Marjorie A. Phillips

Arsenite Suppresses Notch1 Signaling in Human Keratinocytes

Arsenic is a well-known human skin carcinogen whose mechanism of action remains to be elucidated. In this work using cultured human epidermal cells, arsenite suppressed accumulation of the transcriptionally active intracellular domain of Notch1. The cells responded to an active peptide from the Notch1 ligand, Jagged1, with increased levels of differentiation marker mRNAs and decreased colony-forming ability. Arsenite suppressed Jagged1 effects and expression of Jagged1 mRNA as well. Moreover, exposure of the cells to a gamma-secretase inhibitor prevented Notch1 processing, decreased cell size and differentiation marker expression, and increased proliferative potential, all effects that occur with arsenite treatment. Thus, arsenite action in suppressing keratinocyte differentiation while maintaining germinative capability could be due to inhibition of Notch1 signaling subsequent to ligand binding. This work also revealed that such arsenite action depends upon epidermal growth factor receptor kinase activity. These findings may help to explain how arsenite, by decreasing generation of the tumor suppressor Notch1, contributes to skin carcinogenesis.

Arsenite suppression of BMP signaling in human keratinocytes

Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression.

Identification of an involucrin promoter transcriptional response element with activity restricted to keratinocytes.

The involucrin proximal promoter was examined for response elements that confer cell-type specificity. Using a segment spanning positions -157 to +41, three possible response elements were identified by their protein-binding activity using DNase I footprinting. From distal to proximal, they were: an activator protein-1 (AP-1) site (previously identified) overlapping an Ets-like site; a second Ets-like site located 13 bp more proximally; and an extended region designated footprinted site A (FPA). Mutation of the distal Ets-like site had essentially no effect on the transcriptional activity in transfections, while mutation of the proximal site reduced the activity by half. FPA was shown by electrophoretic mobility-shift assay (EMSA) to be comprised of two separable binding sites, FPA1 (distal) and FPA2 (proximal). While mutation of FPA2 had only a modest effect on transcriptional activity in transient transfections, mutation of FPA1 reduced transcriptional activity to approx. 20% of that obtained with the intact promoter. Additional mutations of FPA1 indicated that the active region comprises positions -85 to -73 (GTGGTGAAACCTGT). The molecular masses of the major proteins binding to this site were shown by UV cross-linking to be approx. 40 and 50 kDa, while minor bands were observed at 80 and 110 kDa. Since the involucrin promoter exhibits much higher transcriptional activity in keratinocytes than in other cell types in transfection assays (indicating that cell type specificity of expression is retained), the comparative influence of FPA1 was examined. While mutation of the AP-1 site affected transcriptional activity similarly in all cell lines tested, mutation of FPA1 decreased activity substantially in keratinocytes, but not in NIH-3T3 and HeLa cells, evidence for a contribution to cell-type specificity of expression. Furthermore, a correlation between the sensitivity to FPA1 mutation and amount of involucrin expression in different keratinocyte cell lines was evident. EMSA showed that NIH-3T3 and HeLa cells lacked the same FPA1 DNA-protein complex as keratinocytes. However, the amount of complex formed with nuclear extracts from several keratinocyte lines did not correlate well with the level of involucrin expression. Other factors, such as differences in post-translational modification or co-activators, must account for varied transcriptional response mediated by this site among keratinocyte lines.

Arsenite suppression of involucrin transcription through AP1 promoter sites in cultured human keratinocytes

While preserving keratinocyte proliferative ability, arsenite suppresses cellular differentiation markers by preventing utilization of AP1 transcriptional response elements. In present experiments, arsenite had a dramatic effect in electrophoretic mobility supershift analysis of proteins binding to an involucrin promoter AP1 response element. Without arsenite treatment, binding of JunB and Fra1 was readily detected in nuclear extracts from preconfluent cultures and was not detected a week after confluence, while c-Fos was detected only after confluence. By contrast, band shift of nuclear extracts from arsenite treated cultures showed only JunB and Fra1 binding in postconfluent as well as preconfluent cultures. Immunoblotting of cell extracts showed that arsenite treatment prevented the loss of Fra1 and the increase in c-Fos proteins that occurred after confluence in untreated cultures. Chromatin immunoprecipitation assays demonstrated substantial reduction of c-Fos and acetylated histone H3 at the proximal and distal AP1 response elements in the involucrin promoter and of coactivator p300 at the proximal element. Alteration of AP1 transcription factors was also examined in response to treatment with four metal containing compounds (chromate, vanadate, hemin, divalent cadmium) that also suppress involucrin transcription. These agents all influenced transcription at AP1 elements in a transcriptional reporter assay, but exhibited less effect than arsenite on binding activity assessed by mobility shift and chromatin immunoprecipitation and displayed variable effects on AP1 protein levels. These findings help trace a mechanism by which transcriptional effects of arsenite become manifest and help rationalize the unique action of arsenite, compared to the other agents, to preserve proliferative ability.

