Norbert Wikonkál

Escaping the stem cell compartment: Sustained UVB exposure allows p53-mutant keratinocytes to colonize adjacent epidermal proliferating units without incurring additional mutations

Once mutated, a single cell must expand into a clone before becoming significant for carcinogenesis. The forces driving clonal expansion and the obstacles that must be overcome are poorly understood. In a genetic mechanism, acquiring a second mutation conferring a proliferative advantage would enable the cell to expand autonomously. If carcinogen exposure instead induced a physiological change, clonal expansion would require the carcinogens continued presence. To determine which is the case, we studied microscopic clones of keratinocytes mutated in the p53 tumor suppressor gene. Carcinogen exposure was controlled by irradiating mice with 280–320 nm UV radiation (UVB), sunlights principal carcinogenic component; expansion of mutant clones was observed in epidermal sheets. p53-mutant clones grew only during chronic UVB exposure. Therefore, clonal expansion was not triggered by a proliferative mutation but was instead continually driven by UVB. Unexpectedly, the clone size distribution showed periodicity with maxima at estimated intervals of 16 ± 6 cells, the size of the epidermal proliferating unit in murine dorsal skin. In the absence of UVB, rare “imprisoned clones” increased in cell number without increasing in area. We conclude that: stem cell compartments act as physical barriers to clonal expansion of a p53-mutant keratinocyte; a rate-limiting step in clonal expansion is the colonization of an adjacent compartment; and sustained UVB enables the p53-mutant keratinocyte to colonize without incurring an additional mutation.

Inactivating E2f1 reverts apoptosis resistance and cancer sensitivity in Trp53-deficient mice

The E2f1 transcription factor, which regulates genes required for S-phase entry, also induces apoptosis by transcriptional and post-translational mechanisms. As E2f1 is inducible by DNA damage we investigated its importance in vivo in ultraviolet (UV)-induced apoptosis, a protective mechanism that prevents the epidermis from accumulating UV-induced mutations. Contrary to expectation, E2f1−/− mice demonstrated enhanced keratinocyte apoptosis after UVB exposure, whereas apoptosis was suppressed by epidermis-specific overexpression of human E2F1. Apoptosis induced by γ-radiation was also repressed by E2f1. E2f1−/−;Trp53−/− double knockout mice exhibited the elevated UVB-induced apoptosis of E2f1−/− alone, rather than the profound apoptosis defect seen in Trp53−/− mice, indicating that Trp53 (p53) lies functionally upstream of E2f1. Transfecting E2F1 into E2f1−/−;Trp53−/− primary fibroblasts suppressed UVB-induced apoptosis and this suppression was relieved by Trp53. The double knockout also reverted the abnormal sex ratio and early-onset tumours of Trp53−/− mice. These results imply that E2f1 functions as a suppressor of an apoptosis pathway that is initiated by DNA photoproducts and perhaps genetic abnormalities; p53 relieves this suppression.

Low-level laser (light) therapy (LLLT) for treatment of hair loss.

Alopecia is a common disorder affecting more than half of the population worldwide. Androgenetic alopecia, the most common type, affects 50% of males over the age of 40 and 75% of females over 65. Only two drugs have been approved so far (minoxidil and finasteride) and hair transplant is the other treatment alternative. This review surveys the evidence for low‐level laser therapy (LLLT) applied to the scalp as a treatment for hair loss and discusses possible mechanisms of actions.

