Protein profiling of forehead epidermal corneocytes distinguishes frontal fibrosing from androgenetic alopecia.

Protein profiling offers an effective approach to characterizing how far epidermis departs from normal in disease states. The present pilot investigation tested the hypothesis that protein expression in epidermal corneocytes is perturbed in the forehead of subjects exhibiting frontal fibrosing alopecia. To this end, samples were collected by tape stripping from subjects diagnosed with this condition and compared to those from asymptomatic control subjects and from those exhibiting androgenetic alopecia. Unlike the latter, which exhibited only 3 proteins significantly different from controls in expression level, forehead samples from frontal fibrosing alopecia subjects displayed 72 proteins significantly different from controls, nearly two-thirds having lower expression. The results demonstrate frontal fibrosing alopecia exhibits altered corneocyte protein expression in epidermis beyond the scalp, indicative of a systemic condition. They also provide a basis for quantitative measures of departure from normal by assaying forehead epidermis, useful in monitoring response to treatment while avoiding invasive biopsy.

Epidermal cell cultures from white and green sturgeon (Acipenser transmontanus and medirostris): Expression of TGM1-like transglutaminases and CYP4501A.

Using a system optimized for propagating human keratinocytes, culture of skin samples from white and green sturgeons generated epithelial cells capable of making cross-linked protein envelopes. Two distinct forms of TGM1-like mRNA were molecularly cloned from the cells of white sturgeon and detected in green sturgeon cells, accounting for their cellular envelope forming ability. The protein translated from each displayed a cluster of cysteine residues resembling the membrane anchorage region expressed in epidermal cells of teleosts and tetrapods. One of the two mRNA forms (called A) was present at considerably higher levels than the other (called B) in both species. Continuous lines of white sturgeon epidermal cells were established and characterized. Size measurements indicated that a substantial fraction of the cells became enlarged, appearing similar to squames in human epidermal keratinocyte cultures. The cultures also expressed CYP1A, a cytochrome P450 enzyme inducible by activation of aryl hydrocarbon receptor 2 in fish. The cells gradually improved in growth rate over a dozen passages while retaining envelope forming ability, TGM1 expression and CYP1A inducibility. These cell lines are thus potential models for studying evolution of fish epidermis leading to terrestrial adaptation and for testing sturgeon sensitivity to environmental stresses such as pollution.

Elucidation of familial relationships using hair shaft proteomics.

This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedness. Proteomic profiling and amino acid sequence analysis provide information for quantitative and statistically-based analysis of individualization and sample similarity. Protein expression levels are a function of cell-specific transcriptional and translational programs. These programs are greatly influenced by an individual's genetic background, and are therefore influenced by familial relatedness as well as ancestry and genetic disease. Proteomic profiles should therefore be more similar among related individuals than unrelated individuals. Likewise, profiles of genetically variant peptides that contain single amino acid polymorphisms, the result of non-synonymous SNP alleles, should behave similarly. The proteomically-inferred SNP alleles should also provide a basis for calculation of combined paternity and sibship indices. We test these hypotheses using matching proteomic and genetic datasets from a family of two adults and four siblings, one of which has a genetic condition that perturbs hair structure and properties. We demonstrate that related individuals, compared to those who are unrelated, have more similar proteomic profiles, profiles of genetically variant peptides and higher combined paternity indices and combined sibship indices. This study builds on previous analyses of hair shaft protein profiling and genetically variant peptide profiles in different real-world scenarios including different human hair shaft body locations and pigmentation status. It also validates the inclusion of proteomic information with other biomolecular substrates in forensic hair shaft analysis, including mitochondrial and nuclear DNA.

Off-target effects of protein tyrosine phosphatase inhibitors on oncostatin M-treated human epidermal keratinocytes: the phosphatase targeting STAT1 remains unknown.

