In an effort to identify psoriasis-associated genes, we compared gene expression in normal and psoriatic skin, using differential display RT-PCR technique. Sequence analysis of a 650-bp cDNA fragment (clone 110) that was highly up-regulated in lesional skin revealed homology to a noncoding cDNA (NICE-2). By subsequent cDNA cloning, using RNA from psoriatic skin, we have identified two alternatively spliced mRNA-isoforms (0.5 and 4.4 kb), which differ in composition of their untranslated regions. By sequence comparison, we have mapped the novel gene, named S100A15, to the S100 gene cluster within the epidermal differentiation complex (chromosome 1q21). Analysis of the deduced amino acid sequence revealed a protein of 101 amino acids containing two potential EF-hand motifs with high homology to the S100A7. Northern blot hybridization and semiquantitative RT-PCR analysis confirmed the S100A15 overexpression in psoriasis, showing different levels of expression of the S100A15 mRNA isoforms. In situ hybridization of the S100A15 revealed a markedly increased staining of basal and suprabasal epidermal layers of psoriatic skin compared with healthy tissue. Our data suggest an involvement of the novel S100A15 in epidermal differentiation and inflammation and might therefore be important for the pathogenesis of psoriasis and other diseases.

E. coli is a gram-negative bacterium rarely found on human skin. We investigated whether direct interaction of E. coli with keratinocytes might induce an innate immune response through recognition by pattern recognition receptors. The capacity of E. coli to activate innate immune responses and IL-8 induction was investigated. We found that E. coli significantly induced human S100A7 and S100A15 transcript abundance and IL-8 release in cultured primary human keratinocytes. S100A15 is a member of the S100 protein family with previously unknown function. E. coli induced effects could be inhibited by neutralizing Toll-like receptor 4 (TLR4) antibodies, suggesting that E. coli-induced IL-8 and S100A15 expression in human keratinocytes are TLR4 dependent. TLR4-/- mice lacked elevated mS100A15 expression after infection with E. coli in contrast to wild-type mice. In vitro, human S100A15 displayed antimicrobial activity against E. coli. Our findings suggest that E. coli modulates S100A15 and IL-8 expression of keratinocytes by recognition through TLR4.

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. macrospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora.

Psoriasis is a chronic inflammatory disease characterized by epidermal hyperplasia and an inflammatory infiltrate. The normal differentiation from basal to granular keratinocytes with subsequent apoptosis and cornification is disturbed in the akanthotic epidermis. This could be due to both an excess of mitogenic stimuli with hyperproliferation and/or resistance to apoptosis. By mRNA differential display we found HAX-1 to be overexpressed in lesional psoriatic skin. The overexpression of HAX-1 was verified at the mRNA level by Northern blot and in situ hybridization, as well as at the protein level by Western blot and immunohistochemistry. Detection of HAX-1 in mRNA from different tissues showed strong expression in the brain, pancreas, skeletal muscle, and heart. In contrast to primary keratinocytes and melanocytes we found HAX-1 highly expressed in human immortalized keratinocytes (HaCaT) and different melanoma cell lines. In HaCaT cells as a model for psoriatic keratinocytes we found an increased ultraviolet-induced apoptosis after expression of HAX-1 antisense mRNA. In psoriasis, the epidermal differentiation could be disturbed due to the increased expression of HAX-1 and hence a prolonged resistance to terminal differentiation. Antiapoptotic mechanisms are an emerging concept for the understanding of the pathogenesis of this disease possibly leading to clinical applications.

The human calcium-binding protein (hS100A15) was first identified in inflamed hyperplastic psoriatic skin, where the S100A15 gene is transcribed into two mRNA splice variants, hS100A15-S and hS100A15-L. To compare the contribution of the human S100A15 (hS100A15) isoforms in skin inflammation and differentiation, we determined the expression, distribution and regulation of hS100A15-S and hS100A15-L in psoriasis and chronic atopic eczema compared with normal skin. We found that both hS100A15 transcripts were mainly distributed in the epidermis of normal and inflamed skin with hS100A15-L being the predominantly expressed mRNA isoform in both psoriasis and atopic eczema. In cultured keratinocytes, IL-1beta and Th1 cytokines significantly induced hS100A15-L compared with hS100A15-S. In contrast, Th2-derived cytokines had no influence on the expression of either hS100A15 splice variant. Differentiation of human keratinocytes induced by 1.2 mm calcium resulted in the upregulation of both hS100A15 mRNA isoforms. Our data show that both hS100A15 splice variants are differentially regulated and expressed with epidermal differentiation and skin inflammation. Overexpression of hS100A15 in chronic inflammatory skin diseases and regulation by inflammatory cytokines and calcium suggest that hS100A15 is involved in Th1-associated epithelial responses and epidermal maturation in normal and diseased human skin.

