Franz Trautinger

UVA and UVB radiation differentially regulate vascular endothelial growth factor expression in keratinocyte-derived cell lines and in human keratinocytes.

Vascular endothelial growth factor (VEGF) is a central regulator of neoangiogenesis in inflammatory and neoplastic conditions. Ultraviolet irradiation is one of the mainstays of dermatological therapy for various inflammatory skin diseases. In the present study we have compared the effects of UV irradiation on the production of VEGF by keratinocytes (KC) and by the KC‐derived cell lines A431 and HaCaT. Irradiation of A431 and HaCaT cells with both UVA (10 J/cm2and 20 J/cm2) and UVB (8 mj/cm2and 16 mj/cm2) led to strong upregulation of VEGF mRNA and protein. Induction of VEGF by UVA and UVB in these cells was mediated by different pathways, i.e. the generation of free radicals and the secretion of (a) soluble factor(s), respectively. Unlike KC‐derived cell lines, no increase in VEGF production was observed in KC in primary culture after irradiation with the same UV doses. Increasing the irradiation dose in these cells of UVA to 40 J/cm2led to a marked decrease in soluble VEGF, whereas doses as high as 32 mj/cm2UVB only minimally affected VEGF levels. Reduction of VEGF production by KC might contribute to the effect of UVA irradiation in inflammatory skin diseases. The differential response of primary KC and autonomously growing KC‐derived cell lines to the induction of VEGF by UV light could favor neoangiogenesis in the vicinity of epidermal tumor cells in vivo, thereby endowing them with a growth advantage over normal cells.

The hsp27 kD heat shock protein and p38-MAPK signaling are required for regular epidermal differentiation

BACKGROUNDnIn human epidermal keratinocytes the expression of hsp27 is closely related to differentiation in vitro and in situ.nnnOBJECTIVEnWe aimed to gain further insight into the role of hsp27 in epidermal differentiation by specific inhibition through siRNA and inhibition of p38-MAPK, the key enzyme of hsp27 phosphorylation.nnnMETHODSnNormal human keratinocytes (KC) and organotypic skin cultures (SE-skin equivalents) were used. Expression and phosphorylation of hsp27 was inhibited in these models by siRNA and SB203580, a specific inhibitor of p38-MAPK, respectively. Modification of morphology and expression of hsp27 and other differentiation associated proteins was investigated by immunofluorescence, western blot, and RT-PCR.nnnRESULTSnInhibition of p38-MAPK resulted in a downregulation of hsp27 in KC and SE. Additionally, in the presence of SB203580 Ca(2+) induced expression of pro-filaggrin and loricrin was inhibited at the protein level and expression of filaggrin, keratin 10, and transglutaminase 1 at the mRNA level. Addition of SB203580 to SE, as well as hsp27 knockdown in this model resulted in identical patterns of irregular differentiation, disturbance of epidermal layers, and delayed expression of K10.nnnCONCLUSIONnThese results provide evidence that the expression of hsp27 and its phosphorylation by p38-MAPK are required for keratinocyte differentiation and for the formation of a regularly stratified epidermis.

Heat shock proteins in the skin.

Heat shock proteins (hsp) are expressed in all cells and organisms. Their expression is induced by heat shock (temperatures above 42°C) and other forms of pathophysiological stress. Elevated levels of hsp protect cells from further stress exposure. Hsp are expressed intracellularly. They are highly conserved throughout evolution indicating hsp being necessary for survival under potentially harmful environmental conditions. Hsp are divided into families according to their molecular weight. The majority of hsp function as molecular chaperones. Chaperone function is characterized by binding to other proteins and mediating their folding, transport and interaction with other molecules. In human epidermis hsp are abundantly expressed and have been linked with functions in cell differentiation and photobiology. Recent research has mainly focused on the 27 and 72u2003kD hsp that are constitutively expressed in human keratinocytes. ultraviolet radiation (UV)‐induced cell death and sunburn cell formation can be inhibited by previous heat shock exposure and UV itself can induce hsp expression. The expression of the 27u2003kD hsp (hsp27) in epidermal keratinocytes in situ and in culture correlates with differentiation. Expression of hsp27 increases simultaneously with keratinocyte differentiation. For that reason, hsp27 is described as a marker of epidermal differentiation. Changes in the expression and inducibility of hsp have been linked with ageing. In the skin, recent data indicate that hsp72 expression remains remarkably stable with intrinsic ageing. In contrast, levels of hsp27 have been found to be elevated in sun‐protected aged skin indicating a link between hsp27 expression and age‐dependent epidermal alterations. Regulation of hsp can be modified by pharmacological intervention and the development of safe topical and systemic treatments for the prevention of skin damage and disorders of keratinocyte differentiation can be expected for the future.