Cytokine signaling in the epidermis has an important role in maintaining barrier function and is perturbed in pathological conditions. Environmental exposures, such as to metal compounds, are of interest for their potential contribution to skin disease. Present work explores the possibility that vanadate is a more effective protein tyrosine phosphatase inhibitor in human keratinocytes than previously observed in fibroblasts. It focuses on the state of phosphorylation of signal transducer and activator of transcription 1 (STAT1) on tyrosine 701 upon treatment of cultured human keratinocytes with the cytokine oncostatin M, a cutaneous inflammatory mediator that is highly effective in suppressing several differentiation markers and in preserving proliferative potential of keratinocytes. Exposure to sodium vanadate in the medium greatly prolonged the phosphorylation of STAT1, but only at high concentration (>30 µM). Inhibitors of protein tyrosine phosphatases known to dephosphorylate STAT1 (SHP2, TCPTP, PTP1B) were ineffective in mimicking the action of vanadate. The irreversible protein tyrosine phosphatase inhibitor phenyl vinyl sulfonate alone induced STAT1 phosphorylation and appeared to induce its limited cleavage. It also inhibited cross-linked envelope formation, a characteristic step of keratinocyte terminal differentiation, likely due to its reaction with the active site cysteine of keratinocyte transglutaminase. Thus, the key protein tyrosine phosphatase responsible for STAT1 dephosphorylation remains to be identified, and an off-target effect of a potential inhibitor was revealed.

Optimal processing for proteomic genotyping of single human hairs.

The use of hair evidence for human identification is undergoing considerable improvement through the adoption of proteomic genotyping. Unlike traditional microscopic comparisons, protein sequencing provides quantitative and empirically based estimates for random match probability. Non-synonymous SNPs are translated as single amino acid polymorphisms and result in genetically variant peptides. Using high resolution mass spectrometry, these peptides can be detected in hair shaft proteins and used to infer the genotypes of corresponding SNP alleles. We describe experiments to optimize the proteomic genotyping approach to individual identification from a single human scalp hair 2 cm in length (∼100 µg). This is a necessary step to develop a protocol that will be useful to forensic investigators. To increase peptide yield from hair, and to maximize genetically variant peptide and ancestral information, we examined the conditions for reduction, alkylation, and protein digestion that specifically address the distinctive chemistry of the hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6 h of reduction with 100 mM dithiothreitol at room temperature, alkylation with 200 mM iodoacetamide for 45 min, and 6 h of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times increased the average number of genetically variant peptides from 20 ±â€¯5 to 73 ±â€¯5 (p = 1 × 10-13), excluding intractable hair samples. Random match probabilities reached up to 1 in 620 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original hair protein extraction method. Ancestral information was also present in the data. While the number of genetically variant peptides detected were equivalent for both European and African subjects, the estimated random match probabilities for inferred genotypes of European subjects were considerably smaller in African reference populations and vice versa, resulting in a difference in likelihood ratios of 6.8 orders of magnitude. This research will assure uniformity in results across different biogeographic backgrounds and enhance the use of novel peptide analysis in forensic science by helping to optimize genetically variant peptide yields and discovery. This work also introduces two algorithms, GVP Finder and GVP Scout, which facilitate searches, calculate random match probabilities, and aid in discovery of genetically variant peptides.

Age-Related Changes in Hair Shaft Protein Profiling and Genetically Variant Peptides.

Recent reports highlight possible improvements in individual identification using proteomic information from human hair evidence. These reports have stimulated investigation of parameters that affect the utility of proteomic information. In addition to variables already studied relating to processing technique and anatomic origin of hair shafts, an important variable is hair ageing. Present work focuses on the effect of age on protein profiling and analysis of genetically variant peptides (GVPs). Hair protein profiles may be affected by developmental and physiological changes with age of the donor, exposure to different environmental conditions and intrinsic processes, including during storage. First, to explore whether general trends were evident in the population at different ages, hair samples were analyzed from groups of different subjects in their 20's, 40's and 60's. No significant differences were seen as a function of age, but consistent differences were evident between European American and African American hair profiles. Second, samples collected from single individuals at different ages were analyzed. Mostly, these showed few protein expression level differences over periods of 10 years or less, but samples from subjects at 44 and 65 year intervals were distinctly different in profile. The results indicate that use of protein profiling for personal identification, if practical, would be limited to decadal time intervals. Moreover, batch effects were clearly evident in samples processed by different staff. To investigate the contribution of storage (at room temperature) in affecting the outcomes, the same proteomic digests were analyzed for GVPs. In samples stored over 10 years, GVPs were reduced in number in parallel with the yield of identified proteins and unique peptides. However, a very different picture emerged with respect to personal identification. Numbers of GVPs sufficed to distinguish individuals despite the age differences of the samples. As a practical matter, three hair samples per person provided nearly the maximal number obtained from 5 or 6 samples. The random match probability (where the log increased in proportion to the number of GVPs) reached as high as 1 in 108. The data indicate that GVP results are dependent on the single nucleotide polymorphism profile of the donor genome, where environmental/processing factors affect only the yield, and thus are consistent despite the ages of the donors and samples and batchwise effects in processing. This conclusion is critical for application to casework where the samples may be in storage for long periods and used to match samples recently collected.