The calcium-binding proteins of the human S100A7/A15 (hS100A7/A15) subfamily are differentially expressed in normal and pathological epidermis. The hS100A7 (psoriasin) and S100A15 reside in a chromosomal cluster of highly similar paralogs. To exploit the power of mouse models for determining functions of gene products, the corresponding S100A7/A15 ortholog was cloned and examined in murine skin. The single mouse S100A15 (mS100A15) gene encodes a protein of 104 amino acids with a predicted molecular weight of 12,870 Da and two EF-hand calcium binding sites. Using gene-specific primers and specific antibodies, expression of mS100A15 in both skin and isolated keratinocytes is confined to differentiating granular and cornified epidermal cells. Immunoblotting of epidermal extracts revealed a series of high molecular weight bands that are also recognized by an antibody for transglutaminase-mediated protein crosslinks. mS100A15 expression is upregulated in cultured keratinocytes induced to differentiate by calcium or phorbol esters. Maximal induction occurs concordantly with expression of late differentiation markers. Induction is enhanced in keratinocytes overexpressing protein kinase Calpha and is dependent on activator protein-1 transcription factors. The regulation, expression pattern and crosslinking of mS100A15 are consistent with the characteristics of the human orthologs, providing a valid surrogate model to study changes in these proteins associated with cutaneous pathologies.

Cathepsin (Cat) L is an important lysosomal proteinase involved in a variety of cellular functions including intracellular protein turnover, epidermal homeostasis and hair development. Hurpin (serpinB13) is a cross-class specific serine protease inhibitor of Cat L. We have analysed the expression and localization of Cat L and hurpin in various inflammatory and neoplastic diseases by immunohistochemistry. Whereas Cat L expression in normal skin was below detection limit, immunoreactivity was detected in chronic inflammatory dermatoses. The highest expression of Cat L was found in psoriasis, atopic eczema and squamous cell carcinoma (SCC) samples. Samples of Lupus erythematosus, folliculitis, acne inversa, chronic leg ulcers and pyoderma gangrenosum demonstrated similar but lower expression for Cat L. In malignant cells of SCC, basal cell carcinoma and malignant melanoma, characteristic staining patterns were observed for Cat L, with more abundant expression at the periphery of the tumor. Expanding our previous work, we found that the expression of hurpin was confined mainly to the basal layer in normal skin samples, whereas hurpin was overexpressed and redistributed in diseased skin. The localization of hurpin in dermatoses and neoplasias differed from that in normal skin in that the highest expression was found in the outermost layers of the granular and upper spinous layers. Similarly to Cat L, the highest expression for hurpin was found in psoriasis and SCC. The results presented here summarize for the first time the expression of the protease Cat L and its inhibitor hurpin in a broad spectrum of skin diseases.

The cephalosporin C-producing fungus Acremonium chrysogenum was transformed to hygromycin B resistance using different vector constructs. These constructs contain sequences of the pcbC gene from A. chrysogenum, encoding isopenicillin N synthetase. Detailed analysis of transformants, including pulsed-field gel electrophoresis (PFGE), suggests that integration of multiple vector copies takes place predominantly via non-homologous integration. By increasing the length of vector-DNA homologous to genomic DNA, integration occurs more frequently into chromosome VI, carrying the endogenous pcbC gene copy. In gene disruption experiments, the length of vector homology required to obtain cephalosporin C-minus transformants was investigated. Inactivation of the pcbC gene was observed only when homologous fragments of more than 3.0 kb were used on both sites of the resistance cassette. Southern analysis indicated homologous, as well as heterologous, integration of recombinant DNA. The integration of multiple vector copies leads to the appearance of truncated pcbC transcripts.