Photodegradation of folic acid during extracorporeal photopheresis

Backgroundu2002 Photodegradation of folic acid (FA) by ultraviolet (UV) radiation is a well‐documented photochemical reaction, and decreased serum levels of FA have been found in patients receiving photochemotherapy (psoralen plus UVA). During extracorporeal photopheresis (ECP) leucocytes and plasma are subjected to 8‐methoxypsoralen (8‐MOP) plus UVA.

UVA activated 8-MOP and chlorpromazine inhibit release of TNF-α by post-transcriptional regulation

There is evidence that regulation of inflammatory cytokines is among the immunomodulatory effects of photochemotherapy with 8-MOP and UVA. We have recently demonstrated that in the monocytoid cell line U937 incubation with 8-MOP and subsequent exposure to UVA is able to efficiently downregulate the release of TNF-alpha into the culture supernatant. Chlorpromazine, a well known photosensitising drug, was even more potent with regard to this effect. Based on these observations, in this study we further investigate the mechanisms of TNF-alpha inhibition by 8-MOP and CPZ photosensitization. For this purpose we determined intracellular protein levels and gene expression of TNF-alpha by western blot and quantitative real-time PCR, respectively. Our results indicate that the observed inhibition of TNF-alpha secretion after photochemotherapy is not due to downregulation of gene transcription but rather to a post-transcriptional mechanism. The observed decrease of intracellular TNF-alpha with CPZ and 8-MOP points to decreased protein synthesis or enhanced degradation. These findings demonstrate that posttranscriptional regulation of cytokine expression is a possible mechanism of action of photochemotherapy.

Ultraviolet‐A and ‐B Differentially Modify the Tyrosine‐Kinase Profile of Human Keratinocytes and Induce the Expression of Arg†

To investigate the expression profile of protein tyrosine kinases (PTKs) in normal human epidermal keratinocytes (NHEK) in response to UVA and UVB we employed a reversed transcriptase polymerase chain reaction (PCR) approach using degenerate primers derived from the conserved catalytic domain of PTKs. Quantitative real‐time PCR with specific primers was used to confirm the influence of UV on the expression of the identified PTKs. Arg (Abelson‐related gene, Abl2) was the PTK with the highest prevalence (30% of all PTKs) and UVA led to a further induction of Arg expression reaching nine‐fold mRNA baseline expression at 17u2003h after irradiation. UVB was followed by an initial downregulation and a subsequent increase in Arg mRNA reaching five‐fold baseline levels after 24u2003h. We conclude that UVA and UVB differentially modify the expression of PTKs in NHEK, and that Arg appears to have a major role in the response of keratinocytes to UV. These results provide a basis for further studies of PTK in UV‐induced signaling that regulates protective responses, cell growth and carcinogenesis in the skin.

Photo- und Photochemotherapie des kutanen T-Zell-Lymphoms

Unter den kutanen T-Zell-Lymphomen (CTCL) versteht man eine Gruppe verschiedener Non-Hodgkin-Lymphome, die sich primar an der Haut manifestieren und deren Infiltrate zum Grosteil aus malignen T-Lymphozyten bestehen. Die haufigste klinische CTCL-Variante ist die sogenannte Mycosis fungoides (MF). Die MF gehort zu den niedrigmalignen Non-Hodgkin-Lymphomen, deren klinischer Verlauf durch eine langsame Progression von einem fruhen Plaquestadium uber ein Tumorstadium bis hin zur Lymphknoten- und Organbeteiligung gekennzeichnet ist. Haufig uberlappen sich jedoch die klinischen Stadien, oder es kommt zum Uberspringen einzelner Stadien. Das Sezary-Syndrom (SS) ist eine Sonderform der MF mit generalisiertem Hautbefall im Sinne einer Ery-throdermie und einer Leukozytose mit malignen Lymphozyten. Weitere typische klinische Zeichen sind Befall der Handflachen und Fussohlen mit palmoplantarer Hyperkeratose und tumorose Infiltrate des Gesichtes (Facies leonina). MF und SS sind histopathologisch durch infiltrierende und/oder zirkulierende kleine T-Lymphozyten mit zerebriformen Zellkernen gekennzeichnet, die typischerweise den T-Helferphanotyp aufweisen (CD3+, CD4+, CD8+) .