TINCR is not a non-coding RNA but encodes a protein component of cornified epidermal keratinocytes.

Long non-coding RNAs have been implicated in the regulation of a plethora of biological processes, yet it has been challenging to verify that they are truly not coding for proteins. Terminal differentiation-induced non-coding RNA (TINCR) is a 3.7-kilobase mRNA that is highly abundant in epidermal keratinocytes prior to cornification. Here, we report the presence of an evolutionarily conserved open reading frame in TINCR and the identification of peptides derived from this open reading frame in the proteome of human stratum corneum. Our results demonstrate that TINCR is a protein-coding RNA and suggest that the TINCR-encoded protein is involved in keratinocyte cornification.

Deducing signaling pathways from parallel actions of arsenite and antimonite in human epidermal keratinocytes.

Inorganic arsenic oxides have been identified as carcinogens in several human tissues, including epidermis. Due to the chemical similarity between trivalent inorganic arsenic (arsenite) and antimony (antimonite), we hypothesized that common intracellular targets lead to similarities in cellular responses. Indeed, transcriptional and proteomic profiling revealed remarkable similarities in differentially expressed genes and proteins resulting from exposure of cultured human epidermal keratinocytes to arsenite and antimonite in contrast to comparisons of arsenite with other metal compounds. These data were analyzed to predict upstream regulators and affected signaling pathways following arsenite and antimonite treatments. A majority of the top findings in each category were identical after treatment with either compound. Inspection of the predicted upstream regulators led to previously unsuspected roles for oncostatin M, corticosteroids and ephrins in mediating cellular response. The influence of these predicted mediators was then experimentally verified. Together with predictions of transcription factor effects more generally, the analysis has led to model signaling networks largely accounting for arsenite and antimonite action. The striking parallels between responses to arsenite and antimonite indicate the skin carcinogenic risk of exposure to antimonite merits close scrutiny.

Sebaceous gland abnormalities in fatty acyl CoA reductase 2 (Far2) null mice result in primary cicatricial alopecia.

In a large scale screen for skin, hair, and nail abnormalities in null mice generated by The Jackson Laboratory's KOMP center, homozygous mutant Far2tm2b(KOMP)Wtsi/2J (hereafter referrred to as Far2-/-) mice were found to develop focal areas of alopecia as they aged. As sebocytes matured in wildtype C57BL/NJ mice they became pale with fine, uniformly sized clear lipid containing vacuoles that were released when sebocytes disintegrated in the duct. By contrast, the Far2-/- null mice had sebocytes that were similar within the gland but become brightly eosinophilic when the cells entered the sebaceous gland duct. As sebocytes disintegrated, their contents did not readily dissipate. Scattered throughout the dermis, and often at the dermal hypodermal fat junction, were dystrophic hair follicles or ruptured follicles with a foreign body granulomatous reaction surrounding free hair shafts (trichogranuloma). The Meibomian and clitoral glands (modified sebaceous glands) of Far2-/- mice showed ducts dilated to various degrees that were associated with mild changes in the sebocytes as seen in the truncal skin. Skin surface lipidomic analysis revealed a lower level of wax esters, cholesterol esters, ceramides, and diacylglycerols compared to wildtype control mice. Similar changes were described in a number of other mouse mutations that affected the sebaceous glands resulting in primary cicatricial alopecia.

