To Control Site-Specific Skin Gene Expression, Autocrine Mimics Paracrine Canonical Wnt Signaling and Is Activated Ectopically in Skin Disease.

Despite similar components, the heterogeneity of skin characteristics across the human body is enormous. It is classically believed that site-specific fibroblasts in the dermis control postnatal skin identity by modulating the behavior of the surface-overlying keratinocytes in the epidermis. To begin testing this hypothesis, we characterized the gene expression differences between volar (ventral; palmoplantar) and nonvolar (dorsal) human skin. We show that KERATIN 9 (KRT9) is the most uniquely enriched transcript in volar skin, consistent with its etiology in genetic diseases of the palms and soles. In addition, ectopic KRT9 expression is selectively activated by volar fibroblasts. However, KRT9 expression occurs in the absence of all fibroblasts, although not to the maximal levels induced by fibroblasts. Through gain-of-function and loss-of-function experiments, we demonstrate that the mechanism is through overlapping paracrine or autocrine canonical WNT-ß-catenin signaling in each respective context. Finally, as an in vivo example of ectopic expression of KRT9 independent of volar fibroblasts, we demonstrate that in the human skin disease lichen simplex chronicus, WNT5a and KRT9 are robustly activated outside of volar sites. These results highlight the complexities of site-specific gene expression and its disruption in skin disease.

dsRNA Released by Tissue Damage Activates TLR3 to Drive Skin Regeneration.

Regeneration of skin and hair follicles after wounding--a process known as wound-induced hair neogenesis (WIHN)--is a rare example of adult organogenesis in mammals. As such, WIHN provides a unique model system for deciphering mechanisms underlying mammalian regeneration. Here, we show that dsRNA, which is released from damaged skin, activates Toll-Like Receptor 3 (TLR3) and its downstream effectors IL-6 and STAT3 to promote hair follicle regeneration. Conversely, TLR3-deficient animals fail to initiate WIHN. TLR3 activation promotes expression of hair follicle stem cell markers and induces elements of the core hair morphogenetic program, including ectodysplasin A receptor (EDAR) and the Wnt and Shh pathways. Our results therefore show that dsRNA and TLR3 link the earliest events of mammalian skin wounding to regeneration and suggest potential therapeutic approaches for promoting hair neogenesis.

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro.

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution.

Hypersensitivities for acetaldehyde and other agents among cancer cells null for clinically relevant Fanconi anemia genes.

Large-magnitude numerical distinctions (>10-fold) among drug responses of genetically contrasting cancers were crucial for guiding the development of some targeted therapies. Similar strategies brought epidemiological clues and prevention goals for genetic diseases. Such numerical guides, however, were incomplete or low magnitude for Fanconi anemia pathway (FANC) gene mutations relevant to cancer in FANC-mutation carriers (heterozygotes). We generated a four-gene FANC-null cancer panel, including the engineering of new PALB2/FANCN-null cancer cells by homologous recombination. A characteristic matching of FANCC-null, FANCG-null, BRCA2/FANCD1-null, and PALB2/FANCN-null phenotypes was confirmed by uniform tumor regression on single-dose cross-linker therapy in mice and by shared chemical hypersensitivities to various inter-strand cross-linking agents and γ-radiation in vitro. Some compounds, however, had contrasting magnitudes of sensitivity; a strikingly high (19- to 22-fold) hypersensitivity was seen among PALB2-null and BRCA2-null cells for the ethanol metabolite, acetaldehyde, associated with widespread chromosomal breakage at a concentration not producing breaks in parental cells. Because FANC-defective cancer cells can share or differ in their chemical sensitivities, patterns of selective hypersensitivity hold implications for the evolutionary understanding of this pathway. Clinical decisions for cancer-relevant prevention and management of FANC-mutation carriers could be modified by expanded studies of high-magnitude sensitivities.

DNA-intercalators causing rapid re-expression of methylated and silenced genes in cancer cells.

