Keratinocyte differentiation marker suppression by arsenic: mediation by AP1 response elements and antagonism by tetradecanoylphorbol acetate.

Culture models of target cells are anticipated to help elucidate the mechanism by which inorganic arsenic acts as a carcinogen in humans. Present work characterizes the response of human keratinocytes, a target cell type, to arsenic suppression of their differentiation program. Four representative differentiation marker mRNAs (involucrin, keratinocyte transglutaminase, small proline-rich protein 1, and filaggrin) were suppressed by both arsenate and arsenite in normal, spontaneously immortalized (premalignant), and malignant keratinocytes with EC50 values in the low micromolar range. The suppression was almost completely reversed 9 days after removal of arsenate from the culture medium. In the case of the involucrin gene, suppression was mediated primarily by two functional AP1 response elements in the gene promoter. Both glucocorticoid and serum stimulation of differentiation occurred to a similar extent in the presence and absence of arsenic, indicating neither stimulation was a specific target of arsenic action and neither agent could overcome arsenic suppression. In contrast, 12-O-tetradecanoylphorbol-13-acetate prevented the suppression of keratinocyte transglutaminase, suggesting that arsenic acts upstream of protein kinase C.

Functional AP1 and CRE response elements in the human keratinocyte transglutaminase promoter mediating Whn suppression.

Expression of keratinocyte transglutaminase (TGM1) is critical for maturation of mammalian epidermis and occurs during squamous metaplasia. Examination of the TGM1 5'-flanking region in transient transfections of human epidermal cells revealed an AP1 site approximately 1.5kb upstream from the transcription start site and a CRE site approximately 0.5kb upstream that, combined, accounted for as much as 90% of the transcriptional activity. Upon incubation with nuclear extract, three electrophoretically separable protein complexes were formed by a CRE site oligonucleotide, one of which was competed by an AP1 response element. In super-shift analysis, c-Jun and JunD formed complexes with both the AP1 and CRE sequences. The AP1 and CRE sites were found to mediate the suppressive effects of the Whn transcription factor on the activity of the TGM1 promoter. Similarly, two previously described AP1 sites mediated Whn suppression of involucrin promoter activity.

Role of Sp1 response element in transcription of the human transglutaminase 1 gene.

This study addresses the contribution of an Sp1 response element in the proximal promoter of the transglutaminase 1 gene to transcription in normal epidermis and in a case of lamellar ichthyosis lacking transglutaminase 1 activity. The latter exhibited an Sp1 promoter mutation previously hypothesized to suppress transcription. In this study, several experiments indicated that the native Sp1 response element was functional, but it had only a small influence on transcription, and the previously observed mutation had no effect. These experiments involved mobility shift assays and transfections of promoter constructs in which the Sp1 site was mutated or lacking altogether. In addition the proximal 1.6 kb of the promoter from the affected individual was as active in transfections as the promoter from unaffected individuals. A search for sequence alterations in mRNA transcribed in keratinocytes from the patient revealed a novel single base mutation in codon 661 of the transglutaminase coding region predicted to result in premature termination of protein translation. The presence of this mutation in parental genomic DNA was confirmed by restriction digestion. Thus the lamellar ichthyosis phenotype in this case is likely attributable to a novel non-sense mutation in the coding region leading to reduced transglutaminase 1 mRNA levels rather than mutation of the Sp1 site.

Keratinocyte transglutaminase promoter analysis. Identification of a functional response element.

Keratinocyte transglutaminase catalyzes isopeptide bond formation to yield the highly insoluble cross-linked envelope during terminal differentiation of epidermal cells. Transcriptional response elements were identified in the 5'-flanking DNA of the gene for this enzyme by a combination of transient transfection and electrophoretic mobility shift analyses. Since human keratinocytes transcribed ineffectively transfected transglutaminase flanking DNA, a key feature of these experiments was the use of rat bladder epithelial cells as recipients. Serial deletion experiments identified by transient transfection an important response region containing three putative AP2-like response elements approximately 0.5 kilobases from the transcription initiation site. Oligonucleotides, each containing a single one of the elements, formed specific complexes with keratinocyte nuclear proteins. Two of the response elements were found to be functional by transfection in site-specific deletion experiments. Of these one formed specific DNA-protein complexes with nuclear proteins only from cells exhibiting keratinocyte differentiation. UV cross-linking experiments estimated the protein component of the complex to be approximately 85 kDa. This response element alone increased substantially the transcription of a minimal transglutaminase promoter in transient transfections. Further characterization of the putative transcription factor binding to this response element may provide insight into the regulation of keratinocyte transglutaminase.