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.

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.

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.

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.

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.

Spontaneous immortalization of human epidermal cells with naturally elevated telomerase.

This work explores spontaneous immortalization in keratinocytes, derived from two skin samples, that display naturally elevated telomerase activity. Serially passaged with 3T3 feeder layer support, the keratinocytes were examined for colony-forming ability, telomerase activity, telomere length, and finally gene expression using Affymetrix DNA microarrays. The cells initially exhibited normal karyotypes and low colony-forming efficiencies typical of normal epidermal cells, but after 40 passages (approximately 400 generations) colony-forming ability increased markedly, yielding immortalized lines exhibiting a small number of chromosomal aberrations and functionally normal p53. An improved protocol for analysis of microarray data permitted detection of 707 transcriptional changes accompanying immortalization including reduced p16(INK4A) mRNA. Telomerase activity was clearly elevated in cells even at low passage from both samples, and telomerase catalytic subunit mRNA was greatly elevated in those with elevated colony-forming ability. The data raise the possibility of an unusual natural phenotype in which aberrant telomerase regulation extends keratinocyte lifespan until rare variants evade senescence. In addition to revealing a potential tumor-prone syndrome, the findings emphasize the desirability of carefully minimizing the degree or timing of elevated expression of telomerase used to immortalize cells for therapeutic purposes.

Arsenite maintains germinative state in cultured human epidermal cells.

Arsenic is a well-known carcinogen for human skin, but its mechanism of action and proximal macromolecular targets remain to be elucidated. In the present study, low micromolar concentrations of sodium arsenite maintained the proliferative potential of epidermal keratinocytes, decreasing their exit from the germinative compartment under conditions that promote differentiation of untreated cells. This effect was observed in suspension and in post-confluent surface cultures as measured by colony-forming ability and by proportion of rapidly adhering colony-forming cells. Arsenite-treated cultures exhibited elevated levels of beta1-integrin and beta-catenin, two proteins enriched in cells with high proliferative potential. Levels of phosphorylated (inactive) glycogen synthase kinase 3beta were higher in the treated cultures, likely accounting for the increased levels of transcriptionally available beta-catenin. These findings suggest that arsenic could have co-carcinogenic and tumor co-promoting activities in the epidermis as a result of increasing the population and persistence of germinative cells targeted by tumor initiators and promoters. These findings also identify a critical signal transduction pathway meriting further exploration in pursuit of this phenomenon.

A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes.

BACKGROUND: TGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. METHODS: In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. RESULTS: In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity. CONCLUSIONS: A distal region of the TGM1 gene promoter, containing AP1 and Sp1 binding sites, is evolutionarily conserved and responsible for high level expression in transgenic mice and in transfected keratinocyte cultures.

Global alteration of gene expression in human keratinocytes by inorganic arsenic.

Alteration of gene expression by inorganic arsenic has been studied in cultured human keratinocytes derived from normal epidermis, a premalignant lesion and a malignant tumor. The purpose was to find whether these cells displayed common alterations in gene expression that might elucidate the mechanism of arsenic action. Global analysis of approximately 12 000 genes by microarray showed that approximately 30% were expressed. Of these, transcription of a substantial fraction (up to 12%) was altered, nearly twice as many being suppressed as stimulated by 2-fold or more at 2 micro M sodium arsenite or 6 micro M arsenate, which did not affect cell growth. At 0.67 micro M arsenite (50 p.p.b.), effects on transcription were less pronounced but clearly evident. Genes whose transcription was altered in common among all the treated keratinocytes included those induced by reactive oxygen, of which heme oxygenase-1 displayed the highest fold induction. Genes indicative of reactive oxygen generation were detected at the earliest time examined, raising the possibility this feature drives subsequent cellular responses. Unlike some agents that produced transient induction of heme oxygenase-1, arsenicals produced sustained induction. Comparison with other agents producing reactive oxygen in the cells, as reflected in heme oxygenase-1 induction, suggested cellular differentiation was suppressed by sustained but not transient generation of reactive oxygen. Sustained global changes in gene expression were seen in target cells treated chronically with inorganic arsenic at concentrations consumed by millions of humans in contaminated drinking water.