Epigenetic inactivation of tumor-suppressor and other regulatory genes plays a critical role in carcinogenesis. Transcriptional silencing is often maintained by DNA methyl transferase (DNMT)-mediated hypermethylation of CpG islands in promoter DNA. Nucleoside analogs including azacytidine and decitabine have been used to inhibit DNMT and re-activate genes, and are clinically used. Their shortcomings include a short half-life and a slow onset of action due to required nucleotide incorporation during DNA replication, which may limit clinical utility. It might be useful to begin to identify lead compounds having novel properties, specifically distinct and fast-acting gene desilencing. We previously identified chemicals augmenting gene expression in multiple reporter systems. We now report that a subset of these compounds that includes quinacrine re-expresses epigenetically silenced genes implicated in carcinogenesis. p16, TFPI2, the cadherins E-cadherin and CDH13, and the secreted frizzle-related proteins (SFRPs) SFRP1 and SFRP5 were desilenced in cancer cell lines. These lead compounds were fast-acting: re-expression occurred by 12-24 hours. Reactivation of silenced genes was accompanied by depletion of DNMT1 at the promoters of activated genes and demethylation of DNA. A model compound, 5175328, induced changes more rapidly than decitabine. These gene desilencing agents belonged to a class of acridine compounds, intercalated into DNA, and inhibited DNMT1 activity in vitro. Although to define the mechanism would be outside the scope of this initial report, this class may re-activate silenced genes in part by intercalating into DNA and subsequently inhibiting full DNMT1 activity. Rapid mechanisms for chemical desilencing of methylated genes therefore exist.

Biological clues to potent DNA-damaging activities in food and flavoring.

Population differences in age-related diseases and cancer could stem from differences in diet. To characterize DNA strand-breaking activities in selected foods/beverages, flavorings, and some of their constituent chemicals, we used p53R cells, a cellular assay sensitive to such breaks. Substances testing positive included reference chemicals: quinacrine (peak response, 51×) and etoposide (33×); flavonoids: EGCG (19×), curcumin (12×), apigenin (9×), and quercetin (7×); beverages: chamomile (11×), green (21×), and black tea (26×) and coffee (3-29×); and liquid smoke (4-28×). Damage occurred at dietary concentrations: etoposide near 5µg/ml produced responses similar to a 1:1000 dilution of liquid smoke, a 1:20 dilution of coffee, and a 1:5 dilution of tea. Pyrogallol-related chemicals and tannins are present in dietary sources and individually produced strong activity: pyrogallol (30×), 3-methoxycatechol (25×), gallic acid (21×), and 1,2,4-benzenetriol (21×). From structure-activity relationships, high activities depended on specific orientations of hydroxyls on the benzene ring. Responses accompanied cellular signals characteristic of DNA breaks such as H2AX phosphorylation. Breaks were also directly detected by comet assay. Cellular toxicological effects of foods and flavorings could guide epidemiologic and experimental studies of potential disease risks from DNA strand-breaking chemicals in diets.

Analysis of polymorphisms and haplotype structure of the human thymidylate synthase genetic region: a tool for pharmacogenetic studies.

5-Fluorouracil (5FU), a widely used chemotherapeutic drug, inhibits the DNA replicative enzyme, thymidylate synthase (Tyms). Prior studies implicated a VNTR (variable numbers of tandem repeats) polymorphism in the 5'-untranslated region (5'-UTR) of the TYMS gene as a determinant of Tyms expression in tumors and normal tissues and proposed that these VNTR genotypes could help decide fluoropyrimidine dosing. Clinical associations between 5FU-related toxicity and the TYMS VNTR were reported, however, results were inconsistent, suggesting that additional genetic variation in the TYMS gene might influence Tyms expression. We thus conducted a detailed genetic analysis of this region, defining new polymorphisms in this gene including mononucleotide (poly A:T) repeats and novel single nucleotide polymorphisms (SNPs) flanking the VNTR in the TYMS genetic region. Our haplotype analysis of this region used data from both established and novel genetic variants and found nine SNP haplotypes accounting for more than 90% of the studied population. We observed non-exclusive relationships between the VNTR and adjacent SNP haplotypes, such that each type of VNTR commonly occurred on several haplotype backgrounds. Our results confirmed the expectation that the VNTR alleles exhibit homoplasy and lack the common ancestry required for a reliable marker of a linked adjacent locus that might govern toxicity. We propose that it may be necessary in a clinical trial to assay multiple types of genetic polymorphisms in the TYMS region to meaningfully model linkage of genetic markers to 5FU-related toxicity. The presence of multiple long (up to 26 nt), polymorphic monothymidine repeats in the promoter region of the sole human thymidylate synthetic enzyme is intriguing.

T cell factor 1 initiates the T helper type 2 fate by inducing the transcription factor GATA-3 and repressing interferon-gamma.