Parallel responses of human epidermal keratinocytes to inorganic SbIII and AsIII

SbIII and AsIII are known to exhibit similar chemical properties, but the degree of similarity in their effects on biological systems merits further exploration. Present work compares the responses of human epidermal keratinocytes, a known target cell type for arsenite-induced carcinogenicity, to these metalloids after treatment for a week at environmentally relevant concentrations. Previous work with these cells has shown that arsenite and antimonite have parallel effects in suppressing differentiation, altering levels of several critical enzymes and maintaining colony forming ability. More globally, protein profiling now reveals parallels in SbIII and AsIII effects. The more sensitive technique of transcriptional profiling also shows considerable parallels. Thus, gene expression changes were almost entirely in the same directions for the two treatments, although the degree of change was sometimes significantly different. Inspection of the changes revealed that RYR1 and LRIG1 were among the genes strongly suppressed, consistent with reduced calcium-dependent differentiation and maintenance of EGF-dependent proliferative potential. Moreover, levels of miRNAs in the cells were altered in parallel, with nearly 90% of the 198 most highly expressed ones being suppressed. Among these was miR-203, which is known to decrease proliferative potential. Finally, both SbIII and AsIII were seen to attenuate bone morphogenetic protein 6 induction of dual specificity phosphatases 2 and 14, consistent with maintaining epidermal growth factor receptor signaling. These findings raise the question whether SbIII, like AsIII, could act as a human skin carcinogen.

Localization of Hair Shaft Protein VSIG8 in the Hair Follicle, Nail Unit, and Oral Cavity

The molecular bases of diseases of the epidermal appendages are gradually being elucidated as genes encoding their constituents and regulation are defined. A direct correspondence between altered protein sequence and aberrant hair shaft structure is evident in cases such as monilethrix (Schweizer, 2006). In others, gene defects lead to altered development and thus perturbed regulation of components. Identification of prominent constituents will speed recognition of genes whose defects contribute directly to aberrant structure or indirectly by exacerbating effects of deficiencies in other genes. Present work helps characterize the novel component V-set and immunoglobulin domain containing 8 (VSIG8) in hair shaft and nail plate to assist understanding its possible relation to disease states.

Opposing Actions of Insulin and Arsenite Converge on PKCδ to Alter Keratinocyte Proliferative Potential and Differentiation

When cultured human keratinocytes reach confluence, they undergo a program of changes replicating features of differentiation in vivo, including exit from the proliferative pool, increased cell size, and expression of specialized differentiation marker proteins. Previously, we showed that insulin is required for some of these steps and that arsenite, a human carcinogen in skin and other epithelia, opposes the differentiation process. In present work, we show that insulin signaling, probably through the IGF‐I receptor, is required for the increase in cell size accompanying differentiation and that this is opposed by arsenite. We further examine the impact of insulin and arsenite on PKCδ, a known key regulator of keratinocyte differentiation, and show that insulin increases the amount, tyrosine phosphorylation, and membrane localization of PKCδ. All these effects are prevented by exposure of cells to arsenite or to inhibitors of downstream effectors of insulin (phosphotidylinositol 3‐kinase and mammalian target of rapamycin). Retrovirally mediated expression of activated PKCδ resulted in increased loss of proliferative potential after confluence and greatly increased formation of cross‐linked envelopes, a marker of keratinocyte terminal differentiation. These effects were prevented by removal of insulin, but not by arsenite addition. We further demonstrate a role for src family kinases in regulation of PKCδ. Finally, inhibiting epidermal growth factor receptor kinase activity diminished the ability of arsenite to prevent cell enlargement and to suppress insulin‐dependent PKCδ amount and tyrosine 311 phosphorylation. Thus suppression of PKCδ signaling is a critical feature of arsenite action in preventing keratinocyte differentiation and maintaining proliferative capability.

THE INVOLUCRIN GENE OF THE TREE SHREW : RECENT REPEAT ADDITIONS AND THE RELOCATION OF CYSTEINE CODONS

The coding region of the involucrin gene of Tupaia glis has been cloned and sequenced. It resembles the involucrin coding region of other non-anthropoid mammals in possessing a segment of related, short tandem repeats at a defined location, but in Tupaia, there has been recent serial duplication of a repeat into which a cysteine codon had earlier been introduced. As a result of the duplication, there is a total of as many as six cysteine codons in the segment of repeats, a number larger than for any other species yet examined. In Ratttus there has been a comparable but independent addition of cysteine codons, and both Tupaia and Rattus have eliminated an otherwise conserved cysteine codon 75 located close to but outside the segment of repeats. In Tupaia, this elimination probably occurred by gene conversion. Also independently, the gene of Canis has added cysteine codons to the segment of repeats but has not yet lost cysteine 75. It is proposed that the gain and the loss of cysteine codons are parts of a multi-stage program of cysteine relocation.

Tgm1-like transglutaminases in tilapia (Oreochromis mossambicus)

Among the adaptations of aquatic species during evolution of terrestrial tetrapods was the development of an epidermis preventing desiccation. In present day mammals, keratinocytes of the epidermis, using a membrane-bound transglutaminase (Tgm1), accomplish this function by synthesizing a scaffold of cross-linked protein to which a lipid envelope is attached. This study characterizes the abilities of two homologous transglutaminase isozymes in the teleost fish tilapia to form cross-linked protein structures and their expression in certain tissues. Results indicate they are capable of membrane localization and of generating cellular structures resistant to detergent solubilization. They are both expressed in epithelial cells of the lip, buccal cavity and tips of gill filaments. Adaptation of transglutaminase use in evolution of terrestrial keratinocytes evidently involved refinements in tissue expression, access to suitable substrate proteins and activation of cross-linking during terminal differentiation.