Günter Michel

1,25-(OH)2-VITAMIN D3 AND CALCIPOTRIOL INDUCE IL-10 RECEPTOR GENE EXPRESSION IN HUMAN EPIDERMAL CELLS

Abstract. Psoriasis is a common hyperproliferative and inflammatory skin disease with a prevalence of 0.5–3%. Lesional skin is characterized by pathological overexpression of proinflammatory cytokines such as IL-8 and its receptor and the decreased presence of negative regulatory control factors like the anti-oncogene p53. The expression of these genes can be modulated in the opposite direction by antipsoriatic drugs. Another possible candidate gene is the receptor for the anti-inflammatory cytokine IL-10 (IL-10R). Recently, vitamin D3 and its analogues have attracted interest as new therapeutic agents in the treatment of psoriasis. In extension of these findings we studied here the effect of the physiologically active metabolite, 1,25-dihydroxyvitamin D3 (calcitriol) and its synthetic analogue calcipotriol (MC 903) on the expression of the IL-10R in HaCaT cells by RT-PCR. IL-10 receptor gene expression was effectively induced in the range of 10-8–10 -9 M. Upregulation by calcitriol was about 10-fold, by calcipotriol 12-fold. Induction of the receptor for the anti-inflammatory cytokine IL-10 may be involved in the antipsoriatic action of vitamin D derivatives.

Increased Expression of the Epidermal Growth Factor Receptor in Human Epidermal Keratinocytes after Exposure to Ionizing Radiation

The effect of exposure of human epidermal keratinocytes to ionizing radiation, both in vivo and in vitro, on the expression of the epidermal growth factor receptor (EGF-R) was studied on the protein, mRNA, and functional levels. Quantitative fluorometry of short-term organ cultures incubated with a monoclonal antibody against human EGF-R revealed a dose-dependent increase of EGF-R expression 24 h after irradiation with 4 and 6 Gy, with an additional increase after 48 h. In biopsy specimens from patients undergoing radiation therapy a markedly increased expression could be determined by quantitative fluorometry during radiation therapy which wa still considerably above the baseline level 4 weeks after termination of treatment. Radioligand binding assays demonstrated a 50% increase in 125I-EGF binding to primary keratinocytes and A431 cells, at doses of 1 Gy, with a further increase after 72 and 96 h. Northern blots were performed with total RNA from two human epidermal cell lines (SCLII and A431). In A431 cells, increased EGF-R transcript levels could be detected 48 h after irradiation. In cells of the SCLII cell line, EGF-R expression was not affected by irradiation. These results were confirmed by semiquantitative polymerase chain reaction of primary cultured keratinocytes, demonstrating an increase of transcripts of EGF-R 24 h after irradiation with single doses of 6 Gy. Thus exposure to ionizing radiation leads to an increased expression of functionally intact EGF-R in human keratinocytes, at the protein and mRNA levels, both in vitro and in vivo; we hypothesize that this effect is part of a stress program of epidermal cells in response to ionizing radiation, ensuring rapid repopulation of irradiated areas.

Molecular cloning and characterization of alternatively spliced mRNA isoforms from psoriatic skin encoding a novel member of the S100 family

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.

FK506 in the treatment of inflammatory skin disease: promises and perspectives

The immunosuppressive macrolide drug FK506 is currently gaining increasing importance in dermatopharmacology. Here, Gunter Michel and colleagues summarize the current state of research into the molecular mechanisms responsible for the functional modulation of cell types other than T cells, particularly epidermal cells, by this drug.

Effects of the Neuropeptides Substance P, Calcitonin Gene-Related Peptide and α-Melanocyte-Stimulating Hormone on the IL-8/IL-8 Receptor System in a Cultured Human Keratinocyte Cell Line and Dermal Fibroblasts

The neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP) and α-melanocyte-stimulating hormone (α-MSH) are known to be able to regulate the production of cytokines in the skin. Since IL-8 plays an important role in cutaneous inflammation, the effects of SP, CGRP and α-MSH on the IL-8/IL-8 receptor (IL-8RA) systems of these cell types were studied. Cultures of human dermal fibroblasts and an immortalized keratinocyte cell line HaCaT were treated with 10−8 M SP, CGRP or α-MSH. The results demonstrated that these neuropeptides have different effects on the IL-8 and IL-8RA expressions of the cells. SP and CGRP upregulated the IL-8RA mRNA expression in HaCaT cells, but had no influence on their IL-8 production, whereas, α-MSH had no effect on either the IL-8 or the IL-8RA mRNA expression in HaCaT cells. In contrast, α-MSH resulted in a time-dependent induction of the IL-8 mRNA expression in dermal fibroblasts. This induction was already detectable after 6 h, and after 12 h there was a 5-fold change in comparison with the controls. The IL-8 content of the supernatant was also increased, with a maximum at 48 h after α-MSH treatment. The data established in the present study support the notion that neuropeptides can directly modulate the IL-8/IL-8RA system of keratinocytes and fibroblasts.