The differentiation of activated CD4(+) T cells into the T helper type 1 (T(H)1) or T(H)2 fate is regulated by cytokines and the transcription factors T-bet and GATA-3. Whereas interleukin 12 (IL-12) produced by antigen-presenting cells initiates the T(H)1 fate, signals that initiate the T(H)2 fate are not completely characterized. Here we show that early GATA-3 expression, required for T(H)2 differentiation, was induced by T cell factor 1 (TCF-1) and its cofactor beta-catenin, mainly from the proximal Gata3 promoter upstream of exon 1b. This activity was induced after T cell antigen receptor (TCR) stimulation and was independent of IL-4 receptor signaling through the transcription factor STAT6. Furthermore, TCF-1 blocked T(H)1 fate by negatively regulating interferon-gamma (IFN-gamma) expression independently of beta-catenin. Thus, TCF-1 initiates T(H)2 differentiation of activated CD4(+) T cells by promoting GATA-3 expression and suppressing IFN-gamma expression.

Sustained expression of pre-TCR induced beta-catenin in post-beta-selection thymocytes blocks T cell development.

Pre-TCR and IL-7R signals regulate beta-selection of thymocytes and then must be down-regulated for further development. However, the molecular events that control down-regulation remain unknown. We and others have previously shown that beta-catenin in cooperation with TCF regulates beta-selection. In this paper, we demonstrate that beta-catenin expression is stringently regulated by intrathymic signals, it is expressed at the highest levels in the pre-TCR signaled thymocytes, and is down-regulated in post-beta-selection thymocytes. Pre-TCR-induced beta-catenin regulates initial stages of pre-TCR signaling including expression of early growth response (Egr) genes but must be down-regulated to express RORgammat, which is essential for maturation to the CD4+CD8+ double positive (DP) stage. Sustained expression of beta-catenin results in the generation of IL-7R-, Egr-, and TGFbeta-expressing pre-DP thymocytes that are blocked in development. These data are consistent with a model in which post-beta-selection, pre-TCR-induced beta-catenin expression must return to background levels for efficient transition to the DP stage.

ICAT expression disrupts beta-catenin-TCF interactions and impairs survival of thymocytes and activated mature T cells.

T cell factor (TCF) family of transcription factors and beta-catenin critically regulate T cell development as demonstrated by the deletion of the tcf gene, which results in a block early in development that becomes complete in mice bearing tcf/lef double deletion. However, the role of beta-catenin, a major TCF cofactor, remains controversial. To directly address this, we have generated transgenic mice expressing Inhibitor of beta-catenin and TCF (ICAT), a naturally occurring inhibitor that specifically disrupts TCF and beta-catenin interactions. In this report, we demonstrate that disrupting the interaction of beta-catenin with TCF renders adult thymocytes and activated T cells highly susceptible to apoptosis. In contrast to previously reported observations during fetal thymocyte development, these data show that in adult mice, survival and not differentiation of thymocytes, depends on transcription by TCF and beta-catenin. Indeed, we demonstrate that expression of ICAT impedes thymocyte survival by reducing the expression of Bcl(xL) in thymocytes below a critical threshold. Survival of activated mature T cells was also impaired due to diminished expression of activation-induced Bcl(xL). Accordingly, expression of transgenic Bcl-2 rescued activated ICAT-Tg CD4 T cells from apoptosis. Thus, disruption of TCF-beta-catenin interactions specifically impairs the survival of thymocytes and activated T cells.

Ancestral organization of the MHC revealed in the amphibian Xenopus.

With the advent of the Xenopus tropicalis genome project, we analyzed scaffolds containing MHC genes. On eight scaffolds encompassing 3.65 Mbp, 122 MHC genes were found of which 110 genes were annotated. Expressed sequence tag database screening showed that most of these genes are expressed. In the extended class II and class III regions the genomic organization, excluding several block inversions, is remarkably similar to that of the human MHC. Genes in the human extended class I region are also well conserved in Xenopus, excluding the class I genes themselves. As expected from previous work on the Xenopus MHC, the single classical class I gene is tightly linked to immunoproteasome and transporter genes, defining the true class I region, present in all nonmammalian jawed vertebrates studied to date. Surprisingly, the immunoproteasome gene PSMB10 is found in the class III region rather than in the class I region, likely reflecting the ancestral condition. Xenopus DMalpha, DMbeta, and C2 genes were identified, which are not present or not clearly identifiable in the genomes of any teleosts. Of great interest are novel V-type Ig superfamily (Igsf) genes in the class III region, some of which have inhibitory motifs (ITIM) in their cytoplasmic domains. Our analysis indicates that the vertebrate MHC experienced a vigorous rearrangement in the bony fish and bird lineages, and a translocation and expansion of the class I genes in the mammalian lineage. Thus, the amphibian MHC is the most evolutionary conserved MHC so far analyzed.