Antigen-specific immunity does not mediate acute regression of UVB-induced p53 -mutant clones

Chronic irradiation of human or murine epidermis with ultraviolet B (UVB) induces clones of p53-mutant keratinocytes. Clones precede and parallel the induction of carcinomas, suggesting that they are an early stage of UVB carcinogenesis. In the absence of UVB, these clones rapidly regress. For UVB-induced murine skin tumors and papillomas, regression is known to involve antigen-specific immunity. To determine whether antigen-specific immunity influences the creation, expansion, or regression of p53-mutant clones, we studied Rag1 knockout mice deficient in the recombination activating gene 1 required for development of B, αβT, γδT, and natural killer T cells. Since tissue homeostasis could affect proliferation or persistence of clones, we also examined the effect of Rag1 on UVB-induced hyperplasia and apoptosis. Mice were irradiated with UVB daily for 7–11 weeks to create p53-mutant clones, and then retained in the absence of UV. After UV ended, epidermal thickness decreased and p53-mutant clones observed in the epidermal sheets regressed, with no significant differences between Rag1−/− and wild type. During the initial chronic UVB irradiation, increasing irradiation time increased both the number and size of p53-mutant clones, with no significant difference between genotypes. We conclude that antigen-specific immunity is not involved in the initiation, expansion, or acute regression of p53-mutant clones.

Reduced inflammatory threshold indicates skin barrier defect in transglutaminase 3 knockout mice

Recently, a transglutaminase 3 knockout (TGM3/KO) mouse was generated that showed impaired hair development, but no gross defects in the epidermal barrier, although increased fragility of isolated corneocytes was demonstrated. Here we investigated the functionality of skin barrier in vivo by percutaneous sensitization to FITC in TGM3/KO (n=64) and C57BL/6 wild-type (WT) mice (n=36). Cutaneous inflammation was evaluated by mouse ear swelling test (MEST), histology, serum IgE levels, and by flow cytometry from draining lymph nodes. Inflammation-induced significant MEST difference (P<0.0001) was detected between KO and WT mice and was supported also by histopathology. A significant increase of CD4+ CD25+-activated T cells (P<0.01) and elevated serum IgE levels (P<0.05) in KO mice indicated more the development of FITC sensitization than an irritative reaction. Propionibacter acnes-induced intracutaneous inflammation showed no difference (P=0.2254) between the reactivity of WT and KO immune system. As in vivo tracer, FITC penetration from skin surface followed by two-photon microscopy demonstrated a more invasive percutaneous penetration in KO mice. The clinically uninvolved skin in TGM3/KO mice showed impaired barrier function and higher susceptibility to FITC sensitization indicating that TGM3 has a significant contribution to the functionally intact cutaneous barrier.

Eosinophilic fasciitis associated with Mycoplasma arginini infection

ABSTRACT Eosinophilic fasciitis (EF) with generalized sclerodermiform skin lesions developed over a 19-month period in a previously healthy 23-year-old man. Although we confirmed EF by skin histology and laboratory tests, the recurrent fevers and the clinical observation of sclerotic prepuce with urethritis indicated further bacteriological analysis by conventional microbiological and DNA-based tests. Urethra cultures were positive for an arginine-hydrolyzing mycoplasma and Ureaplasma urealyticum. The patient also had serum IgM antibodies to Mycoplasma pneumoniae using enzyme-linked immunosorbent assay (ELISA)-based qualitative detection. Mycoplasma arginini was isolated from two independent venous blood serum samples and was identified by conventional microbiological tests and sequencing of the 16S rRNA and rpoB genes (GenBank sequence accession numbers HM179555 and HM179556, respectively). M. arginini genomic DNA also was detected by species-specific PCR in the skin lesion biopsy sample. Treatment with corticosteroids and long-term courses of selected antibiotics led to remission of skin symptoms and normalization of laboratory values. This report provides the first evidence of EF associated with mycoplasma infection and the second report of human infection with M. arginini and therefore suggests that this mycoplasma infection might have contributed to the pathogenesis of the disease.

