Laboratories

Research

Main Scientific Achievements

• Elucidating the molecular mechanism of action of flavonoids and nonsteroidal antiinflammatory drugs on cellular processes.
• Decoding the exact mode of genistein’s actions on the human body, including positive modulation and activity of the lysosome biogenesis factor TFEB and negative regulation of the serine-threonine kinase (mammalian/mechanistic target of rapamycin) pathway.
• Defining the role of the lysosome in Ps. Lysosomal signaling in Ps was associated with the modulation of immune-mediated inflammation that accompanies this dermatosis.

Research Description

• Work on the molecular mechanism of action of isoflavone G, which has properties that may be responsible for correcting cellular changes that accompany the immuno-genetic disorder psoriasis (Ps)

The study investigated the anti-Ps activity of G in vitro (model of “Ps-like” keratinocytes) and in vivo (Ps patients). Gene expression analyses revealed transcriptional changes that confirmed known disease-associated pathways and highlighted many Ps-related genes. Our results suggested that the aberrant activity of genes that contribute to the progression of Ps could be improved by the actions of the tested isoflavone. These in vitro investigations are ongoing and will be followed by studies of the dysregulation of intercellular communication (including mitogen-activated protein kinase signal transduction pathway and its downstream cascades) by G. Because investigations of other groups supported our findings on G as a potential treatment for Ps, the impact of the isoflavone on clinical scores in moderate-to-severe Ps patients was further explored in clinical trials and in vivo tests. We sought to develop innovative technologies for G production and its use as an oral pharmaceutical formulation. Safety, tolerability, pharmacodynamics effects, and efficacy in treating Ps patients were evaluated, together with the determination of cytokine levels in serum. We did find evidence of G-dependent improvements in Ps clinical severity scores. A certain regression of the Ps phenotype was observed, based on photo-documented Ps lesions. This isoflavone was well tolerated by 85% of patients, and no serious adverse events or discontinuations of treatment occurred. No dose-limiting toxicity or substantial changes in pharmacodynamic parameters were observed. Subtle modulation of the cytokine profile in serum was found in all tested patients. The positive results of our research became the basis of performing in vivo tests to determine changes in gene expression in patients with Ps as a result of the action of G. We tested the effects of this isoflavone on transcript levels in both skin specimens and peripheral blood cells in Ps subjects and found that this compound modulated the activity of genes that encode anti-Ps members and antiinflammatory mediators of inflammation. Isoflavone G impaired the activity of certain genes which are overexpressed in Ps and stimulated the expression of other transcripts that are repressed in dermatosis.

• Work on the molecular mechanism of the pathogenesis and treatment of Ps

The aim of our work was to reveal the molecular basis of the regulation of lysosomal activity, the potential effects of the therapeutic intervention in inflammatory skin diseases, and the participation of lysosomal signaling in the modulation of inflammatory response on the example of Ps. Of all the components of the cell, the lysosome was an obvious candidate to investigate its role in the inflammatory process. Our research included work on the key role of EB transcription factor (TFEB) in cellular metabolism. The latest findings of TFEB as a master regulator of lysosome function raises the possibility that this factor may be crucially important in linking lysosome dysfunction with immune-mediated inflammatory disorders. In vitro and in situ studies in this area are ongoing and will be followed by end-point validation studies in in vivo animal models. In parallel, we will establish and evaluate psoriasiform inflammatory models in vitro using monocultures of keratinocytes and co-cultures of keratinocytes plus immune cells that are stimulated by a combination of specific factors that mimic processes leading to Ps lesion formation and the skin phenotype arising from this disease. Overall, we aim to contribute to the research related to the molecular pathogenesis of Ps, in order to identify key pathogenic skin-specific molecules that can be targeted for treatment. This work will be validated with meta-data and expression patterns of GEO profiles with regard to both Ps skin lesions and in vitro cultures of Ps keratinocytes.

• Work on the calcium signaling network in T lymphocytes that involves nuclear and lysosomal transcription factors in the context of Ps

This ongoing investigation is analyzing details of lysosomal metabolism in CD4+ Th lymphocytes in Ps patients and evaluating the role of cellular Ca²⁺ levels in activation and regulation of the inflammation process that accompanies this disease. Our scientific hypothesis is that the calcium signaling network in T cells involves NFAT and TFEB transcription factors that are stimulated in parallel. NFAT T-cell activation and lysosomal biogenesis that are stimulated by TFEB are processes that interact and have common effects on cytokine production and release to sustain the inflammatory state that is associated with Ps and are regulated by intracellular Ca²⁺ levels.