DUOX expression in human keratinocytes and bronchial epithelial cells: Influence of vanadate.

Dual oxygenases (DUOX) 1 and 2, expressed in many animal tissues, participate in host defense at mucosal surfaces and may have important signaling roles through generation of reactive oxygen. Present work addresses their expression in cultured human epidermal keratinocytes and effects of cytokines and metal/metalloid compounds. Both DUOX1 and 2 were expressed at much higher levels after confluence than in the preconfluent state. Maximal DUOX1 mRNA levels were 50 fold those of DUOX2. DUOX1 and 2 were induced ≈3 fold by interleukin 4, but only DUOX1 was induced by interferon gamma (IFNγ). In human bronchial HBE1 cells, by contrast, interleukin 4 induced only DUOX 1, and IFNγ induced only DUOX2. A survey in the keratinocytes of metal/metalloid compounds showed that arsenite, antimonite, chromate, cadmium, copper, lead and vanadate suppressed DUOX1 levels but did not prevent interleukin 4 stimulation. Effects on DUOX2 were less dramatic, except that vanadate potentiated the stimulation by IFNγ up to 7 fold. The results indicate that epithelial cell types of different tissue origins can differ in their cytokine regulation and that epidermal cells can exhibit striking alterations in response due to certain metal/metalloid exposures.

Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides.

Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence.

Proteomic profiling reveals candidate markers for arsenic-induced skin keratosis.

Proteomics technology is an attractive biomarker candidate discovery tool that can be applied to study large sets of biological molecules. To identify novel biomarkers and molecular targets in arsenic-induced skin lesions, we have determined the protein profile of arsenic-affected human epidermal stratum corneum by shotgun proteomics. Samples of palm and foot sole from healthy subjects were analyzed, demonstrating similar protein patterns in palm and sole. Samples were collected from the palms of subjects with arsenic keratosis (lesional and adjacent non-lesional samples) and arsenic-exposed subjects without lesions (normal). Samples from non-exposed healthy individuals served as controls. We found that three proteins in arsenic-exposed lesional epidermis were consistently distinguishably expressed from the unaffected epidermis. One of these proteins, the cadherin-like transmembrane glycoprotein, desmoglein 1 (DSG1) was suppressed. Down-regulation of DSG1 may lead to reduced cell-cell adhesion, resulting in abnormal epidermal differentiation. The expression of keratin 6c (KRT6C) and fatty acid binding protein 5 (FABP5) were significantly increased. FABP5 is an intracellular lipid chaperone that plays an essential role in fatty acid metabolism in human skin. This raises a possibility that overexpression of FABP5 may affect the proliferation or differentiation of keratinocytes by altering lipid metabolism. KRT6C is a constituent of the cytoskeleton that maintains epidermal integrity and cohesion. Abnormal expression of KRT6C may affect its structural role in the epidermis. Our findings suggest an important approach for future studies of arsenic-mediated toxicity and skin cancer, where certain proteins may represent useful biomarkers of early diagnoses in high-risk populations and hopefully new treatment targets. Further studies are required to understand the biological role of these markers in skin pathogenesis from arsenic exposure.

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.

Crisp1 and alopecia areata in C3H/HeJ mice.

Alopecia areata (AA), a cell mediated autoimmune disease, is the second most common form of hair loss in humans. While the autoimmune disease is responsible for the underlying pathogenesis, the alopecia phenotype is ultimately due to hair shaft fragility and breakage associated with structural deficits. Quantitative trait genetic analyses using the C3H/HeJ mouse AA model identified cysteine-rich secretory protein 1 (Crisp1), a hair shaft structural protein, as a candidate gene within the major AA locus. Crisp1 transcripts in the skin at various times during disease development were barely detectable. In situ hybridization identified Crisp1 expression within the medulla of hair shafts from clinically normal strains of mice but not C3H/HeJ mice with AA. Follow-up work with 5-day-old C3H/HeJ mice with normal hair also had essentially no expression of Crisp1. Other non-inflammatory based follicular dystrophy mouse models with similar hair shaft abnormalities also have little or no Crisp1 expression. Shotgun proteomics, used to determine strain difference in hair proteins, confirmed that there was very little CRISP1 within normal C3H/HeJ mouse hair in comparison to 11 other strains. However, mutant mice with hair medulla defects also had undetectable levels of CRISP1 in their hair. Crisp1 null mice had normal skin, hair follicles, and hair shafts indicating that the lack of the CRISP1 protein does not translate directly into defects in the hair shaft or hair follicle. These results suggest that CRISP1 may be an important structural component of mouse hair and that its strain-specific dysregulation may indicate a predisposition to hair shaft disease such as AA.

Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

Metabolic activation of the antibacterial agent triclocarban by cytochrome P450 1A1 yielding glutathione adducts.

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is an antibacterial agent used in personal care products such as bar soaps. Small amounts of chemical are absorbed through the epidermis. Recent studies show that residues of reactive TCC metabolites are bound covalently to proteins in incubations with keratinocytes, raising concerns about the potential toxicity of this antimicrobial agent. To obtain additional information on metabolic activation of TCC, this study characterized the reactive metabolites trapped as glutathione conjugates. Incubations were carried out with (14)C-labeled TCC, recombinant CYP1A1 or CYP1B1, coexpressed with cytochrome P450 reductase, glutathione-S-transferases (GSH), and an NADPH-generating system. Incubations containing CYP1A1, but not 1B1, led to formation of a single TCC-GSH adduct with a conversion rate of 1% of parent compound in 2 hours. Using high-resolution mass spectrometry and diagnostic fragmentation, the adduct was tentatively identified as 3,4-dichloro-3'-glutathionyl-4'-hydroxycarbanilide. These findings support the hypothesis that TCC is activated by oxidative dehalogenation and oxidation to a quinone imine. Incubations of TCDD-induced keratinocytes with (14)C-TCC yielded a minor radioactive peak coeluting with TCC-GSH. Thus, we conclude that covalent protein modification by TCC in TCDD-induced human keratinocyte incubations is mainly caused by activation of TCC by CYP1A1 via a dehalogenated TCC derivative as reactive species.

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.

Opposing actions of insulin and arsenite converge on PKCdelta 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 PKCdelta, a known key regulator of keratinocyte differentiation, and show that insulin increases the amount, tyrosine phosphorylation, and membrane localization of PKCdelta. 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 PKCdelta 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 PKCdelta. Finally, inhibiting epidermal growth factor receptor kinase activity diminished the ability of arsenite to prevent cell enlargement and to suppress insulin-dependent PKCdelta amount and tyrosine 311 phosphorylation. Thus suppression of PKCdelta signaling is a critical feature of arsenite action in preventing keratinocyte differentiation and maintaining proliferative capability.

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.

Generation of oxidant response to copper and iron nanoparticles and salts: Stimulation by ascorbate.

The present work describes a two-stage approach to analyzing combustion-generated samples for their potential to produce oxidant stress. This approach is illustrated with the two commonly encountered transition metals, copper and iron. First, their abilities to generate hydroxyl radical were measured in a cell-free, phosphate-buffered saline solution containing ascorbate and/or citrate. Second, their abilities to induce heme oxygenase-1 in cultured human epidermal keratinocytes were assessed in cell culture. Combustion-generated copper oxide nanoparticles were active in both assays and were found to be soluble in culture medium. Depletion of glutathione in the cells or loading the cells with ascorbate greatly increased heme oxygenase-1 induction in the presence of copper. By contrast, iron oxide nanoparticles were active in the phosphate-buffered saline but not in cell culture, and they aggregated in culture medium. Soluble salts of copper and iron exhibited the same contrast in activities as the respective combustion-generated particles. The results suggest that the capability of combustion-generated environmental samples to produce oxidant stress can be screened effectively in a two step process, first in phosphate-buffered saline with ascorbate and subsequently in epithelial cell culture for those exhibiting activity initially. The results also point to an unanticipated interaction in cells of oxidant stress-generating metals with an antioxidant (ascorbate) that is usually missing in culture medium formulations. Thus, ascorbate supplementation of cultured human cells is likely to improve their ability to model the in vivo effects of particulate matter containing copper and other redox-active metals.