Novel sphingolipid derivatives promote keratinocyte differentiation

Abstract:  Sphingolipids are important components of the water permeability barrier of the skin. Moreover, ceramides were also shown to influence keratinocyte differentiation and regulate cellular signalling. A confluence‐induced differentiation model of normal human keratinocytes was established to allow evaluation of pro‐ and anti‐differentiation effects of exogenous compounds. The effects of phytosphingosine (PS), sphingosine (SO), sphinganine (SA) and their hexanoyl (–C6), stearoyl (–C18) and salicyl (–SLC) derivatives, C12‐alkylamine‐salicylate (C12‐SLC), salicylate (SLC) along with vitamin D3 (VD3) and retinol as control substances were tested in this system. Cytotoxicity assays were carried out to optimize the incubation conditions of compounds and whole genome expression changes were monitored by DNA‐microarray on days 0, 1 and 4. Geometric means of gene expression levels of a subset of known keratinocyte differentiation‐related genes were calculated from the microarray data to compare effects of the sphingolipid derivatives. Compound treatment‐induced transcriptional changes were analysed by the ExPlain™ software (BIOBASE GmbH). Five of the assayed substances (SA, SO‐C6, PS‐C6, SO‐SLC, PS‐SLC) were found to be potent promoters of keratinocyte differentiation compared with VD3, and C12‐SLC revealed potential anti‐differentiation properties. ExPlain™ analysis found a different regulatory profile in the computed transcriptional networks of the sphingoid bases versus their –C6 and especially –SLC derivatives suggesting that the change in their keratinocyte differentiation modifying potential is due to a unique effect of the covalent attachment of the salicylic acid. Taken together, these results demonstrate the gene regulatory potential of sphingolipid species that could be valuable for dermatological or cosmetic applications.

UVB induces a biphasic response of HIF‐1α in cultured human keratinocytes

Abstract:  Hypoxia in the skin is important in chronic degenerative dermo‐epidermal changes, inflammation, photoageing and carcinogenesis. In these processes, vascular endothelial growth factor (VEGF) plays a crucial role and is known to be affected by ultraviolet radiation (UVR). Hypoxia‐inducible factor‐1 (HIF‐1) closely regulates the expression of VEGF in several experimental settings. We set out to study the impact of acute UVB irradiation on the level of HIF‐1 as a major regulator of hypoxia‐induced genes. Effects of UVB exposure on HIF‐1α expression were investigated in HaCaT cells after a single irradiation by Western blots. Downstream target gene expression was measured by quantitative real‐time polymerace chair reaction (PCR). UVB treatment resulted in an initial decrease of the HIF‐1α protein level followed by a subsequent prolonged increase. If cells were exposed to additional UVB irradiation, another decrease in HIF‐1α was provoked, similar to the original effect. The observed changes followed a strict timeline and were dose‐dependent. The role of the PI3K/AKT pathway was examined. No change in the total level of AKT after UVB treatment was seen; however, its phosphorylation level was found to be markedly higher. In accordance with these observations, wortmannin, an inhibitor of PI3‐kinase effectively blocked the UVB‐induced increase in HIF‐1α. In agreement with previous findings, UVB irradiation increased VEGF and haem oxygenase‐1 mRNA levels determined by quantitative real‐time PCR. It is concluded that changes in HIF‐1α expression underlie the alterations in expression of VEGF upon UVB irradiation. Our findings indicate the involvement of PI3K in UVB‐mediated HIF‐1α upregulation.

Dirofilaria repens infection case in Hungary: a case report

A 51‐year‐old female developed urticarial lesions of her right forearm which progressed into transient edema and subcutaneous swelling. Later a small infiltrated subcutaneous nodule also appeared and was removed in toto. Histopathological examination revealed the presence of Dirofilaria repens.This worm is the cause of an endemic zoonosis in the Mediterranean area. In the past decade many cases have been reported worldwide, but the condition appears rare in Hungary and skin findings have not been described.

Whole genome transcriptional profiling identifies novel differentiation regulated genes in keratinocytes

Please cite this paper as: Whole genome transcriptional profiling identifies novel differentiation regulated genes in keratinocytes. Experimental Dermatology 2010; 19: 100–107.