• Work on the connection between endosomal-lysosomal system dysregulation and the antigen presentation process that is mediated by MHC class II molecules in relation to Ps

This study was initiated recently to analyze quantitative changes in acidic organelles in keratinocytes with a Ps phenotype and qualitative disturbances of these organelles that affect the abnormal process of antigen endocytosis and further processing that involves lysosomal proteolytic enzymes. The formation of abnormal antigen fragments that are presented by MHC class II molecules and may be the cause of excessive T-cell activation and intensification of the immune response leading to chronic inflammation that is observed in Ps are examined.

Methodology

• In vitro, in situ, and in vivo work, cell cultures, the handling of patient samples, clinical trials, skin biopsies, blood sample work, cell transfections, RNA and DNA work, electrophoresis, gene expression analysis (real-time quantitative reverse transcription polymerase chain reaction [qRT-PCR], microarrays, RNA sequencing, and gene silencing), protein expression analysis (electrophoresis and immunoblotting tests, including Western blot, enzyme-linked immunosorbent assay ELISA, MagPix, and immunohistochemistry IHC), the estimation of enzymatic activity, microscopic analyses (transmission electron microscopy TEM and immunofluorescence IF), and statistical data analysis.
• Equipment: Our laboratory is equipped with CO₂ incubators, laminar flow chambers, centrifuges, water baths, heat shakers, freezers, deep-freezers, a real-time qRT-PCR LightCycler System 480, Agilent 2100 Bioanalyzer, the Luminex equipment MagPix, light inverted microscope, fluorescent inverted microscope, electron microscope, Merck Millipore Muse Cell Analyzer, Perkin Elmer spectrophotometer (Victor 3 1420-012), NanoDrop UV-Vis spectrophotometer, QuadroMACS Separator, and a broad range of basic laboratory equipment.

Selected Publications

• The phytoestrogen genistein modulates lysosomal metabolism and transcription factor EB (TFEB) activation. Moskot M, Montefusco S, Jakóbkiewicz-Banecka J, Mozolewski P, Węgrzyn A, Bernardo D, Węgrzyn G, Medina LD, Ballabio A, Gabig-Cimińska M. Journal of Biological Chemistry. 2014. doi: 10.1074/jbc.M114.555300.
• Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids. Moskot M, Jakóbkiewicz-Banecka J, Kloska A, Smolińska E, Mozolewski P, Malinowska M, Rychłowski M, Banecki B, Węgrzyn G, Gabig-Cimińska M. Scientific Reports. doi: 10.1038/SREP09378.
• Nonsteroidal anti-inflammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways. Mozolewski P, Moskot M, Jakóbkiewicz-Banecka J, Węgrzyn G, Bocheńska K, Banecki B, Gabig-Cimińska M. Scientific Reports. 2017. doi: 10.1038/srep43157.
• Molecular action of isoflavone genistein in the human epithelial cell line HaCaT. Smolińska E, Moskot M, Jakóbkiewicz-Banecka J, Węgrzyn G, Banecki B, Szczerkowska-Dobosz A, Purzycka-Bohdan D, Gabig-Cimińska M. PLoS ONE. 2018. doi: 10.1371/journal.pone.0192297.
• A new potential mode of cardiorenal protection of KLOTHO gene variability in type 1 diabetic adolescents. Słomiński B, Ryba-Stanisławowska M, Skrzypkowska M, Gabig-Cimińska M, Myśliwiec M Journal of Molecular Medicine. 2020. doi: 10.1007/s00109-020-01918-7.

Collaborations

• Andrea Ballabio, Dr. Diego Medina, Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy; Texas Children Hospital and Baylor College of Medicine in Houston, USA.
• Aneta Szczerkowska-Dobosz, Medical University of Gdansk (MUG), Faculty of Medicine, Department of Dermatology, Venereology and Allergology, Gdansk, Poland.

Prizes and Awards

• Marta Moskot. Scholarship for excellent PhD students. 2014. Ministry of Science and Higher Education, Poland.
• Magdalena Gabig-Cimińska. Award of PGS. 2010. Polish Genetic Society, Poland.
• Magdalena Gabig-Cimińska, Marta Moskot. Award of Division II: Biological and Agricultural Sciences of PAS. 2016. Polish Academy of Sciences, Poland.
• Marta Moskot. Scholarship for outstanding young scientists. 2016. Ministry of Science and Higher Education, Poland.
• Marta Moskot. Award for outstanding PhD dissertation. 2017. Prime Minister of Poland, Poland.