No CpG loci achieved genome-wide significance, although a small number of weakly suggestive associations with Bayley-III Receptive Communication scales were noted. While umbilical cord blood is a convenient resource for genetic analyses of birth outcomes, our results do not provide conclusive evidence that its use for DNA methylation profiling yields epigenetic markers that are directly related to postnatal neurocognitive outcomes at 1 year of age.

Combined treatment with sodium butyrate and PD153035 enhances keratinocyte differentiation.

Epidermal growth factor (EGF) receptor (EGFR) signalling is a critical determinant of keratinocyte proliferation and differentiation in both normal and diseased skin. Here we explore the effects of combined treatment with the differentiation-promoting agent sodium butyrate (SB) and the EGFR inhibitor (EGFRI) PD153035 on terminal differentiation of normal human epidermal keratinocytes (NHEKs). Cells treated with SB showed increased expression of the levels of mRNA and protein of the differentiation markers filaggrin and transglutaminase 1. Cotreatment with EGF significantly blunted these effects of SB. Combined treatment with SB and PD153035 alleviated these inhibitory actions of EGF, resulting in improved effects of decreased cell growth and increased terminal differentiation, relative to the individual treatments. These results indicate that the combined use of a differentiation-promoting agent and an EGFR inhibitor may offer an additional approach to the management of hyperproliferative skin diseases.

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.

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.