Antioncogene P53 and mitogenic cytokine interleukin-8 aberrantly expressed in psoriatic skin are inversely regulated by the antipsoriatic drug tacrolimus (FK506)

Uncontrolled proliferation of epidermal cells is the most prominent characteristic of psoriasis. This widespread skin disease can be effectively treated with the microbial substance FK506, which acts by modulating gene expression. We, therefore, asked if the drug changes the expression of genes involved in growth regulation (the mitogenic cytokine interleukin-8 (IL-8) and p53, a negative cell cycle regulator) and signal transduction (protooncogenes c-ras, c-raf, and HER-2). Gene expression was monitored by semiquantitative mRNA-PCR and for p53 by immunocytochemistry in cultured primary keratinocytes (KC). In addition, p53 expression was analysed in skin biopsies of psoriatic patients. After 1-3 hr, IL-8 mRNA levels were dose-dependently decreased in tacrolimus (FK506)-treated cells. Protooncogene expression was not significantly altered. Interestingly, p53 transcription was clearly induced by FK506 treatment. This tendency could be verified on the protein level by immunocytochemistry. In contrast, p53 expression was decreased in lesional psoriatic as compared to normal skin, providing evidence that not only posttranslational modification of the p53 protein, but also transcriptional modulation of the p53 gene, are involved in pathological processes and pharmacological drug action in skin. Together with earlier results showing downmodulation for IL-8 receptor type A expression in cultured KC treated with FK506, these results suggest that both the mitogenic IL-8/IL-8R system and the cell cycle inhibitor p53 represent potential targets for the antipsoriatic action of the drug, whereas protooncogenes acting downstream in mitogenic signal transduction cascades are unaffected. The differential modulation of an entire set of genes provides evidence for the specificity of the drug effects and rules out nonspecific toxic effects on KC.

Molecular basis of basal cell carcinoma: analysis of differential gene expression by differential display PCR and expression array.

Basal cell carcinoma (BCC) is the most common tumor in the Caucasian population. Although BCC rarely metastasize and cause death, they are problematic due to their destructive growth and the frequent localization on the face. Until now the knowledge of genes differentially expressed in BCC has been incomplete. To elucidate the complex alterations in BCC‐associated gene expression, we took advantage of 2 techniques: the differential display RT‐PCR (DD‐PCR) and the differential hybridization of cDNA arrays. Using DD‐PCR, we showed differential expression of genes known from other biological contexts (e.g., rac, ubiquitin hydrolase), which could now be associated with BCC. In addition, we detected unknown genes possibly contributing to the carcinogenesis of BCC. Of the 588 genes screened by differential hybridization of the Atlas™ human cDNA array, differences in the expression levels of BCC were observed for 10 genes. These data were obtained with RNA probes pooled from several BCC of different donors and were subsequently confirmed by semiquantitative RT‐PCR for Janus protein tyrosine kinase 3 (Jak3), microsomal glutathione S‐transferase 1 (GST 12), teratocarcinoma‐derived growth factor cripto, glutaredoxin and the monocyte chemoattractant protein 1 (MCP‐1) in 10 individual BCC specimens, 2 squamous cell carcinoma (SCC), the cell line HaCaT and cultured normal human keratinocytes (NHK) in comparison to normal skin. These genes are candidates from gene families with known association to tumors, but they have not been reported in the carcinogenesis of BCC yet. In summary, both approaches allow the detection of differentially expressed genes possibly involved in the carcinogenesis of BCC.

Role of lnterleukin-8 Receptor in Skin

Interleukin-8 (IL-8) is a potent chemotactic and proinflammatory cytokine, produced in the skin by a variety of cells in response to inflammatory stimuli. Recent studies suggest that in addition to its potent actions on leukocytes, IL-8 exerts a direct influence on several functions of human epidermal cells such as chemotaxis, Candida albicans killing activity or proliferation. The effects of IL-8 are mediated by binding to two types of specific high-affinity receptors which contain seven transmembrane domains typical of guanine nucleotide binding protein (G protein)-coupled receptors. In the skin, a broad spectrum of cells such as neutrophils, T lymphocytes, mast cells, dermal macrophages, endothelial cells and keratinocytes possess binding sites for IL-8. Recently, increased expression of epidermal IL-8 receptors has been observed in psoriasis an inflammatory and hyperproliferative skin disease. Since the effects of IL-8 may be modulated at the receptor level, the pharmacological manipulation of the IL-8 receptor may prove an important target for the therapy of skin diseases with increased IL-8 levels.

Analysis of UVB‐modulated Gene Expression in Human Keratinocytes by mRNA Differential Display Polymerase Chain Reaction

Abstract— EUltraviolet (UV) light is the most important environmental insult to skin. Even a single exposure to UVB radiation can result in inflammation and may also lead to DNA damage and apoptosis in the acute response of the cutaneous tissue. To elucidate the complex alterations of gene expression in human keratinocytes underlying these UV responses we took advantage of differential display polymerase chain reaction (DD‐PCR) technologys ability to detect qualitative and quantitative changes in gene expression in more than two cell populations simultaneously. We demonstrate that low‐dose UVB (100 Jm‐2) leads to both induction and down regulation of different genes during the 24 h after irradiation in a time‐dependent manner. In addition to the identification of known genes as possible effectors or targets in the UV response of human keratinocytes, we here identify a new sequence that is negatively regulated by UVB irradiation and was termed HUR 7 (HaCaT UV repressed). In general our results showed that DD‐PCR is a useful tool in the analysis of quantitative changes of mRNA levels in human keratinocytes after UV irradiation. The identification of new UVB‐repressed genes offers the opportunity to identify unrecognized molecular mechanisms in the UV response of human cells.

The interleukin-8 receptor: a potential target for antipsoriatic therapy?☆

Interleukin-8 is assumed to play a central role in the pathogenesis of psoriasis. Since an increased expression of the interleukin-8 receptor has been observed both in polymorphonuclear leukocytes and in affected psoriatic epidermis, we were interested in whether the interleukin-8 receptor could be a molecular target of antipsoriatic compounds. Cyclosporine, calcitriol, calcipotriol or dithranol caused a dose-dependent decrease in interleukin-8 binding to cultured human keratinocytes, while interleukin-8 binding to granulocytes was not affected. In addition, the interleukin-8-induced human leukocyte antigen-DR (HLA-DR) expression of keratinocytes was nearly completely blocked by treatment of the cells with these substances. The inhibition of the keratinocyte interleukin-8 receptor and its function by antipsoriatic drugs may contribute to their therapeutic action.