Gabriele Klosner

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

BACKGROUND In human epidermal keratinocytes the expression of hsp27 is closely related to differentiation in vitro and in situ. OBJECTIVE We 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. METHODS Normal 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. RESULTS Inhibition 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. CONCLUSION These 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 72 kD 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 27 kD 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.

Dermal infiltrates of cutaneous T‐cell lymphomas with epidermotropism but not other cutaneous lymphomas are abundant with langerin+ dendritic cells

Background  The Langerhans cell (LC) hypothesis suggests that cutaneous T‐cell lymphomas (CTCL) are diseases of chronic T‐cell stimulation by LC‐mediated antigen presentation.

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 17 h after irradiation. UVB was followed by an initial downregulation and a subsequent increase in Arg mRNA reaching five‐fold baseline levels after 24 h. 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.

Abstract 1091: A new image processing approach for functional context-based analysis of tryptase positive mast cells in cutaneous T-cell lymphomas

Quantification data on mast cells is scarce in primary cutaneous T-cell lymphomas (CTCL), a heterogeneous group of non-Hodgkin lymphomas with initial presentation in the skin. The aim of the study was to develop a new whole-slide in-silico analysis method for immunohistochemistry stained skin sections. The design of the analysis method was focused on results about structural context of the cell location distribution and cell morphometry linked to mast cell activation states. Immunohistochemistry staining with a monoclonal anti-mast cell tryptase antibody was performed on paraffin embedded biopsies from patients with different CTCL subtypes and controls (normal skin, inflammatory skin diseases),while hematoxylin was used for counter staining. Slides were scanned with a TissueFAXS 200 Cytometer (TissueGnostics GmbH, Vienna). Whole-slide digital images were analyzed using StrataQuest 5.0 Advanced (TissueGnostics, Vienna). Hematoxylin+ cell nuclei and tryptase+ cells (TRYP + ) objects were automatically detected using the available analysis modules with adaptive segmentation of color-deconvoluted images. Mast cells were first selected as brown-positive cells in a peri-nuclear compartment, then further stratified depending on the spatial distribution of brown-positive dots inside the cellular compartment. In an additional analysis layer, an algorithm pipeline for the identification of the epidermis / dermis area was assembled. Density-based CTCL areas were segmented in a third independent pipeline. Inside dermis, two additional CTCL-proximity masks were defined at 0-30μm away from CTCL (P1 mask), as well as in the 30-60μm interval (P2 mask). Typically, there were more mast cells inside CTCL areas than in P1, which in turn had less than the P2 mask. This distribution was especially noticed in Mycosis fungoides(MF) IIB, where CTCL had 2-3 times more mast cells than P1 mask had. However, this trend was not consistent in all types of lymphoma. Interestingly, MF IIA had a different distribution showing more mast cells in P1 than in CTCL areas. The new algorithms provided a rich data insight on functional context-based characteristics and propose new surrogate markers for further characterization of CTCL. The analysis time needed for statistically relevant large whole-slide tissue sections was drastically reduced when using the computer-aided approach when compared against full manual annotation and counting. Moreover, distance assessment and morphometry parameter quantification for each cell are only feasible in in-silico approaches. Note: This abstract was not presented at the meeting. Citation Format: Radu Rogojanu, Johanna Eder, Waltraud Jerey, Gabriele Klosner, Verene Paulitschke, Isabella Ellinger, Theresia Thalhammer, Dan Kolmer, Franz Trautinger. A new image processing approach for functional context-based analysis of tryptase positive mast cells in cutaneous T-cell lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1091. doi:10.1158/1538-7445.AM2015-1091