LC-MS/MS analysis of lesional and normally looking psoriatic skin reveals significant changes in protein metabolism and RNA processing.

BACKGROUND: Plaque psoriasis is a chronic autoimmune disorder characterized by the development of red scaly plaques. To date psoriasis lesional skin transcriptome has been extensively studied, whereas only few proteomic studies of psoriatic skin are available. AIM: The aim of this study was to compare protein expression patterns of lesional and normally looking skin of psoriasis patients with skin of the healthy volunteers, reveal differentially expressed proteins and identify changes in cell metabolism caused by the disease. METHODS: Skin samples of normally looking and lesional skin donated by psoriasis patients (n = 5) and samples of healthy skin donated by volunteers (n = 5) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After protein identification and data processing, the set of differentially expressed proteins was subjected to protein ontology analysis to characterize changes in biological processes, cell components and molecular functions in the patients' skin compared to skin of the healthy volunteers. The expression of selected differentially expressed proteins was validated by ELISA and immunohistochemistry. RESULTS: The performed analysis identified 405 and 59 differentially expressed proteins in lesional and normally looking psoriatic skin compared to healthy control. In normally looking skin of the patients, we discovered decreased expression of KNG1, APOE, HRG, THBS1 and PLG. Presumably, these changes were needed to protect the epidermis from spontaneous activation of kallikrein-kinin system and delay the following development of inflammatory response. In lesional skin, we identified several large groups of proteins with coordinated expression. Mainly, these proteins were involved in different aspects of protein and RNA metabolism, namely ATP synthesis and consumption; intracellular trafficking of membrane-bound vesicles, pre-RNA processing, translation, chaperoning and degradation in proteasomes/immunoproteasomes. CONCLUSION: Our findings explain the molecular basis of metabolic changes caused by disease in skin lesions, such as faster cell turnover and higher metabolic rate. They also indicate on downregulation of kallikrein-kinin system in normally looking skin of the patients that would be needed to delay exacerbation of the disease. Data are available via ProteomeXchange with identifier PXD021673.

[Genetically predetermined limitation in the use of HaCaT cells that affects their ability to serve as an experimental model of psoriasis].

Proinflammatory cytokines TNF, IFNG and ILl7 play an important role in eruption of psoriasis. The activation of epidermal keratinocytes with the named cytokines alters their terminal differentiation program and causes their hyperproliferation in the diseased skin. HaCaT cells, which are immortalized human keratinocytes, are often used as a cellular model of psoriasis. The aim of this study was to evaluate changes in gene expression and the proliferation rates in cultured HaCaT cells treated with TNF, IFNG and IL17. We found that HaCaT cells decrease their proliferation rate in response to either IL17 or a combination TNF and IF-NG. The analysis of microarray data discovered a group of 12 genes, which were downregulated in HaCaT after treatments with the named cytokines and upregulated in psoriatic lesional skin. Eight genes were important for DNA replication and they also contributed to two larger networks that regulated cell progression through the cell cycle. We conclude that HaCaT cells have a sufficient limitation as a cellular model of psoriasis due to their treatment with proinflammatory cytokines, namely TNF, IFNG and IL17 does not increase their proliferation rate. Thus, the studies of psoriasis based on HaCaT cells as an experimental model shall take in account this important phenomenon.

[Genetically modified animals as model systems of psoriasis].

Psoriasis is a chronic autoimmune skin disorder. Experimental models of psoriasis can be used to study the disease in controlled conditions. Moreover, the experimental models allow to study a certain aspect of the pathological process. Although none of the multiple mouse models reproduces the human disease precisely, lab animals as model systems can be very helpful because of two reasons. First, introduction of new mutations into animal genome allows to reveal the new genes that may play a certain role in pathogenesis of the disease. Second, the experiments that are carried on the lab animals can be used for testing the new drugs and selection of the most efficient chemical agents from a variety of the proposed experimental preparations. The aim of this paper was to summarize the data on the lab animals that serve as experimental models of psoriasis.

Pharmacological control of receptor of advanced glycation end-products and its biological effects in psoriasis.

Receptor for advanced glycation end-products is implicated in a development of chronic inflammatory response. Aim of this paper is to provide a review on commercial and experimental medicines that can interfere with RAGE and signaling through RAGE. We searched three bibliographical databases (PubMed, Web of Science and MEDLINE) for the publications from 2005 to March 2012 and identified 5 major groups of agents that can interfere with RAGE biological effects. In the first part of this paper, we discuss AGE crosslink breakers. These chemicals destroy advanced glycation end products (AGEs) that are crosslinked to the extracellular matrix proteins and can interact with RAGE as ligands. Then, we describe two non-conventional agents SAGEs and KIOM-79 that abolish certain biological effects of RAGE and have a strong anti-inflammatory potential. In the third part, we evaluate the inhibitors of the signaling cascades that underlie RAGE. Particularly, we discuss two groups of kinase inhibitors tyrphostins and the inhibitors of JAK kinases. Considering RAGE as a potential master regulator of processes that are crucial for the pathogenesis of psoriasis, we propose that these medicins may help in controlling the disease by abolishing the chronic inflammation in skin lesions.

[Role of receptor for advanced glycation end-products in pathgenesis of psoriasis].

This review summarizes the existing knowledge regarding the role of receptor for advanced glycation end products which is a key participant of the inflammatory process, in pathogenesis of psoriasis. By interacting with multiple ligands and activating several signaling mechanisms, receptor for advanced glycation end products regulates gene expression via a group transcription factors, that includes NFKB and AP1. According to the published data the expression of receptor for advanced glycation end products in both immune cells and their targets, a high stability of this receptor in complexes with ligands as well as a positive feedback loop, upregulating the expression of its certain ligands, suggest receptor for advanced glycation end products as a possible principal factor that makes the inflammatory response in psoriasis sustainable. Considering receptor for advanced glycation end products as a potential master regulator of several processes that play a crucial role in development of psoriatic plaques, we believe that further experimental studies are needed to elucidate how exactly this receptor converts a transient inflammatory reaction to a sustainable inflammatory response. These studies are also needed for the development of novel medications that target receptor for advanced glycation end products and signaling mechanisms that this receptor activates.

Three-dimensional model of mouse epidermis for experimental studies of psoriasis.

Three-dimensional models of skin and epidermis imitate the structure of real tissues and provide accurate information about certain skin conditions, such as psoriasis. A three-dimensional model of mouse epidermis was generated from the epidermal keratinocytes of newborn mice and treated with cytokines. The aim of this study was to evaluate this model as an experimental model of psoriasis and to assess the changes occurring in its structure and gene expression after the exposure to proinflammatory cytokines. Treatment of the three-dimensional model with either interleukin 17 or a combination of tumor necrosis factor and interferon γ was shown to produce morphological changes, which were similar to acanthosis in psoriatic skin. The observed changes in gene expression of metalloproteinases and certain psoriasis biomarkers, such as mki67, krt16 and fosl1, were similar to the changes in patients' skin. Notably, changes caused by interleukin 17 were less evident than those caused by the combination of interferon γ and tumor necrosis factor. On the contrary, HaCaT cells exhibited no significant changes in the expression of fosl1 and had decreased levels of mki67 after being treated with a combination of TNF and IFNG. Moreover, treatment with IL17 had no significant effect on krt16 and mki67 expression and even reduced the fosl1 levels. The findings suggest that artificially generated three-dimensional models of murine skin can be used to study psoriasis.

Molecular mechanism of lysosomal sialidase deficiency in galactosialidosis involves its rapid degradation.

Galactosialidosis is an inherited lysosomal storage disease caused by the combined deficiency of lysosomal sialidase and beta-galactosidase secondary to the deficiency of cathepsin A/protective protein, which is associated with sialidase and beta-galactosidase in a high-molecular weight (1.27MDa) complex. Clinical phenotypes of patients as well as the composition of compounds which are stored in patient's tissues implicate sialidase deficiency as the underlying pathogenic defect. The recent cloning and sequencing of lysosomal sialidase [Pshezhetsky, Richard, Michaud, Igdoura, Wang, Elsliger, Qu, Leclerc, Gravel, Dallaire and Potier (1997), Nature Genet. 15, 316-320] allowed us to study the molecular mechanism of sialidase deficiency in galactosialidosis. By Western blotting, using antibodies against the recombinant human enzyme, and by NH2-terminal sequencing, we showed that sialidase is synthesized as a 45.5 kDa precursor and after the cleavage of the 47-amino acid signal peptide and glycosylation becomes a 48.3 kDa mature active enzyme present in the 1.27 kDa complex. Transgenic expression of sialidase in cultured skin fibroblasts from normal controls and from galactosialidosis patients, followed by immunofluorescent and immunoelectron microscopy showed that in both normal and affected cells the expressed sialidase was localized on lysosomal and plasma membranes, but the amount of sialidase found in galactosialidosis cells was approximately 5-fold reduced. Metabolic labelling studies demonstrated that the 48.3 kDa mature active form of sialidase was stable in normal fibroblasts (half-life approximately 2.7 h), whereas in galactosialidosis fibroblasts the enzyme was rapidly converted (half-life approximately 30 min) into 38.7 and 24 kDa catalytically inactive forms. Altogether our data provide evidence that the molecular mechanism of sialidase deficiency in galactosialidosis is associated with abnormal proteolytic cleavage and fast degradation.